Single Tube Multiplex PCR, Liquid Hybridization, and ELISA (Multiplex NAT-ELISA) for rapid detection of HIV, HBV, and HCV in the window period.

Single Tube Multiplex PCR, Liquid Hybridization, and ELISA (Multiplex NAT-ELISA) for rapid detection of HIV, HBV, and HCV in the window period. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Single Tube Multiplex PCR, Liquid Hybridization, and ELISA (Multiplex NAT-ELISA) for rapid detection of HIV, HBV, and HCV in the window period. Priyanka Sharma, Gunjan Batheja, Hriday Narayan Verma, Pradeep Seth This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1708998/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: A molecular assay based on single-tube multiplex PCR, liquid hybridization and indirect ELISA has been designed for rapid detection of HIV-1 & 2, HCV, and HBV in plasma samples (multiplex NAT-ELISA) during the window period of these infections. Methods: Nucleic acid extracted from the virus standards and blood donor plasma samples were amplified by conventional reverse transcription-PCR with a mixture of optimized concentrations of forward and biotinylated reverse primers followed by liquid hybridization of immobilized denatured biotinylated amplicons with digoxigenin-labeled specific probes and indirect ELISA to measure hybridization signals colorimetrically. The performance of Multiplex NAT-ELISA was validated by Analytical Sensitivity, Analytical Specificity, and by the “Accuracy” (Trueness) of the assay. Results: Performance characteristics of the assay were established. Analytical sensitivity (LOD95) was 17 IU/ml (10 geq/ml) for HIV-1) 13 IU/ml (73 geq /ml) for HBV, and 15 IU/ml (66 geq/ml) for HCV. A comparison of the assay with the serological screening tests for HIV/HCV antibodies and HBsAg shows a concordance of 98%. Five serologically negative samples yielded HBV DNA in 4 samples and HCV RNA in one sample by NAT-ELISA. Conclusions: Multiplex NAT-ELISA assay described here is performed on individual donations. The main advantage of the assay is the interdiction of HIV/HCV/HBV virus infections offering a significantly higher sensitivity for detecting blood-borne infections at an affordable cost (less than US$ 5 per sample). Moreover, it has analytical sensitivity that matches that of Procleix Ultrio System and Cobas TaqScreen MPX. Virology PCR Liquid hybridization ELISA Blood bank Analytical sensitivity NAT-yield Introduction National AIDS Control Organization (NACO) mandated screening blood donors by serological tests as a part of the National Blood Policy in April 2002. Despite a reduction in the risk of transfusion-transmitted infection (TTI) with pathogenic blood-borne viruses particularly HIV, HCV and HBV, by serological screening of donors’ blood samples for HBV surface antigen (HBsAg) and antibodies to HCV and HIV-1& 2 and by improving donor selection, continued transmission of these viral agents still occurs. This has been attributed to the failure of screening serological tests to identify infected donors in antibody/antigen negative window period [ 1 ]. The window period for HIV is 6–38 days (Av. 22 days), for HCV is 54 to 192 days (Av. 82 days) and HBV is 37–87 days (Av. 59 days) [ 2 ]. A significant number of donated blood units is likely to remain unsafe, as it is not screened for the presence of these viruses during window period. The donations screened only by serological assays are still at risk for HBV, HCV and HIV infections. The prevalence of HIV, HBV, and HCV in blood donors in India is about 0.24%, 1.18% and 0.43% [ 3 ] compared to 0.0097%, 0.3%, and 0.07% in the US blood donors, respectively [ 2 ]. In developed countries screening of donated blood units particularly antibody/antigen negative blood units for viral genetic material by Nucleic Acid test (NAT) has been implemented since 1999 [ 4 ]. The primary benefit of NAT is the ability to reduce residual risk of infectious window period donations by detecting the viruses earlier than the serological screening methods thus, shortening the window period. The estimated reduction of the window period utilizing NAT for HCV is from 82 days to12 days, HIV from 22 to 11 days, and HBV from 59 to 25–30 days [ 5 ]. In addition, since NAT is highly sensitive and specific it adds the benefit of resolving false reactive results of serological methods.[ 6 ]. Currently two commercial NAT assays are used for screening blood donors around the world: Transcription Mediated Amplification (TMA) technology based fully automated Procleix Ultrio System (Gen-Probe, Inc. (USA), and PCR/RT-PCR amplification based fully automated Cobas TaqScreen MPX assay (Roche Diagnostics GmbH, Mannheim, Germany) [ 7 ]. Either system has been used in two formats: to test individual donations (ID-NAT) or minipools (MP-NAT) of different sizes comprising 96, 24, 16, 8 or 6 donations [ 8 , 9 ]. Although testing minipools cuts the cost of detection, it compromises the screening sensitivity of the assay depending upon the size of the pool thus reducing the gains of NAT assays. In addition, once the pool is found reactive, all the members of the pool need to be tested individually to identify the reactive unit(s) thus delaying release of donations. Though in India use of NAT for screening the blood donors has been recommended by NACO, it has not been made mandatory because of higher investment for its implementation. Of 1300 registered blood banks in India less than 5% employ NAT for blood screening for HIV, HCV and HBV. Large majority blood banks in India do not use NAT assays to screen the blood donors because of the exorbitant cost of infrastructure required for these assays. Approximately, 7% of all collected blood units are NAT tested in India [ 10 ]. Thus, the potential for an affordable and simple multiplex molecular test to detect the presence of HIV, HCV and HBV in voluntary blood donors in India is staggering, particularly when one takes notice of the high number of carriers of HIV (2.5 million), HBV (43 million), and HCV (15 million). [ 11 – 12 ]. Here we describe the development of an affordable, technically simple single-tube multiplex NAT ELISA assay in which all the four PCR reactions are carried out in a single tube by multiplex reverse transcriptase (rt)-PCR using a mixture of specific primers of HIV-1, HIV-2, HCV and HBV. This is followed by detection of each amplified product by liquid hybridization and ELISA with digoxigenin labelled target specific probes. Unlike other NAT assays, this assay is performed on individual donor plasma sample. Material And Methods Clinical Samples: Blood samples from healthy voluntary donors were collected under anonymous, unlinked conditions and an informed and written consent was taken from all the blood donors as per National AIDS Control Organization (NACO) guidelines. An ethical clearance for this proposal was obtained from the Institutional Ethics Committee (IEC) of National Institute of Nutrition (ICMR), Hyderabad, Telangana, which permitted to collect blood samples from the donors as per National guidelines. The samples were aliquoted and stored at -80 0 C. Three well-characterized blood samples positive for HIV-1(SRF-11, 2.65x10 5 IU/ml) , HCV (SRF-5, 10 1.62 IU/ml and HBV (SRF-8, 10 1.95 IU/ml) for the respective viral genomes by Transcription Mediated Amplification (TMA) assay system in ID-NAT format (Procleix Ultrio; Novartis Diagnostics, USA) were collected for standardization of assay from AIIMS blood bank. Three ml aliquots of 500 hundred fresh blood samples from healthy volunteer blood donors were collected from Indian Red Cross Society (IRCS) blood bank at New Delhi and were transported to the Seth research Foundation, Gurgaon at room temperature in batches of 50-75 blood samples every week from 15 December 2018 to 23 January 2019. The blood samples were drawn after obtaining informed and written consent from each donor before entry in the study. Donated blood did not carry any identification that can be linked to its donor. At the IRCS blood bank each donated blood was screened for HIV& HCV antibodies as well as for hepatitis B surface antigen (HBsAg) by rapid tests/ ELISA/ p24 antigen test for HIV as per NACO guidelines. At the Seth Research Foundation these blood samples were tested for HIV1 & 2 RNA, HCV RNA and HBV DNA by Multiplex NAT-ELISA assay. One hundred and sixty blood samples from blood donor volunteers, which had been tested for the presence of HIV1 & 2/HCV/HBV by serological screening tests as well as by ID NAT assay at AIIMS blood bank were used for determining Cut-off value for interpreting ELISA results. Viruses: WHO International Standards for HIV1 [Code#10/152, 8.5x10 5 IU/mL]; HIV2 [Code#08/150, 10 3 IU/ml]); HBV [Code#10/264, 8.5x10 5 IU/mL]; and HCV [Code#14/150 1x10 5 IU/mL] were obtained from NIBSC E ngland. These were employed for validation of the performance of the assay. In addition, HIV2 virus isolate CBL20 (Cat. # 600) was obtained as cell-free virus with a titer of 9.12x103 TCID50/ml or 15.7x103 IU/ml from NIH AIDS Reagent Program, German Town, USA. MS2 bacteriophage RNA (Concentration 0.8mg/ml, Catalog# 10165948001, Roche Diagnostics USA) was used as an exogenous internal control. All the viruses were stored at -80 0 C. Primers and Probes: Primers and probes described earlier [13] were used in the study. The conserved regions of HIV1 & 2 ( p-24 gene), HCV ( 5’UTR ) HBV ( S gene) and mat gene of MS2 phage RNA were selected for designing primers and oligonucleotide probes. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. The primer pairs and respective hybridizing specific probes were: HIV-1 sense primer (FP-321) 5’-TGGTCCAAAATGCAAA CCCAGA-3’; antisense primer (RP-321) 5’-TAGCCTGCCTCTCAGTACAGTC-3’; probe (Hyb321) 5’-AAAGGCTGTTGGAAATGTGG-3’; HIV-2 (FP-291) 5’-TTGGAGTCAAAAGAGGGTTGCC-3’; (RP-291) 5’CACATGGGTATAATTGCCGCCT-3’; probe (Hyb291) 5’-GGAACTGCAGAGAAAATGCC-3’; HCV (FP-105) 5’-TTCTTGGAACAACCCGCTCAAT-3’; (RP-105) 5’-CTATCAGGCAGTACCACAAGGC-3’; probe (Hyb105) 5’-GAGATCACTAGCCGAGTAGT-3’; HBV (FP-229) 5’-ACTCGTGGTGGACTTCTCTCAA-3’; (RP-229) 5’-AGAGGACAAACGGGCAACATAC-3’; probe (Hyb229) 5’-TTCCTCTTCATCCTGCTGCT-3’. The primer sets were designed in such a manner that there was no cross hybridization with each other and no possibility of mis-priming with other templates present in the same tube. The number of G or C bases within the last five bases from 3’ end of primers (GC clamp) was kept ≤ 3G’s or C’s`. Designing of primers and probes for multiplex PCR was done on software MPprimer [14]. Primer specificity was established by BLAST and primer specificity evaluation program MFEprimer [15]. Reverse primers were biotinylated at 5’end and the probes were tagged with digoxigenin at 3’ end. Multiplex reverse-transcriptase (rt) PCR: Nucleic acid was extracted from the WHO standards/plasma samples with Viral RNA/DNA extraction kit (Cat. # K0821; Thermofisher Scientific, USA) strictly as per manufacturer protocol. Each sample was spiked with 1000 of copies of MS2 phage RNA (internal control) before extraction. Amplification of extracted nucleic acid was undertaken using Qiagen One Step RT-PCR kit (Cat#210210; Qiagen; Netherland) as per manufacturer’s recommendation except that the concentration of MgCl 2 (2.5mM), annealing temperature (55 0 C) and concentration of each set of sense and antisense primers (0.6µM) were optimized for optimum amplification in multiplex PCR reaction. Briefly, 10µl 5X master mix containing 2.5mM MgCl 2 , 2µl dNTP mix (400µM of each dNTP), 2µl of enzyme mix (reverse transcriptase and Taq DNA polymerase) was mixed with primer mix of all the five pairs of sense and biotinylated antisense primers (0.6µM of each primer), 10 units RNase inhibitor, and 10µl of extracted viral nucleic acid. Molecular grade RNase free water was added to make final volume to 50µl. The tubes were then incubated in pre-heated (50 0 C) thermocycler (Bio Rad Laboratories, USA) first for 30 min at 50 0 C for reverse transcription then for 15min at 95 0 C, followed by 35 cycle of three-step amplification (denaturation at 94 o C for 30sec, annealing at 55 0 C for 1min and extension at 72 0 C for 1min) and final extension for 10min at 72 0 C. The amplicons were analyzed by 1.2% agarose gel electrophoresis and liquid hybridization assay as described below. Appropriate steps were taken to prevent carryover contamination during PCR [16]. Hybridization assay: Liquid Hybridization assay and detection of hybridization signal by indirect ELISA were performed as described by earlier [13] with modifications. Briefly, immobilization of biotinylated amplicon onto streptavidin- coated wells of flat-bottom ELISA plates (Cat # 15120, Thermo Fisher Scientific India) at 37 o C for 30 min in a shaker incubator, was followed by chemical denaturation at 25 o C for 30 min. Thereafter, 200ul of hybridization buffer containing digoxigenin (DIG) labeled specific probes HIV1-Hyb (2fmol)/HIV2-Hyb (4fmol)/HCV-Hyb (2fmol)/HBV-Hyb (10fmol)/MS2-Hyb (2fmol) was added in separate wells containing denatured biotinylated amplicons and incubated at 45 0 C for 120 min in a shaker incubator. Each well was washed with stringent wash buffer at 55 0 C for 30 min. Hybridization signal was detected by indirect ELISA after incubating with HRP conjugated anti-digoxigenin sheep antibody (Cat # 11207733910, Sigma Aldrich, India) at 37 0 C for 30 min, followed by incubating with 3,3’,5,5’ tetramethylbenzidine-substrate (Cat # T0440, Sigma-Aldrich, USA) at room temperature for 15 min in dark. The reaction was stopped with 50µl of 0.16M H 2 SO 4 and OD of each well was taken at 450 nm in ELISA reader. Cut-off value (0.290) was calculated from the multiplex NAT-ELISA results of 160 plasma samples negative for HIV/HCV/HBVas suggested by Classen et al [17]. Validation of Multiplex NAT-ELISA assay: Performance of Multiplex NAT-ELISA was determined by Analytical Sensitivity, Analytical Specificity, and by the Accuracy of the assay [18]. Analytical sensitivity was determined by measuring population variance from replicate testing of low-level sample(s). Analytical specificity of the assay refers to the ability of a molecular assay to detect only the intended target . Accuracy refers to the closeness of the agreement between the results of a single measurement and the true value of the analyte. i. Analytical Sensitivity or “Limit of Detection” (LOD) study: Three well-characterized positive blood samples SRF-11 (HIV-1), SRF-8 (HBV) and SRF-5 (HCV) were employed. Three ten-fold dilutions of SRF11 (10 2.65 , 10 1.65 , 10 0.65 IU/ml) and SRF8 (10 1.95 , 10 0.95 , 10 0.095 IU/ml IU/ml) two dilutions of SRF5 (10 1.6 , 10 0.6 IU/ml) and were analyzed by NAT-ELISA. Extracted nucleic acid from each dilution was amplified by multiplex rt-PCR and six replicates of each amplicon of each virus dilution was analyzed by liquid hybridization with respective digoxigenin labelled virus specific probes followed by indirect ELISA. LOD 50 of each positive sample was determined from the OD 450 data by Reed-Muench method [19]. To determine LOD 95 and LOD 99.7 , four replicates of one LOD 50 /ml concentration of SRF-11, SRF-8 and SRF-5 were amplified, and the amplified products were analyzed by Multiplex NAT-ELISA assay. LOD 95 and LOD 99.7 was determined from the mean values and standard deviation for each sample using the following equation: LOD 95 =(LOD 50 ) log 10 +2 SD LOD 99.5 =(LOD 50 ) log 10 +3 SD ii. Analytical specificity: Analytical specificity was determined by Interference Screen procedure in which sample containing potential interfering material with the analyte is run in the analytical run to see if acceptable amplification has occurred in the presence of interfering substance. Interference by the presence of unrelated viruses nucleotides, and oligonucleotides was studied by spiking three plasma samples SRF-11 (HIV1 +), SRF-2 (HBV+), SRF-3 (HCV+) and HIV2 (CBL-2) with MS2 phage RNA and PCR amplification of the extracted nucleic acid by a primer-mix of five sets of oligonucleotides in a single tube multiplex NAT-ELISA by One step multiplex rt-PCR system. Amplified products were analyzed by ELISA-NAT with digoxigenin labeled probes. In addition, interference by high or low concentration of other amplified products in the detection of low copy number analyte/s present in the same reaction tube by multiplex ELISA-NAT assay was studied. WHO International standards were diluted to high and low copy numbers and the dilutions were then mixed to form five pools of various combinations of high and/low copies of each biological standard as shown in table 3. Viral nucleic acid from each pool was extracted, amplified, and hybridization of amplicons with specific probes followed by detection of hybridization signals by indirect ELISA. The results were statistically analyzed by two-tailed t -test. iii. Accuracy study: Accuracy of NAT-ELISA was evaluated by Comparison of Methods Study . Five hundred donor blood samples were tested in parallel with both NAT-ELISA assay and serological screening tests. Nucleic acid from each 500 donor plasma samples was extracted, amplified by multiplex NAT-ELISA, and the amplified products were analyzed by liquid hybridization, followed by measuring hybridization signals by indirect ELISA. Another aliquot of each sample was screened for HIV & HCV antibodies and HBsAg at the IRCS blood bank as per NACO guidelines. The results were compared and evaluated statistically. Results Multiplex reverse-transcript (rt) PCR: Extracted nucleic acids of MS2 phage RNA spiked positive samples of HIV-1 (SRF-11), HBV (SRF-8), HCV (SRF-5) and HIV2 isolate CBL2 from NIH were amplified by single tube multiplex rt-PCR. The amplicons were analyzed by 1.5% gel-electrophoresis which showed that each pair of the specific primers produced desired size of amplified product: HIV1, HIV2, HBV and HCV amplified products were of 321bp, 291bp, 229bp, and 105bp respectively (data not shown). The amplified products were also analyzed by liquid hybridization assay followed by indirect ELISA with digoxigenin labelled individual probes, Hyb321, Hyb291, Hyb229 and Hyb105. This experiment was done three times and the mean OD 450 values are mentioned in table 1. The data were statistically evaluated by two-tailed independent t -test. Analysis was based on the difference between the means of the test and control samples and the allowable error that is clinically significant for the test. OD 450 values of all the test wells with biotinylated amplified products were significantly higher than that of control wells (table 2 ; p<.05; t (18)=141.796, p value <0.00001 ) ). In addition, the data show MS2 RNA as the internal control was also be detected in each amplified product. Performance Characteristics (Validation) of Multiplex NAT-ELISA assay: Validation of Multiplex NAT-ELISA was done by determination of Analytical Sensitivity, Analytical Specificity, and Accuracy of the assay. i. Analytical Sensitivity: Analytical sensitivity or LOD was determined in ten-fold dilutions of three well-characterized clinical samples. Data presented in table 2 show that LOD 50 by Reed-Muench method [19] for HIV-1 was 14 IU/ml (7 geq/ml), and for HBV and HCV were 10 IU/ml (56 geq/ml) and 13 IU/ml (57 geq/ml), respectively. LOD 95 and LOD 99.7 were also determined by analyzing one LOD 50 concentration of each sample by NAT-ELISA. LOD 95 and LOD 99.7 were 17 IU/ml (10 geq/ml) and 19 IU/ml (11 geq/ml) respectively for HIV-1, 13 IU/ml (73 geq/ml) and 14 IU/ml (78 geq/ml) for HBV, and 15 IU/ml (66 geq/ml) and 17 IU/ml (75 geq/ml) for HCV, respectively. ii. Analytical Specificity: a. Interference by unrelated viruses, nucleotides and/or oligonucleotides : Data presented in table 1 show that the presence of unrelated primer pairs in the same reaction tube of multiplex PCR did not interfere with the amplification of the specific virus genome nor did they cross-react with the unrelated virus to produce unrelated amplified products. In addition, presence of internal control MS2-RNA did not interfere with the specific amplification of the targets. Statistical evaluation of the data by two-tailed independent t -test showed that there was no influence of the presence of unrelated viruses, nucleic acid and oligonucleotides on the performance of NAT-ELISA ( p <.05; t (23) = 41.602, p value = <.00001). b. Interference by high or low concentration of other amplified products in multiplex assay : Data presented in table 3 show that presence of high copy numbers of one or more analyte/s did not interfere with the amplification and detection of low copy number analyte/s present in the same reaction tube. Suggesting thereby there was no interference or inhibitory effect on the detection of analytes by high or low concentration of other analytes present in the same reaction tube in multiplex NAT-ELISA assay ( p>.05 ; t (13) = -1.248, p -value= 0.234). iii. Accuracy study: Comparison of Methods study : Five hundred blood samples from voluntary donors were screened in parallel at IRCS blood bank for serologically screening for HIV1 & 2 and HCV antibodies, p -24 antigen and Hepatitis B surface antigen (HBsAg) and at Seth Research Foundation by multiplex NAT-ELISA and comparative results are presented in table 4. Four hundred and sixty-nine blood samples were negative and 21were positive by both methods. Thus, there was a concordance of 98% between the results of serological screening and NAT-ELISA. Five serologically negative samples yielded HBV DNA in four samples and HCV RNA in one sample by NAT-ELISA (table 4). On the other hand, NAT-ELISA failed to detect HIV-RNA in one, HBV-DNA and HCV-RNA in two blood samples each that were positive by serological tests (table 4). However, NAT-ELISA detected multiple targets in four donor blood samples, which were positive only for one target (HIV1/HCV) by serological assays (table 5). All the four samples yielded HBV DNA in addition to HIV and/or HCV RNA. None of these samples was positive for HBsAg. Discussion Molecular tests to detect infectious agents are now widely used in many clinical laboratories. Molecular testing for infectious diseases includes assays that detect a single target, and multiplexed assays that detect two or more analytes in the same specimen. Most molecular tests used in clinical laboratories are commercially produced and are accredited. Laboratory developed tests though are accepted as being scientifically valid; their performance specifications are required to be determined. [ 18 – 20 ]. Therefore, three important performance requirements for a molecular diagnostic assay namely, Analytical Sensitivity, Analytical Specificity and Accuracy were established for multiplex NAT-ELISA test described here. Analytical sensitivity or LOD, is the ability of the assay to detect the lowest actual concentration of a given substance in a biological specimen [ 21 ]. It is an inherent characteristic of a molecular diagnostic assay. LOD is the lowest concentration of an analyte that can be consistently detected in a defined type of a specimen (e.g., in ≥ 95% of specimens tested) with acceptable precision under routine laboratory conditions [ 16 , 20 ]. It is expressed as a concentration of DNA/RNA (usually copies/ml or geq /ml or IU/ml). Analytical performance of a molecular diagnostic assay defines the ability of the test to diagnose disease and determine treatment endpoints. LOD of multiplex NAT-ELISA was determined empirically, by testing dilutions of a well-characterized sample with known concentration of the target substance in the analytical range of the expected detection limit [ 18 ]. Data presented in table 4 show that mean LOD 95 of multiplex NAT-ELISA assay for HIV1 is 17 IU/ml (10 geq/ml), for HBV it is 13 IU/ml (73 geq/ml) and for HCV 15 IU/ml (66 geq/ml) respectively. Several studies have reported LOD 95 of 14 IU/ml (78 geq/ml), 5IU/ml (22geq/ml) and 32 IU/ml (19 geq/ml) of HBV, HCV, and HIV respectively by Transcription-mediated amplification assay [ 22 – 24 ]. Similarly, other workers have reported LOD 95 of 10 IU (6 geq/ml) for HIV-1 by Ultrasensitive RT-PCR assay [ 25 ], 12 IU (53 geq/ml) for HCV by Taqman RT-PCR assay [ 26 ], and 6 IU (1 geq/ml) for HBV by TaqMan-MGB probe assay [ 27 ]. Meaning thereby, multiplex NAT-ELISA assay described here has a matching analytical sensitivity with more expensive Transcription-mediated amplification tests, RT-PCR assays and MGB probe assay. Although, these values do not reflect the diagnostic or clinical sensitivity of the assay, yet they suggest that NAT-ELISA has a potential for the detection of HIV, HCV and HBV infections in the early phase and determine treatment endpoints like exorbitantly costly NAT assays. Analytical specificity of the assay refers to the ability of a molecular assay to detect only the intended target. Data presented in table-1 show that the presence of unrelated primer pairs in the same reaction tube did not interfere with the amplification of the specific virus genome nor did they cross-react with the unrelated virus to produce unrelated amplified products. Furthermore, co-amplification of analytes, particularly in low concentration was not inhibited by high or low concentrations other analytes (table 3) suggesting thereby there was no interference or inhibitory effect on amplification of analytes in multiplex NAT-ELISA assay ( p > .05 ; t (13)= -1.248, p -value = 0.234), thus, validating the Analytical Specificity on NAT-ELISA. The term Accuracy refers to the closeness of the agreement between the results of a single measurement and the true value of the analyte [ 18 ]. Accuracy studies are the cornerstone of method validation. Comparison method study was used to evaluate accuracy of NAT-ELISA [ 18 ]. In this study, 500 blood samples from healthy blood donors were tested in parallel by serological screening tests for HIV1 & 2 antibodies, HCV antibodies and Hepatitis B surface antigen (HBsAg) and by multiplex NAT-ELISA assay for HIV 1 & 2 RNA/HCV RNA/HBV DNA. There was a concordance of 98% between the results of serological screening and NAT-ELISA (table 4). An international survey undertaken to assess the benefits of NAT testing over serological screening of blood donations from 1999 to 2009 in 37 countries reported a concordance of 97.98% between serological screening tests and NAT [ 4 ]. However, five serologically negative samples yielded discrepant results in our study: four samples were positive for HBV DNA and one was positive for HCV RNA. NAT-ELISA assay was repeated thrice to confirm the positive results. A cumulative NAT-yield in our study was 1: 95, which is too high to draw any inference as the sample size was very small. Cumulative NAT-yield in India is generally high as compared to the NAT-yields reported from Western countries. NAT-yield reported from India varies from 1: 1559 from Gujarat [ 28 ], 1:2622 from New Delhi [ 8 ] and 1:2000 from Andhra Pradesh [ 29 ]. A large study by Makroo and his colleagues [ 30 ] reported that NAT could interdict 3272 infectious donations a year among 5 million annual donations in India (NAT-yield of 1:1528). The NAT-yield reported from western countries is much lower as compared to data from India. In USA a NAT yield of 1:2 million for HIV and 1:270,00 for HCV for 66 million donations was reported [ 31 ]. Another study found a NAT-yield of 1: 600,000 for HCV and 1: 1.8 million for HIV in Europe after screening 3.6 million donations.[ 32 ]. The higher NAT yield in blood banks in India is possibly because of the higher prevalence of HBV and HCV infections among the blood donors. Across the globe and particularly in developing countries, HBV is the most common cause of high NAT yield. [ 3 ]. In India, a prevalence of 1.18% of HBV infection among voluntary blood donors translates to the presence of about 12 HBV positive donors out of 1000 donors, which is alarming. Not all NAT positive samples transmit infection. Studies have shown that 19–83% of NAT positive but seronegative donors may transmit infection with higher risk associated with window period donation [ 33 ]. Interestingly, one HIV antibody positive sample was HIV1 RNA negative by multiplex NAT-ELISA (table 4). It could be a false positive result of HIV screening test. Alternatively, this voluntary blood donor could be an Elite Controller of HIV infection. Elite Controller is a subset of HIV infected seronegative individuals who have readily detectable levels of plasma HIV RNA in the absence of antiretroviral therapy. They comprise less than 1% of the HIV infected population [ 34 – 35 ]. These individuals remain asymptomatic for indefinite period (> 20years) [ 36 ]. Many of these subjects have replication-competent virus and thus represent a model of a functional cure [ 37 ]. In the present study, this donor could not be followed for further evaluation as his donation was collected under anonymous unlinked conditions as per NACO guidelines. Similarly, two HBsAg positive donor samples were negative for HBV DNA (table 4). PCR of these samples was not inhibited as each assay included an internal control (MS2 phage RNA) that was added to each sample and amplified. Other workers too have reported lack of HBV DNA in HBsAg positive donors. The most likely explanation could be either extremely low level of HBV DNA or intermittent viremia. [ 38 , 39 ]. Kuhns and colleagues have reported about 6 percent of HBsAg positive donations were negative by MP-NAT method and about 3 percent of HBsAg positive donations remained undetected even by sensitive ID-NAT [ 40 ]. On the other hand, four HBsAg negative samples yielded HBV-DNA by NAT-ELISA (table 5). It is possible that these four HBsAg negative donors were having occult HBV infection with HBsAg levels below the detection limits of current assays. Occult hepatitis B infection (OBI) is defined as the existence of low-level HBV DNA in the serum (< 200 IU/mL), cells of the lymphatic (immune) system, and/or hepatic tissue and the absence of serum HBsAg in patients. More than 20 percent of patients had no serologic markers because the antibody titer may become undetectable over time, leaving HBV DNA as the only marker of the infection [ 41 – 43 ]. An individual with OBI can transmit HBV through blood transfusion causing classical forms of hepatitis B infection in recipients. Incidentally, these four samples also had HIV-1 and HCV detected by multiplex NAT-ELISA. Thus, HBV window period positive donors and OBI cases pose major challenges in blood screening. Detection frequency of HBV window period positive and OBI patients is directly dependent on sensitivity of assays for either or both HBV markers and HBV prevalence in the populations [ 44 ]. The main advantage of NAT screening is interdiction of HBV window period positive infections and identification of OBI carrier status, offering a significantly higher sensitivity for detecting blood-borne infections [ 45 ]. Similarly, HCV antibody was detected in six blood samples by ELISA. Four of these samples were also positive for HCV RNA. Meaning thereby, these donors had active HCV infection. Two donors were HCV antibody positive but were HCV RNA negative. These donors might have had past HCV infection from which they had recovered. One HCV antibody negative donor had HCV RNA by NAT-ELISA. This donor could have been a recently exposed donor. However, false negative report of HCV antibodies in this individual was not ruled out. None of these six donors were further evaluated as all blood donations in India are anonymous unlinked as per NACO guidelines. The aim of NAT test is primarily to prevent donations tainted with HIV/HCV/HBV made during the window-period from being used in transfusions [ 6 , 46 ]. NAT is performed in two formats: individual donation testing (ID-NAT) in which each individual sample is tested with NAT; or minipool testing (MP-NAT) in which 4/16/24/96 donations are pooled and an aliquot is then screened by NAT. If minipool is found reactive, all the donations constituting that pool are tested individually to identify the reactive unit(s). This would delay the release of donations until NAT testing of all the constituents is complete. In addition, pooling may effectively reduce the viral load of an infectious sample to undetectable levels particularly if the donation has low viral load. Chatterjee and colleagues (8) compared the sensitivity of ID-NAT and MP-NAT on TMA assay system (Procleix). They observed that though samples with high viral load were detected by MP-NAT, 67% of samples of low viral load were missed. Data from other studies have also shown that ID-NAT is more sensitive than MP-NAT [ 47 , 48 ]. Bouchardeau and colleagues [ 48 ] compared MP-NAT and ID-NAT to evaluate performance of NAT in detection of HBV infection in HBsAg positive blood donations. They observed that the sensitivity of ID-NAT was 98% versus 84% for MP-NAT. Viral load in the MP-NAT nonreactive samples was 1,420 copies/mL versus 17,000 copies/mL in MP-NAT reactive samples. Similarly, Gonzalez and colleagues [ 49 ] reported that HBV DNA in HBsAg negative blood donors in the window period is detected at a higher frequency with ID-NAT than with MP-NAT. ID-NAT is significantly more efficacious than MP-NAT in detecting window period donations and provides the highest level of blood safety. Further, risk analysis by Weusten and colleagues [ 9 ] shows that an ID-NAT screening algorithm with duplicate or triplicate repeat test resolution of initially reactive donations is safer than MP-NAT protocols even if the pool size is reduced to two donations. Currently only serological screening of donors’ blood has been made mandatory by NACO. However, this does not address the problem of detection of pathogenic blood-borne viruses particularly HIV, HCV and HBV during window period. Two commercial NAT assays, Procleix Ultrio System and Cobas TaqScreen MPX assay, have been recommended by NACO for screening the donor blood. Since both these tests are outrageously expensive, technically demanding and require advanced resources and trained manpower, they are beyond the affordability of most blood banks in India. Only around 5% of blood banks in our country are doing NAT and approximately 7% of all collected blood units are NAT tested [ 10 ]. Multiplex NAT-ELISA assay described here is performed on individual donations and is most suitable for India and other developing counties. The assay is affordable and requires only PCR thermocycler and ELISA reader to screen the blood samples. Unlike NAT assays, it is not technically demanding. Moreover, it has analytical sensitivity that matches with that of Procleix Ultrio System and Cobas TaqScreen MPX. References Agarwal N, Chatterjee K, Coshic P, Borgohain M. (2013) Nucleic acid testing for blood banks: An experience from a tertiary care centre in New Delhi, India. Transfus Apher Sci 49: 482–484. doi: 10.1016/j.transci.2013.03.004 . Busch MP, Collier A, Gernsheimer T, Carrington JD, Flanigan TP, Kashkari M, Kennedy M, Kumar PN, Lane TA, Mellors JW, Mohandas K, Pollard RB, Viele M, Yomtovian R, Holland PV, McCurdy PR (1996) The Viral Activation Transfusion Study (VATS): rationale, objectives, and design overview. 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Declarations Funds : The Indian Council of Medical Research, New Delhi provided financial support for this project under Project number HIV/50/166/11/2013-ECD-II. Conflict of Interests : The authors have no relevant financial or non-financial interests, directly or indirectly to disclose. CRediT Authors Statement : Following description of contribution of each author is accurate and has been agreed upon by all authors: Priyanka Sharma : Investigation, Data curation, Validation, Formal analysis, Visualization; Writing-original draft; Gunjan Batheja : Investigation, Formal analysis; H.N. Verma : Supervision, editing; Pradeep Seth : Conceptualization, Methodology, Formal Analysis, Resources, Editing original draft and writing final manuscript, Supervision, Project administration, funding acquisition. The first draft of the manuscript was written by Priyanka Sharma, edited by Pradeep Seth and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethical Approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the National Institute of Nutrition (ICMR), Hyderabad, Telangana, (Date 01/01/2015, # 01/I/2015) Consent to participate: Informed consent was obtained from all individual participants included in the study as per National guidelines. Tables Table-1: Detection of Multiplex PCR products of MS2 spiked HIV1/HIV2/HBV/HCV plasma samples by Liquid hybridization assay and Indirect ELISA with digoxigenin (DIG) labelled virus-specific probes and pooled DIG labelled probes (DIG-pool) Probes Amplicons (Mean OD 450 ± SD)* Probe Controls HIV-1+MS2 HIV-2 +MS2 HBV+MS2 HCV+MS2 MS2 HIV1-hyb 3.022±0.096 0.208±0.048 0.189±0.033 0.201±0.046 0.139±0.023 0.080±0.011 HIV2-hyb 0.192±0.049 3.141±0.08 0.172±0.036 0.163±0.039 0.115±0.021 0.068±0.015 HBV-hyb 0.200±0.041 0.186±0.036 3.160±0.089 0.169±0.045 0.145±0.027 0.064±0.005 HCV-hyb 0.143±0.052 0.114±0.029 0.182±0.047 3.047±0.065 0.156±0.036 0.069±0.008 MS2-hyb 3.144±0.119 2.445±0.635 2.711±0.442 2.586±0.231 2.943±0.078 0.091 ± 0.024 Amp-Controls 0.075±0.009 0.078±0.012 0.084±0.011 0.072±0.009 0.096±0.018 Cut-off 0.290 Conjugate control:0.065±0.011 Buffer Control: 0.060±0.001 * Mean and Standard deviation of 3 experiments. Table-2. Determination of lower limit of detection (LoD) of NAT ELISA assay Probe SRF11 (HIV-1) (IU/ml)* SRF8 (HBV) (IU/ml)* SRF5 (HCV) (IU/ml)* 10 2.65 10 1.65 10 0.65 10 1.95 10 0.95 10 0.095 10 1.6 10 0.6 1 HIV1-Hyb 3.084** 1.154 0.208 0.183 0.105 0.123 0.143 0.132 2 HBV-Hyb 0.138 0.137 0.170 1.498 1.198 0.270 0.109 0.117 3 HCV-Hyb 0.255 0.112 0.153 0.266 0.259 0.121 0.899 0.0.178 LOD 50 10 1.15 IU/ml (14 IU/ml or 7 geq/ml)) 10 0.98 IU/ml (10 IU/ml or 56 geq/ml) 10 1.1 IU/ml (13 IU/ml or 57 geq/ml) LOD 95 17 IU/ml or 10 geq/ml 13 IU/ml or 73 geq/ml 15 IU/ml or 66 geq/ml LOD 99.7 19 IU/ml or 11 geq/ml 14 IU/ml or 78 geq/ml 17 IU/ml or 75 geq/ml Cut-off 0.290 * IU=International units. HIV 1 IU/ml = 0.58copies/ml; HBV1 IU/ml = 5.6 copies/ml; HCV1IU/ml = 4.4 copies/ ml; ** OD 450 values of hybridization signal with digoxigenin labelled specific probes (HIV1-Hyb, HCV-Hyb, HBV-Hyb) by indirect ELISA. Table-3: Validation of Analytical Specificity by Co-amplification of HIV-1/HCV/HBV (WHO Standards) in combination by Multiplex NAT-ELISA Pools of positive samples Constituents of Pools HIV-1 (IU/ml) OD 450 * HCV (IU/ml) OD 450 * HBV (IU/ml) OD 450 * 1 High (5.6x10 4 ) 0.955 High (8.6x10 3 ) 0.429 High (8.3x10 3 ) 0.441 2 Low (5.6x10 2 ) 1.907 Low (8.6x10 1 ) 0.411 Low (8.3x10 1 ) 0.542 3 High (5.6x10 4 ) 0.897 High (8.6x10 3 ) 0.399 Low (8.3x10 1 ) 0.523 4 Low (5.6x10 2 ) 1.196 High (8.6x10 3 ) 0.518 Low (8.3x10 1 ) 0.591 5 Low (5.6x10 2 ) 1.512 Low (8.6x10 1 ) 0.508 High (8.3x10 3 ) 0.640 Cut-Off =0.290 * OD450 values of hybridization signal with digoxigenin labelled specific probes (HIV1-Hyb, HCV-Hyb, HBV-Hyb) by indirect ELISA Table 4. Comparative analysis of data of screening serological tests with the data of NAT-ELISA test on 500 donors blood samples from IRC IRC* (Serological Tests) Seth Research Foundation** (NAT-ELISA for HIV1 & 2 RNA, HCV RNA and HBV DNA) HIV1+HIV2+HBV+HCV HIV-1 HIV2 HBV HCV Pos Neg Total Pos Neg Total Pos Neg Total Pos Neg Total Pos Neg Total Positive 21 5 26 5 1 6 0 0 0 12 2 14 4 2 6 Negative 5 469 474 0 494 494 0 500 500 4 482 486 1 493 494 Total 26 474 500 5 495 500 0 500 500 16 484 500 5 495 500 Concordance 490/500 (98%) 499/500 (99.8%) ---- 494/500 (98.8%) 497/500 (99.4%) Discordance 10/500 (2%) 1/500 (0.2%) ----- 6/500 (1.2%) 3/500 (0.6%) * The blood samples were tested for HIV1 & 2 and HCV antibodies, HBsAg and p-24 antigen at Indian Red Cross Blood Bank. ** The blood samples were tested for HIV1 & 2 and HCV RNA and HBV DNA by multiplex NAT-ELISA at Seth Research Foundation. (see text) Table-5. Detection of multiple targets by NAT-ELISA test in 4 donor blood samples positive for single target by serological tests as per National Blood Bank policy (P=positive; N=Negative) # SRF ID IRC Results SRF Results HBV HIV1 HBV HCV HBV HIV 1 HIV2 HCV 1 82 N P N N P P N N 2 91 N P N N P P N P 3 92 N P N N P P N N 5 98 N N N P P N N P Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1708998","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":109884856,"identity":"ad3359db-b5f5-4e38-b0a6-8caee555e6bc","order_by":0,"name":"Priyanka Sharma","email":"","orcid":"https://orcid.org/0000-0001-6384-7346","institution":"Seth Research Foundation","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Priyanka","middleName":"","lastName":"Sharma","suffix":""},{"id":109884857,"identity":"b9bb75e3-3dcf-4c39-8883-772c05e1c973","order_by":1,"name":"Gunjan Batheja","email":"","orcid":"https://orcid.org/0000-0002-7107-837X","institution":"Seth Research Foundation","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gunjan","middleName":"","lastName":"Batheja","suffix":""},{"id":109884858,"identity":"88f8129c-b571-4027-bb09-9456e4f88026","order_by":2,"name":"Hriday Narayan Verma","email":"","orcid":"https://orcid.org/0000-0003-0600-5489","institution":"Jaipur National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hriday","middleName":"Narayan","lastName":"Verma","suffix":""},{"id":109884859,"identity":"ec7c6e7b-ec90-4395-a922-0f09d5330b85","order_by":3,"name":"Pradeep Seth","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDCCA2CSGcJhbLABkY0HiNdysCENrJEkLYeRBHEAvtsH2B5X1FjL8Uu3P3z8ccd5u7Xth4G21NhE49IieS6B3fDMsXRjyTlnjA0OnrmdvO1MIlDLsbTcBhxaDM4wsEk2sB1O3HAjh03iYNvtZLMDQC2MDYcJaPl3uH7/jfTnPw62nUs2O/+QCC2NbYcTDCQSzBgOth2wM7tBwBbJM4ztho196YYz7pwxljjblpxgdgNoSwIev/CdYT72sOGbtTz/7PaHHyrb7OzNzqc/fPChxganFmDEtUFoCQiVCFaZgFM5GLChaLHHr3gUjIJRMApGIgAAl4xqMVnewEoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1912-8322","institution":"Seth Research Foundation","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Pradeep","middleName":"","lastName":"Seth","suffix":""}],"badges":[],"createdAt":"2022-05-30 17:56:25","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false,"coiExplicitlySet":false},"doi":"10.21203/rs.3.rs-1708998/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1708998/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":22310700,"identity":"0f1c8a91-b679-4ec8-a4a5-7b8eff5641f9","added_by":"auto","created_at":"2022-06-06 16:09:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":459825,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1708998/v1/3d210f0e-0cc8-4dec-bb59-80f525619103.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSingle Tube Multiplex PCR, Liquid Hybridization, and ELISA (Multiplex NAT-ELISA) for rapid detection of HIV, HBV, and HCV in the window period.\u003c/p\u003e\u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNational AIDS Control Organization (NACO) mandated screening blood donors by serological tests as a part of the National Blood Policy in April 2002. Despite a reduction in the risk of transfusion-transmitted infection (TTI) with pathogenic blood-borne viruses particularly HIV, HCV and HBV, by serological screening of donors\u0026rsquo; blood samples for HBV surface antigen (HBsAg) and antibodies to HCV and HIV-1\u0026amp; 2 and by improving donor selection, continued transmission of these viral agents still occurs. This has been attributed to the failure of screening serological tests to identify infected donors in antibody/antigen negative window period [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The window period for HIV is 6\u0026ndash;38 days (Av. 22 days), for HCV is 54 to 192 days (Av. 82 days) and HBV is 37\u0026ndash;87 days (Av. 59 days) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A significant number of donated blood units is likely to remain unsafe, as it is not screened for the presence of these viruses during window period. The donations screened only by serological assays are still at risk for HBV, HCV and HIV infections. The prevalence of HIV, HBV, and HCV in blood donors in India is about 0.24%, 1.18% and 0.43% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] compared to 0.0097%, 0.3%, and 0.07% in the US blood donors, respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn developed countries screening of donated blood units particularly antibody/antigen negative blood units for viral genetic material by Nucleic Acid test (NAT) has been implemented since 1999 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The primary benefit of NAT is the ability to reduce residual risk of infectious window period donations by detecting the viruses earlier than the serological screening methods thus, shortening the window period. The estimated reduction of the window period utilizing NAT for HCV is from 82 days to12 days, HIV from 22 to 11 days, and HBV from 59 to 25\u0026ndash;30 days [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, since NAT is highly sensitive and specific it adds the benefit of resolving false reactive results of serological methods.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently two commercial NAT assays are used for screening blood donors around the world: Transcription Mediated Amplification (TMA) technology based fully automated Procleix Ultrio System (Gen-Probe, Inc. (USA), and PCR/RT-PCR amplification based fully automated Cobas TaqScreen MPX assay (Roche Diagnostics GmbH, Mannheim, Germany) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Either system has been used in two formats: to test individual donations (ID-NAT) or minipools (MP-NAT) of different sizes comprising 96, 24, 16, 8 or 6 donations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although testing minipools cuts the cost of detection, it compromises the screening sensitivity of the assay depending upon the size of the pool thus reducing the gains of NAT assays. In addition, once the pool is found reactive, all the members of the pool need to be tested individually to identify the reactive unit(s) thus delaying release of donations.\u003c/p\u003e \u003cp\u003eThough in India use of NAT for screening the blood donors has been recommended by NACO, it has not been made mandatory because of higher investment for its implementation. Of 1300 registered blood banks in India less than 5% employ NAT for blood screening for HIV, HCV and HBV. Large majority blood banks in India do not use NAT assays to screen the blood donors because of the exorbitant cost of infrastructure required for these assays. Approximately, 7% of all collected blood units are NAT tested in India [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, the potential for an affordable and simple multiplex molecular test to detect the presence of HIV, HCV and HBV in voluntary blood donors in India is staggering, particularly when one takes notice of the high number of carriers of HIV (2.5\u0026nbsp;million), HBV (43\u0026nbsp;million), and HCV (15\u0026nbsp;million). [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere we describe the development of an affordable, technically simple single-tube multiplex NAT ELISA assay in which all the four PCR reactions are carried out in a single tube by multiplex reverse transcriptase (rt)-PCR using a mixture of specific primers of HIV-1, HIV-2, HCV and HBV. This is followed by detection of each amplified product by liquid hybridization and ELISA with digoxigenin labelled target specific probes. Unlike other NAT assays, this assay is performed on individual donor plasma sample.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003e\u003cstrong\u003eClinical Samples:\u0026nbsp;\u003c/strong\u003eBlood samples from healthy voluntary donors were collected under anonymous, unlinked conditions and an informed and written consent was taken from all the blood donors as per National AIDS Control Organization (NACO) guidelines. An ethical clearance for this proposal was obtained from the Institutional Ethics Committee (IEC) of National Institute of Nutrition (ICMR), Hyderabad, Telangana, which permitted to collect blood samples from the donors as per National guidelines. The samples were aliquoted and stored at -80\u003csup\u003e0\u003c/sup\u003eC.\u003c/p\u003e\n\u003col class=\"decimal_type\" start=\"1\"\u003e\n \u003cli\u003eThree well-characterized blood samples positive for HIV-1(SRF-11, 2.65x10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003eIU/ml)\u0026nbsp;, HCV (SRF-5, 10\u003csup\u003e1.62\u003c/sup\u003e IU/ml and HBV (SRF-8, 10\u003csup\u003e1.95\u003c/sup\u003e IU/ml) for the respective viral genomes\u0026nbsp;by Transcription Mediated Amplification (TMA) assay system in\u0026nbsp;ID-NAT format (Procleix Ultrio;\u0026nbsp;Novartis Diagnostics, USA)\u0026nbsp;were collected for standardization of assay from AIIMS blood bank.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThree ml aliquots of 500 hundred fresh blood samples from healthy volunteer blood donors were collected from Indian Red Cross Society (IRCS) blood bank at New Delhi and were transported to the Seth research Foundation, Gurgaon at room temperature in batches of 50-75 blood samples every week from 15 December 2018 to 23 January 2019. The blood samples were drawn after obtaining informed and written consent from each donor before entry in the study. Donated blood did not carry any identification that can be linked to its donor. At the IRCS blood bank each donated blood was screened for HIV\u0026amp; HCV antibodies as well as for hepatitis B surface antigen (HBsAg) by rapid tests/ ELISA/ p24 antigen test for HIV as per NACO guidelines. At the Seth Research Foundation these blood samples were tested for HIV1 \u0026amp; 2 RNA, HCV RNA and HBV DNA by Multiplex NAT-ELISA assay.\u003c/li\u003e\n \u003cli\u003eOne hundred and sixty blood samples from blood donor volunteers, which had been tested for the presence of HIV1 \u0026amp; 2/HCV/HBV by serological screening tests as well as by ID NAT assay at AIIMS blood bank were used for determining Cut-off value for interpreting ELISA results.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eViruses:\u0026nbsp;\u003c/strong\u003eWHO International Standards for HIV1 [Code#10/152,\u0026nbsp;8.5x10\u003csup\u003e5\u003c/sup\u003e IU/mL]; HIV2 [Code#08/150, 10\u003csup\u003e3\u003c/sup\u003e IU/ml]); HBV [Code#10/264, 8.5x10\u003csup\u003e5\u003c/sup\u003e IU/mL]; and HCV [Code#14/150\u0026nbsp;1x10\u003csup\u003e5\u003c/sup\u003e IU/mL] were obtained from NIBSC\u003cstrong\u003e\u0026nbsp;E\u003c/strong\u003england. These were employed for validation of the performance of the assay. In addition, HIV2 virus isolate CBL20 (Cat. # 600) was obtained as cell-free virus with a titer of 9.12x103 TCID50/ml or 15.7x103 IU/ml from NIH AIDS Reagent Program, German Town, USA. MS2 bacteriophage RNA (Concentration 0.8mg/ml, Catalog# 10165948001, Roche Diagnostics USA) was used as an exogenous internal control. All the viruses were stored at -80\u003csup\u003e0\u003c/sup\u003eC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimers and Probes:\u003c/strong\u003e Primers and probes described earlier [13] were used in the study. The conserved regions of HIV1 \u0026amp; 2 (\u003cem\u003ep-24\u003c/em\u003e gene), HCV (\u003cem\u003e5\u0026rsquo;UTR\u003c/em\u003e) HBV (\u003cem\u003eS\u003c/em\u003e gene) and \u003cem\u003emat\u003c/em\u003e gene of MS2 phage RNA were selected for designing primers and oligonucleotide probes. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. The primer pairs and respective hybridizing specific probes were: HIV-1 sense primer (FP-321) 5\u0026rsquo;-TGGTCCAAAATGCAAA CCCAGA-3\u0026rsquo;; antisense primer (RP-321) 5\u0026rsquo;-TAGCCTGCCTCTCAGTACAGTC-3\u0026rsquo;; probe (Hyb321) 5\u0026rsquo;-AAAGGCTGTTGGAAATGTGG-3\u0026rsquo;; HIV-2 (FP-291) 5\u0026rsquo;-TTGGAGTCAAAAGAGGGTTGCC-3\u0026rsquo;; (RP-291) 5\u0026rsquo;CACATGGGTATAATTGCCGCCT-3\u0026rsquo;; probe (Hyb291) 5\u0026rsquo;-GGAACTGCAGAGAAAATGCC-3\u0026rsquo;; HCV (FP-105) 5\u0026rsquo;-TTCTTGGAACAACCCGCTCAAT-3\u0026rsquo;; (RP-105) 5\u0026rsquo;-CTATCAGGCAGTACCACAAGGC-3\u0026rsquo;; probe (Hyb105) 5\u0026rsquo;-GAGATCACTAGCCGAGTAGT-3\u0026rsquo;; HBV (FP-229) 5\u0026rsquo;-ACTCGTGGTGGACTTCTCTCAA-3\u0026rsquo;; (RP-229) 5\u0026rsquo;-AGAGGACAAACGGGCAACATAC-3\u0026rsquo;; probe (Hyb229) 5\u0026rsquo;-TTCCTCTTCATCCTGCTGCT-3\u0026rsquo;. The primer sets were designed in such a manner that there was no cross hybridization with each other and no possibility of mis-priming with other templates present in the same tube. The number of G or C bases within the last five bases from 3\u0026rsquo; end of primers (GC clamp) was kept \u0026le; 3G\u0026rsquo;s or C\u0026rsquo;s`. Designing of primers and probes for multiplex PCR was done on software \u003cem\u003eMPprimer\u003c/em\u003e [14]. Primer specificity was established by \u003cem\u003eBLAST\u003c/em\u003e and primer specificity evaluation program \u003cem\u003eMFEprimer\u003c/em\u003e [15]. Reverse primers were biotinylated at 5\u0026rsquo;end and the probes were tagged with digoxigenin at 3\u0026rsquo; end.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultiplex reverse-transcriptase (rt) PCR:\u0026nbsp;\u003c/strong\u003eNucleic acid was extracted from the WHO standards/plasma samples with\u0026nbsp;Viral RNA/DNA extraction kit (Cat. # K0821; Thermofisher Scientific, USA) strictly as per manufacturer protocol. Each sample was spiked with 1000 of copies of MS2 phage RNA (internal control) before extraction. Amplification of extracted nucleic acid was undertaken using\u0026nbsp;Qiagen One Step RT-PCR kit (Cat#210210; Qiagen; Netherland) as per manufacturer\u0026rsquo;s recommendation except that the concentration of MgCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(2.5mM), annealing temperature (55\u003csup\u003e0\u003c/sup\u003eC) and concentration of each set of sense and antisense primers (0.6\u0026micro;M) were optimized for optimum amplification in multiplex PCR reaction.\u0026nbsp;Briefly, 10\u0026micro;l 5X master mix containing 2.5mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2\u0026micro;l dNTP mix (400\u0026micro;M of each dNTP), 2\u0026micro;l of enzyme mix (reverse transcriptase and \u003cem\u003eTaq\u0026nbsp;\u003c/em\u003eDNA polymerase) was mixed with primer mix of all the five pairs of sense and biotinylated antisense primers (0.6\u0026micro;M of each primer), 10 units RNase inhibitor, and 10\u0026micro;l of extracted viral nucleic acid. Molecular grade RNase free water was added to make final volume to 50\u0026micro;l. The tubes were then incubated in pre-heated (50\u003csup\u003e0\u003c/sup\u003eC) thermocycler (Bio Rad Laboratories, USA) first for 30 min at 50\u003csup\u003e0\u003c/sup\u003eC for reverse transcription then for 15min at 95\u003csup\u003e0\u003c/sup\u003eC, followed by 35 cycle of three-step amplification (denaturation at 94\u003csup\u003eo\u003c/sup\u003eC for 30sec, annealing at 55\u003csup\u003e0\u003c/sup\u003eC for 1min and extension at 72\u003csup\u003e0\u003c/sup\u003eC for 1min) and final extension for 10min at 72\u003csup\u003e0\u003c/sup\u003eC.\u0026nbsp;The amplicons were analyzed by 1.2% agarose gel electrophoresis and liquid hybridization assay as described below. Appropriate steps were taken to prevent carryover contamination during PCR [16]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHybridization assay:\u0026nbsp;\u003c/strong\u003eLiquid\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHybridization assay and detection of hybridization signal by indirect ELISA were performed as described by earlier [13] with modifications. Briefly,\u0026nbsp;immobilization of biotinylated amplicon\u0026nbsp;onto streptavidin- coated wells of\u0026nbsp;flat-bottom ELISA plates (Cat # 15120, Thermo Fisher Scientific India) at 37\u003csup\u003eo\u003c/sup\u003eC for 30 min in a shaker incubator, was followed by chemical denaturation at 25\u003csup\u003eo\u003c/sup\u003eC for 30 min. Thereafter, 200ul of hybridization buffer containing\u0026nbsp;digoxigenin (DIG) labeled specific probes\u0026nbsp;HIV1-Hyb (2fmol)/HIV2-Hyb (4fmol)/HCV-Hyb (2fmol)/HBV-Hyb (10fmol)/MS2-Hyb (2fmol)\u0026nbsp;was added in separate wells containing denatured biotinylated amplicons and\u0026nbsp;incubated at 45\u003csup\u003e0\u003c/sup\u003eC for 120 min in a shaker incubator. Each well was washed with stringent wash buffer at 55\u003csup\u003e0\u003c/sup\u003eC for 30 min. Hybridization signal was detected by indirect ELISA after incubating with HRP conjugated anti-digoxigenin sheep antibody (Cat # 11207733910, Sigma Aldrich, India)\u0026nbsp;at 37\u003csup\u003e0\u003c/sup\u003eC for 30 min, followed by incubating with 3,3\u0026rsquo;,5,5\u0026rsquo; tetramethylbenzidine-substrate (Cat # T0440, Sigma-Aldrich, USA) at room temperature for 15 min in dark. The reaction was stopped with 50\u0026micro;l of 0.16M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eand OD of each well was taken at 450 nm in ELISA reader. Cut-off value (0.290) was calculated from the multiplex NAT-ELISA results of 160\u0026nbsp;plasma samples negative for HIV/HCV/HBVas suggested by Classen et al [17].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of Multiplex NAT-ELISA assay:\u0026nbsp;\u003c/strong\u003ePerformance of Multiplex NAT-ELISA was determined by Analytical Sensitivity, Analytical Specificity, and by the Accuracy of the assay [18]. Analytical sensitivity was determined by measuring population variance from replicate testing of low-level sample(s).\u0026nbsp;Analytical specificity of the assay refers to the ability of a molecular assay to detect only the intended target\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eAccuracy refers to the closeness of the agreement between the results of a single measurement and the true value of the analyte.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei. Analytical Sensitivity or \u0026ldquo;Limit of Detection\u0026rdquo; (LOD) study:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThree well-characterized positive blood samples SRF-11 (HIV-1), SRF-8 (HBV) and SRF-5 (HCV) were employed. Three ten-fold dilutions of SRF11 (10\u003csup\u003e2.65\u003c/sup\u003e, 10\u003csup\u003e1.65\u003c/sup\u003e, 10\u003csup\u003e0.65\u0026nbsp;\u003c/sup\u003eIU/ml) and SRF8 (10\u003csup\u003e1.95\u003c/sup\u003e, 10\u003csup\u003e0.95\u003c/sup\u003e, 10\u003csup\u003e0.095\u0026nbsp;\u003c/sup\u003eIU/ml IU/ml) two dilutions of SRF5 (10\u003csup\u003e1.6\u003c/sup\u003e, 10\u003csup\u003e0.6\u003c/sup\u003e IU/ml) and were analyzed by NAT-ELISA. Extracted nucleic acid from each dilution was amplified by multiplex rt-PCR and six replicates of each amplicon of each virus dilution was analyzed by liquid hybridization with respective digoxigenin labelled virus specific probes followed by indirect ELISA. LOD\u003csub\u003e50\u003c/sub\u003e of each positive sample was determined from the OD\u003csub\u003e450\u003c/sub\u003e data by Reed-Muench method [19].\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003eTo determine LOD\u003csub\u003e95\u003c/sub\u003e and LOD\u003csub\u003e99.7\u003c/sub\u003e, four replicates of one LOD\u003csub\u003e50\u003c/sub\u003e/ml concentration of SRF-11, SRF-8 and SRF-5 were amplified, and the amplified products were analyzed by Multiplex NAT-ELISA assay. LOD\u003csub\u003e95\u003c/sub\u003e and LOD\u003csub\u003e99.7\u003c/sub\u003e was determined from the mean values and standard deviation for each sample using the following equation:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eLOD\u003csub\u003e95\u003c/sub\u003e=(LOD\u003csub\u003e50\u003c/sub\u003e) log\u003csub\u003e10\u0026nbsp;\u003c/sub\u003e+2 SD\u003c/li\u003e\n \u003cli\u003eLOD\u003csub\u003e99.5\u003c/sub\u003e=(LOD\u003csub\u003e50\u003c/sub\u003e) log\u003csub\u003e10\u0026nbsp;\u003c/sub\u003e+3 SD\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eii. Analytical specificity:\u0026nbsp;\u003c/strong\u003eAnalytical specificity was determined by Interference Screen procedure in which sample containing potential interfering material with the analyte is run in the analytical run to see if acceptable amplification has occurred in the presence of interfering substance. Interference by the presence of unrelated viruses nucleotides, and oligonucleotides was studied by spiking three plasma samples SRF-11 (HIV1 +), SRF-2 (HBV+), SRF-3 (HCV+) and HIV2 (CBL-2) with MS2 phage RNA and PCR amplification of the extracted nucleic acid by a primer-mix of five sets of oligonucleotides in a single tube multiplex NAT-ELISA by One step multiplex rt-PCR system. Amplified products were analyzed by ELISA-NAT with digoxigenin labeled probes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, interference by high or low concentration of other amplified products in the detection of low copy number analyte/s present in the same reaction tube by multiplex ELISA-NAT assay was studied. WHO International standards were diluted to high and low copy numbers and the dilutions were then mixed to form five pools of various combinations of high and/low copies of each biological standard as shown in table 3. Viral nucleic acid from each pool was extracted, amplified, and hybridization of amplicons with specific probes followed by detection of hybridization signals by indirect ELISA. \u0026nbsp;The results were statistically analyzed by two-tailed \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiii. Accuracy study:\u0026nbsp;\u003c/strong\u003eAccuracy of NAT-ELISA was evaluated by \u003cem\u003eComparison of Methods Study\u003c/em\u003e.\u0026nbsp;Five hundred donor blood samples were tested in parallel with both\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNAT-ELISA assay and serological screening tests. Nucleic acid from each 500 donor plasma samples was extracted, amplified by multiplex NAT-ELISA, and the amplified products were analyzed by liquid hybridization, followed by measuring hybridization signals by indirect ELISA. Another aliquot of each sample was screened for HIV \u0026amp; HCV antibodies and HBsAg at the IRCS blood bank as per NACO guidelines. The results were compared and evaluated statistically.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMultiplex reverse-transcript (rt) PCR:\u0026nbsp;\u003c/strong\u003eExtracted nucleic acids of MS2 phage RNA spiked positive samples of HIV-1 (SRF-11), HBV (SRF-8), HCV (SRF-5) and HIV2 isolate CBL2 from NIH were amplified by single tube multiplex rt-PCR. The amplicons were analyzed by 1.5% gel-electrophoresis which showed that each pair of the specific primers produced desired size of amplified product: HIV1, HIV2, HBV and HCV amplified products were of 321bp, 291bp, 229bp, and 105bp respectively (data not shown). \u0026nbsp;The amplified products were also analyzed by liquid hybridization assay followed by indirect ELISA with digoxigenin labelled individual probes, Hyb321, Hyb291, Hyb229 and Hyb105. This experiment was done three times and the mean OD\u003csub\u003e450\u003c/sub\u003e values are mentioned in table 1. The data were statistically evaluated by two-tailed independent \u003cem\u003et\u003c/em\u003e-test. Analysis was based on the difference between the means of the test and control samples and the allowable error that is clinically significant for the test. OD\u003csub\u003e450\u0026nbsp;\u003c/sub\u003evalues of all the test wells with biotinylated amplified products were significantly higher than that of control wells (table 2\u003cem\u003e; p\u0026lt;.05;\u0026nbsp;\u003c/em\u003et (18)=141.796,\u003cem\u003e\u0026nbsp;p\u0026nbsp;\u003c/em\u003evalue \u0026lt;0.00001\u003cem\u003e)\u003c/em\u003e). \u003csub\u003e\u0026nbsp;\u003c/sub\u003eIn addition, the data show MS2 RNA as the internal control was also be detected in each amplified product.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerformance Characteristics (Validation) of Multiplex NAT-ELISA assay:\u003c/strong\u003e Validation of Multiplex NAT-ELISA was done by determination of Analytical Sensitivity, Analytical Specificity, and Accuracy of the assay. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei. Analytical Sensitivity:\u0026nbsp;\u003c/strong\u003eAnalytical sensitivity or LOD was determined in ten-fold dilutions of three well-characterized clinical samples. Data presented in table 2 show that LOD\u003csub\u003e50\u003c/sub\u003e by Reed-Muench method [19] for HIV-1 was 14 IU/ml (7 geq/ml), and for HBV and HCV were 10 IU/ml (56 geq/ml) and 13 IU/ml (57 geq/ml), respectively. LOD\u003csub\u003e95\u0026nbsp;\u003c/sub\u003eand LOD\u003csub\u003e99.7\u003c/sub\u003e were also determined by analyzing one LOD\u003csub\u003e50\u0026nbsp;\u003c/sub\u003econcentration of each sample by NAT-ELISA. LOD\u003csub\u003e95\u0026nbsp;\u003c/sub\u003eand LOD\u003csub\u003e99.7\u003c/sub\u003e were 17 IU/ml (10 geq/ml) and 19 IU/ml (11 geq/ml) respectively for HIV-1, 13 IU/ml (73 geq/ml) and 14 IU/ml (78 geq/ml) for HBV, and 15 IU/ml (66 geq/ml) and 17 IU/ml (75 geq/ml) for HCV, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eii. Analytical Specificity:\u0026nbsp;\u003c/strong\u003ea.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eInterference by unrelated viruses, nucleotides and/or oligonucleotides\u003c/em\u003e:\u0026nbsp;Data presented in table 1 show that the presence of unrelated primer pairs in the same reaction tube of multiplex PCR did not interfere with the amplification of the specific virus genome nor did they cross-react with the unrelated virus to produce unrelated amplified products. In addition, presence of internal control MS2-RNA did not interfere with the specific amplification of the targets. Statistical evaluation of the data by two-tailed independent\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test showed that there was no influence of the presence of unrelated viruses, nucleic acid and oligonucleotides on the performance of NAT-ELISA (\u003cem\u003ep\u003c/em\u003e\u0026lt;.05; \u003cem\u003et\u003c/em\u003e (23) = 41.602, \u003cem\u003ep\u003c/em\u003e value = \u0026lt;.00001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eb.\u0026nbsp;\u003cem\u003eInterference by high or low concentration of other amplified products in multiplex assay\u003c/em\u003e: Data presented in table 3 show that presence of high copy numbers of one or more analyte/s did not interfere with the amplification and detection of low copy number analyte/s present in the same reaction tube. Suggesting thereby there was no interference or inhibitory effect on the detection of analytes by high or low concentration of other analytes present in the same reaction tube in multiplex NAT-ELISA assay (\u003cem\u003ep\u0026gt;.05\u003c/em\u003e; \u003cem\u003et\u003c/em\u003e (13) = -1.248, \u003cem\u003ep\u003c/em\u003e-value= 0.234).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiii. Accuracy study:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eComparison of Methods study\u003c/em\u003e:\u0026nbsp;Five hundred blood samples from voluntary donors were screened in parallel at IRCS blood bank for serologically screening for HIV1 \u0026amp; 2 and HCV antibodies, \u003cem\u003ep\u003c/em\u003e-24 antigen and Hepatitis B surface antigen (HBsAg) and at Seth Research Foundation by multiplex NAT-ELISA and comparative results are presented in table 4. Four hundred and sixty-nine blood samples were negative and 21were positive by both methods. Thus, there was a concordance of 98% between the results of serological screening and NAT-ELISA. Five serologically negative samples yielded HBV DNA in four samples and HCV RNA in one sample by NAT-ELISA (table 4). On the other hand, NAT-ELISA failed to detect HIV-RNA in one, HBV-DNA and HCV-RNA in two blood samples each that were positive by serological tests (table 4). However, NAT-ELISA detected multiple targets in four donor blood samples, which were positive only for one target (HIV1/HCV) by serological assays (table 5). All the four samples yielded HBV DNA in addition to HIV and/or HCV RNA. None of these samples was positive for HBsAg. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMolecular tests to detect infectious agents are now widely used in many clinical laboratories. Molecular testing for infectious diseases includes assays that detect a single target, and multiplexed assays that detect two or more analytes in the same specimen. Most molecular tests used in clinical laboratories are commercially produced and are accredited. Laboratory developed tests though are accepted as being scientifically valid; their performance specifications are required to be determined. [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, three important performance requirements for a molecular diagnostic assay namely, Analytical Sensitivity, Analytical Specificity and Accuracy were established for multiplex NAT-ELISA test described here.\u003c/p\u003e \u003cp\u003eAnalytical sensitivity or LOD, is the ability of the assay to detect the lowest actual concentration of a given substance in a biological specimen [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It is an inherent characteristic of a molecular diagnostic assay. LOD is the lowest concentration of an analyte that can be consistently detected in a defined type of a specimen (e.g., in \u0026ge;\u0026thinsp;95% of specimens tested) with acceptable precision under routine laboratory conditions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It is expressed as a concentration of DNA/RNA (usually copies/ml or geq /ml or IU/ml). Analytical performance of a molecular diagnostic assay defines the ability of the test to diagnose disease and determine treatment endpoints. LOD of multiplex NAT-ELISA was determined empirically, by testing dilutions of a well-characterized sample with known concentration of the target substance in the analytical range of the expected detection limit [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Data presented in table 4 show that mean LOD\u003csub\u003e95\u003c/sub\u003e of multiplex NAT-ELISA assay for HIV1 is 17 IU/ml (10 geq/ml), for HBV it is 13 IU/ml (73 geq/ml) and for HCV 15 IU/ml (66 geq/ml) respectively. Several studies have reported LOD\u003csub\u003e95\u003c/sub\u003e of 14 IU/ml (78 geq/ml), 5IU/ml (22geq/ml) and 32 IU/ml (19 geq/ml) of HBV, HCV, and HIV respectively by Transcription-mediated amplification assay [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similarly, other workers have reported LOD\u003csub\u003e95\u003c/sub\u003e of 10 IU (6 geq/ml) for HIV-1 by Ultrasensitive RT-PCR assay [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], 12 IU (53 geq/ml) for HCV by Taqman RT-PCR assay [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and 6 IU (1 geq/ml) for HBV by TaqMan-MGB probe assay [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Meaning thereby, multiplex NAT-ELISA assay described here has a matching analytical sensitivity with more expensive Transcription-mediated amplification tests, RT-PCR assays and MGB probe assay. Although, these values do not reflect the diagnostic or clinical sensitivity of the assay, yet they suggest that NAT-ELISA has a potential for the detection of HIV, HCV and HBV infections in the early phase and determine treatment endpoints like exorbitantly costly NAT assays.\u003c/p\u003e \u003cp\u003eAnalytical specificity of the assay refers to the ability of a molecular assay to detect only the intended target. Data presented in table-1 show that the presence of unrelated primer pairs in the same reaction tube did not interfere with the amplification of the specific virus genome nor did they cross-react with the unrelated virus to produce unrelated amplified products. Furthermore, co-amplification of analytes, particularly in low concentration was not inhibited by high or low concentrations other analytes (table 3) suggesting thereby there was no interference or inhibitory effect on amplification of analytes in multiplex NAT-ELISA assay (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;.05\u003c/em\u003e; \u003cem\u003et\u003c/em\u003e (13)= -1.248, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;=\u0026thinsp;0.234), thus, validating the Analytical Specificity on NAT-ELISA.\u003c/p\u003e \u003cp\u003eThe term Accuracy refers to the closeness of the agreement between the results of a single measurement and the true value of the analyte [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Accuracy studies are the cornerstone of method validation. Comparison method study was used to evaluate accuracy of NAT-ELISA [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, 500 blood samples from healthy blood donors were tested in parallel by serological screening tests for HIV1 \u0026amp; 2 antibodies, HCV antibodies and Hepatitis B surface antigen (HBsAg) and by multiplex NAT-ELISA assay for HIV 1 \u0026amp; 2 RNA/HCV RNA/HBV DNA. There was a concordance of 98% between the results of serological screening and NAT-ELISA (table 4). An international survey undertaken to assess the benefits of NAT testing over serological screening of blood donations from 1999 to 2009 in 37 countries reported a concordance of 97.98% between serological screening tests and NAT [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, five serologically negative samples yielded discrepant results in our study: four samples were positive for HBV DNA and one was positive for HCV RNA. NAT-ELISA assay was repeated thrice to confirm the positive results. A cumulative NAT-yield in our study was 1: 95, which is too high to draw any inference as the sample size was very small. Cumulative NAT-yield in India is generally high as compared to the NAT-yields reported from Western countries. NAT-yield reported from India varies from 1: 1559 from Gujarat [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], 1:2622 from New Delhi [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and 1:2000 from Andhra Pradesh [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A large study by Makroo and his colleagues [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] reported that NAT could interdict 3272 infectious donations a year among 5\u0026nbsp;million annual donations in India (NAT-yield of 1:1528). The NAT-yield reported from western countries is much lower as compared to data from India. In USA a NAT yield of 1:2\u0026nbsp;million for HIV and 1:270,00 for HCV for 66\u0026nbsp;million donations was reported [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Another study found a NAT-yield of 1: 600,000 for HCV and 1: 1.8\u0026nbsp;million for HIV in Europe after screening 3.6\u0026nbsp;million donations.[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe higher NAT yield in blood banks in India is possibly because of the higher prevalence of HBV and HCV infections among the blood donors. Across the globe and particularly in developing countries, HBV is the most common cause of high NAT yield. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In India, a prevalence of 1.18% of HBV infection among voluntary blood donors translates to the presence of about 12 HBV positive donors out of 1000 donors, which is alarming. Not all NAT positive samples transmit infection. Studies have shown that 19\u0026ndash;83% of NAT positive but seronegative donors may transmit infection with higher risk associated with window period donation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, one HIV antibody positive sample was HIV1 RNA negative by multiplex NAT-ELISA (table 4). It could be a false positive result of HIV screening test. Alternatively, this voluntary blood donor could be an \u003cem\u003eElite Controller\u003c/em\u003e of HIV infection. \u003cem\u003eElite Controller\u003c/em\u003e is a subset of HIV infected seronegative individuals who have readily detectable levels of plasma HIV RNA in the absence of antiretroviral therapy. They comprise less than 1% of the HIV infected population [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These individuals remain asymptomatic for indefinite period (\u0026gt;\u0026thinsp;20years) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Many of these subjects have replication-competent virus and thus represent a model of a functional cure [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, this donor could not be followed for further evaluation as his donation was collected under anonymous unlinked conditions as per NACO guidelines.\u003c/p\u003e \u003cp\u003eSimilarly, two HBsAg positive donor samples were negative for HBV DNA (table 4). PCR of these samples was not inhibited as each assay included an internal control (MS2 phage RNA) that was added to each sample and amplified. Other workers too have reported lack of HBV DNA in HBsAg positive donors. The most likely explanation could be either extremely low level of HBV DNA or intermittent viremia. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Kuhns and colleagues have reported about 6 percent of HBsAg positive donations were negative by MP-NAT method and about 3 percent of HBsAg positive donations remained undetected even by sensitive ID-NAT [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, four HBsAg negative samples yielded HBV-DNA by NAT-ELISA (table 5). It is possible that these four HBsAg negative donors were having occult HBV infection with HBsAg levels below the detection limits of current assays. Occult hepatitis B infection (OBI) is defined as the existence of low-level HBV DNA in the serum (\u0026lt;\u0026thinsp;200 IU/mL), cells of the lymphatic (immune) system, and/or hepatic tissue and the absence of serum HBsAg in patients. More than 20 percent of patients had no serologic markers because the antibody titer may become undetectable over time, leaving HBV DNA as the only marker of the infection [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. An individual with OBI can transmit HBV through blood transfusion causing classical forms of hepatitis B infection in recipients. Incidentally, these four samples also had HIV-1 and HCV detected by multiplex NAT-ELISA. Thus, HBV window period positive donors and OBI cases pose major challenges in blood screening. Detection frequency of HBV window period positive and OBI patients is directly dependent on sensitivity of assays for either or both HBV markers and HBV prevalence in the populations [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The main advantage of NAT screening is interdiction of HBV window period positive infections and identification of OBI carrier status, offering a significantly higher sensitivity for detecting blood-borne infections [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilarly, HCV antibody was detected in six blood samples by ELISA. Four of these samples were also positive for HCV RNA. Meaning thereby, these donors had active HCV infection. Two donors were HCV antibody positive but were HCV RNA negative. These donors might have had past HCV infection from which they had recovered. One HCV antibody negative donor had HCV RNA by NAT-ELISA. This donor could have been a recently exposed donor. However, false negative report of HCV antibodies in this individual was not ruled out. None of these six donors were further evaluated as all blood donations in India are anonymous unlinked as per NACO guidelines.\u003c/p\u003e \u003cp\u003eThe aim of NAT test is primarily to prevent donations tainted with HIV/HCV/HBV made during the window-period from being used in transfusions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. NAT is performed in two formats: individual donation testing (ID-NAT) in which each individual sample is tested with NAT; or minipool testing (MP-NAT) in which 4/16/24/96 donations are pooled and an aliquot is then screened by NAT. If minipool is found reactive, all the donations constituting that pool are tested individually to identify the reactive unit(s). This would delay the release of donations until NAT testing of all the constituents is complete. In addition, pooling may effectively reduce the viral load of an infectious sample to undetectable levels particularly if the donation has low viral load. Chatterjee and colleagues (8) compared the sensitivity of ID-NAT and MP-NAT on TMA assay system (Procleix). They observed that though samples with high viral load were detected by MP-NAT, 67% of samples of low viral load were missed. Data from other studies have also shown that ID-NAT is more sensitive than MP-NAT [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Bouchardeau and colleagues [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] compared MP-NAT and ID-NAT to evaluate performance of NAT in detection of HBV infection in HBsAg positive blood donations. They observed that the sensitivity of ID-NAT was 98% versus 84% for MP-NAT. Viral load in the MP-NAT nonreactive samples was 1,420 copies/mL versus 17,000 copies/mL in MP-NAT reactive samples. Similarly, Gonzalez and colleagues [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] reported that HBV DNA in HBsAg negative blood donors in the window period is detected at a higher frequency with ID-NAT than with MP-NAT. ID-NAT is significantly more efficacious than MP-NAT in detecting window period donations and provides the highest level of blood safety. Further, risk analysis by Weusten and colleagues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] shows that an ID-NAT screening algorithm with duplicate or triplicate repeat test resolution of initially reactive donations is safer than MP-NAT protocols even if the pool size is reduced to two donations.\u003c/p\u003e \u003cp\u003eCurrently only serological screening of donors\u0026rsquo; blood has been made mandatory by NACO. However, this does not address the problem of detection of pathogenic blood-borne viruses particularly HIV, HCV and HBV during window period. Two commercial NAT assays, Procleix Ultrio System and Cobas TaqScreen MPX assay, have been recommended by NACO for screening the donor blood. Since both these tests are outrageously expensive, technically demanding and require advanced resources and trained manpower, they are beyond the affordability of most blood banks in India. Only around 5% of blood banks in our country are doing NAT and approximately 7% of all collected blood units are NAT tested [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Multiplex NAT-ELISA assay described here is performed on individual donations and is most suitable for India and other developing counties. The assay is affordable and requires only PCR thermocycler and ELISA reader to screen the blood samples. Unlike NAT assays, it is not technically demanding. Moreover, it has analytical sensitivity that matches with that of Procleix Ultrio System and Cobas TaqScreen MPX.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgarwal N, Chatterjee K, Coshic P, Borgohain M. (2013) Nucleic acid testing for blood banks: An experience from a tertiary care centre in New Delhi, India. 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Vox Sang 102: 82\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1423-0410.2011.01506.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1423-0410.2011.01506.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiedmann M, Kluwick S, Walter M, Fauchald G, Howe J, Bronold M, Zauke M (2007) HIV-1, HCV and HBV seronegative window reduction by the new Roche cobas TaqScreen MPX test in seroconverting donors. 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Transfusion 50:221\u0026ndash;230. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1537-2995.2009.02343.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1537-2995.2009.02343.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunds\u003c/strong\u003e: The Indian Council of Medical Research, New Delhi provided financial support for this project under Project number HIV/50/166/11/2013-ECD-II. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e: The authors have no relevant financial or non-financial interests, directly or indirectly to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT Authors Statement\u003c/strong\u003e: Following description of contribution of each author is accurate and has been agreed upon by all authors: \u003cstrong\u003ePriyanka Sharma\u003c/strong\u003e: Investigation, Data curation, Validation, Formal analysis, Visualization; Writing-original draft; \u003cstrong\u003eGunjan Batheja\u003c/strong\u003e: Investigation, Formal analysis; \u003cstrong\u003eH.N. Verma\u003c/strong\u003e: Supervision, editing; \u003cstrong\u003ePradeep Seth\u003c/strong\u003e: Conceptualization, Methodology, Formal Analysis, Resources, Editing original draft and writing final manuscript, Supervision, Project administration, funding acquisition. \u0026nbsp;The first draft of the manuscript was written by Priyanka Sharma, edited by Pradeep Seth and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eThis study was performed in line with the principles of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe Declaration of Helsinki. Approval was granted by the Ethics Committee of the National Institute of Nutrition (ICMR), Hyderabad, Telangana, (Date 01/01/2015, # 01/I/2015)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Informed consent was obtained from all individual participants included in the study as per National guidelines.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTable-1:\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003eDetection of Multiplex PCR products of MS2 spiked HIV1/HIV2/HBV/HCV plasma samples by Liquid hybridization assay and Indirect ELISA with digoxigenin (DIG) labelled virus-specific probes and pooled DIG labelled probes (DIG-pool)\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 64.35pt;border: 1pt solid windowtext;padding: 0in 5.4pt;height: 13.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Probes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 313.4pt;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Amplicons (Mean OD\u003csub\u003e450\u0026nbsp;\u003c/sub\u003e\u0026plusmn; SD)*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 67.5pt;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eProbe Controls\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 61.4pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 13.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-2.55pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHIV-1+MS2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 13.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-2.55pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHIV-2 +MS2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 13.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-2.55pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHBV+MS2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 13.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-2.55pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHCV+MS2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 13.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-2.55pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New 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none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.182\u0026plusmn;0.047\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e3.047\u0026plusmn;0.065\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e3.144\u0026plusmn;0.119\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e2.445\u0026plusmn;0.635\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e2.711\u0026plusmn;0.442\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e2.586\u0026plusmn;0.231\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e2.943\u0026plusmn;0.078\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.091\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e0.024\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 64.35pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-3.85pt;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eAmp-Controls\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61.4pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.075\u0026plusmn;0.009\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.078\u0026plusmn;0.012\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.084\u0026plusmn;0.011\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.072\u0026plusmn;0.009\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.096\u0026plusmn;0.018\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 67.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eCut-off\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-1.1pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.290\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 188.75pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eConjugate control:0.065\u0026plusmn;0.011\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 189pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eBuffer Control: 0.060\u0026plusmn;0.001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e* Mean and Standard deviation of 3 experiments.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:1.5in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:135.0pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eTable-2. Determination of lower limit of detection (LoD) of NAT ELISA assay\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.5e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 16.8pt;border: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 68.45pt;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-6.2pt;margin-bottom:0in;margin-left:-6.3pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eProbe\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1.75in;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eSRF11 (HIV-1)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;(IU/ml)*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 139.5pt;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eSRF8 (HBV)\u003c/span\u003e\u003c/p\u003e\n \u003cp 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.117\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.8pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68.45pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-6.2pt;margin-bottom:0in;margin-left:-6.3pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHCV-Hyb\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.3pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.255\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.112\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.2pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.153\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.266\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.259\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.121\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.899\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.0.178\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.8pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68.45pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-6.2pt;margin-bottom:0in;margin-left:-6.3pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLOD\u003csub\u003e50\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1.75in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-4.15pt;margin-bottom:0in;margin-left:-5.3pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e10\u003csup\u003e1.15\u003c/sup\u003eIU/ml (14 IU/ml or \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;7 geq/ml))\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 139.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e10\u003csup\u003e0.98\u003c/sup\u003e IU/ml (10 IU/ml or \u0026nbsp; \u0026nbsp; \u0026nbsp;56 geq/ml)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 103.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e10\u003csup\u003e1.1\u003c/sup\u003e IU/ml (13 IU/ml or 57 geq/ml)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.8pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68.45pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-9.75pt;margin-bottom:0in;margin-left:-4.55pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLOD\u003csub\u003e95\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1.75in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e17 IU/ml or 10 geq/ml\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 139.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e13 IU/ml or 73 geq/ml\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 103.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-.8pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e15 IU/ml or 66 geq/ml\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.8pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68.45pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-9.75pt;margin-bottom:0in;margin-left:-4.55pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLOD\u003csub\u003e99.7\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1.75in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e19 IU/ml or 11 geq/ml\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 139.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e14 IU/ml or 78 geq/ml\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 103.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:-5.3pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family: \"Times New Roman\",serif;'\u003e\u0026nbsp;17 IU/ml or 75 geq/ml\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.8pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68.45pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-6.2pt;margin-bottom:0in;margin-left:-6.3pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eCut-off\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"8\" style=\"width: 369pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.290\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:-4.5pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e* IU=International units. HIV 1 IU/ml = 0.58copies/ml; HBV1 IU/ml = 5.6 copies/ml; HCV1IU/ml = 4.4 copies/ ml;\u003cspan style=\"color:red;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:-4.5pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e** OD\u003csub\u003e450\u0026nbsp;\u003c/sub\u003evalues of hybridization signal with digoxigenin labelled specific probes (HIV1-Hyb, HCV-Hyb, HBV-Hyb) by indirect ELISA.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:1.25in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTable-3: Validation of Analytical Specificity by Co-amplification of HIV-1/HCV/HBV (WHO Standards) in combination by Multiplex NAT-ELISA\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 49.25pt;border: 1pt solid windowtext;padding: 0in 5.4pt;height: 17.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom: 0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003ePools \u0026nbsp;of positive samples\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" style=\"border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 17.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eConstituents of Pools\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 17.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHIV-1 (IU/ml)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 17.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eOD\u003csub\u003e450\u003c/sub\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 17.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHCV (IU/ml)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 17.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eOD\u003csub\u003e450\u003c/sub\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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(8.6x10\u003csup\u003e3\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.518\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLow (8.3x10\u003csup\u003e1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.591\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.25pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLow (5.6x10\u003csup\u003e2\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e1.512\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLow (8.6x10\u003csup\u003e1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.508\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHigh (8.3x10\u003csup\u003e3\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e0.640\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 390.2pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:1.45pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eCut-Off =0.290\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:67.5pt;margin-bottom:0in;margin-left:4.5pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e* OD450 values of hybridization signal with digoxigenin labelled specific probes (HIV1-Hyb, HCV-Hyb, HBV-Hyb) by indirect ELISA\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:202.5pt;margin-bottom:0in;margin-left:4.5pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:63.0pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eTable 4. Comparative analysis of data of screening serological tests with the data of NAT-ELISA test on 500 donors blood samples from IRC\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:63.0pt;margin-bottom:0in;margin-left:9.0pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 49.25pt;border: 1pt solid windowtext;padding: 0in 5.4pt;height: 26.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-9.9pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eIRC*\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e(Serological \u0026nbsp;Tests)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"15\" style=\"width: 355.5pt;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 26.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eSeth Research Foundation**\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e(NAT-ELISA for HIV1 \u0026amp; 2 RNA, HCV RNA and HBV DNA)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eHIV1+HIV2+HBV+HCV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eHIV-1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 67.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eHIV2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eHBV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eHCV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003ePos\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eNeg\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.5in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003ePos\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eNeg\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003ePos\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eNeg\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003ePos\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eNeg\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.5in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003ePos\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eNeg\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e493\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.5in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e494\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.25pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e474\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.5in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e500\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e495\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e500\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e500\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e500\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e484\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.5in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e500\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.25in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e495\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.5in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;height: 4.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e500\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.25pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eConcordance\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom: 0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp; 490/500 (98%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e499/500 (99.8%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 67.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom: 0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;----\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom: 0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;494/500 (98.8%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom: 0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; 497/500 (99.4%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.25pt;border-color: currentcolor windowtext windowtext;border-style: none solid solid;border-width: medium 1pt 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-.05in;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eDiscordance\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom: 0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; 10/500 (2%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom: 0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1/500 (0.2%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 67.5pt;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom:0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e-----\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom: 0in;margin-left:-5.75pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;6/500 \u0026nbsp; \u0026nbsp; (1.2%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 1in;border-color: currentcolor windowtext windowtext currentcolor;border-style: none solid solid none;border-width: medium 1pt 1pt medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.5pt;margin-bottom: 0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp; 3/500 (0.6%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:63.0pt;margin-bottom:0in;margin-left:9.0pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e* The blood samples were tested for HIV1 \u0026amp; 2 and HCV antibodies, HBsAg and p-24 antigen at Indian Red Cross Blood Bank.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:63.0pt;margin-bottom:0in;margin-left:9.0pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e** The blood samples were tested for HIV1 \u0026amp; 2 and HCV RNA and HBV DNA by multiplex NAT-ELISA\u003c/span\u003e \u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eat Seth Research Foundation. (see text)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:63.0pt;margin-bottom:0in;margin-left:9.0pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:1.75in;margin-bottom:0in;margin-left:4.5pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eTable-5. Detection of multiple targets by NAT-ELISA test in 4 donor blood samples positive for single target by serological tests as per National Blood Bank policy (P=positive; N=Negative)\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:1.75in;margin-bottom:0in;margin-left:4.5pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 0.25in;border: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cbr\u003e\u0026nbsp;\u003c/span\u003e\u0026nbsp;\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e#\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 37.8pt;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.25pt;margin-bottom: 0in;margin-left:-5.2pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eSRF ID\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:-5.25pt;margin-bottom: 0in;margin-left:-5.2pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 1.75in;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eIRC Results\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 117pt;border-color: windowtext windowtext windowtext currentcolor;border-style: solid solid solid none;border-width: 1pt 1pt 1pt medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eSRF Results\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:.5in;border-top:none;border-left:none;border-bottom: solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-3.0pt;margin-bottom: 0in;margin-left:-2.95pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHBV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:.5in;border-top:none;border-left:none;border-bottom: solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-3.0pt;margin-bottom: 0in;margin-left:-2.95pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHIV1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.0pt;border-top:none;border-left:none;border-bottom: solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-3.0pt;margin-bottom: 0in;margin-left:-2.95pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHBV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.0pt;border-top:none;border-left:none;border-bottom: solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-3.0pt;margin-bottom: 0in;margin-left:-2.95pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHCV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:31.5pt;border-top:none;border-left:none;border-bottom: solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-3.0pt;margin-bottom: 0in;margin-left:-2.95pt;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHBV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:31.5pt;border-top:none;border-left:none;border-bottom: solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 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Research","awardNumber":"HIV/50/166/11/2013-ECD-II","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Seth Research Foundation","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PCR, Liquid hybridization, ELISA, Blood bank, Analytical sensitivity, NAT-yield","lastPublishedDoi":"10.21203/rs.3.rs-1708998/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1708998/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntroduction: A molecular assay based on single-tube multiplex PCR, liquid hybridization and indirect ELISA has been designed for rapid detection of HIV-1 \u0026amp; 2, HCV, and HBV in plasma samples (multiplex NAT-ELISA) during the window period of these infections.\u003c/p\u003e\u003cp\u003eMethods: Nucleic acid extracted from the virus standards and blood donor plasma samples were amplified by conventional reverse transcription-PCR with a mixture of optimized concentrations of forward and biotinylated reverse primers followed by liquid hybridization of immobilized denatured biotinylated amplicons with digoxigenin-labeled specific probes and indirect ELISA to measure hybridization signals colorimetrically. The performance of Multiplex NAT-ELISA was validated by Analytical Sensitivity, Analytical Specificity, and by the “Accuracy” (Trueness) of the assay.\u003c/p\u003e\u003cp\u003eResults: Performance characteristics of the assay were established. Analytical sensitivity (LOD95) was 17 IU/ml (10 geq/ml) for HIV-1) 13 IU/ml (73 geq /ml) for HBV, and 15 IU/ml (66 geq/ml) for HCV. A comparison of the assay with the serological screening tests for HIV/HCV antibodies and HBsAg shows a concordance of 98%. Five serologically negative samples yielded HBV DNA in 4 samples and HCV RNA in one sample by NAT-ELISA.\u003c/p\u003e\u003cp\u003eConclusions: Multiplex NAT-ELISA assay described here is performed on individual donations. The main advantage of the assay is the interdiction of HIV/HCV/HBV virus infections offering a significantly higher sensitivity for detecting blood-borne infections at an affordable cost (less than US$ 5 per sample). Moreover, it has analytical sensitivity that matches that of Procleix Ultrio System and Cobas TaqScreen MPX.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Single Tube Multiplex PCR, Liquid Hybridization, and ELISA (Multiplex NAT-ELISA) for rapid detection of HIV, HBV, and HCV in the window period.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-06 16:09:22","doi":"10.21203/rs.3.rs-1708998/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"645f1cd0-6e05-4717-b86a-6b98e6852573","owner":[],"postedDate":"June 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":12936369,"name":"Virology"}],"tags":[],"updatedAt":"2022-06-06T16:09:22+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-06 16:09:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1708998","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1708998","identity":"rs-1708998","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}