Validation of Real Star® CMV PCR Kit 1.0 for the detection of cytomegalovirus in Immunocompromised Patients

Validation of Real Star® CMV PCR Kit 1.0 for the detection of cytomegalovirus in Immunocompromised Patients | 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 Validation of Real Star® CMV PCR Kit 1.0 for the detection of cytomegalovirus in Immunocompromised Patients Zainah Shaheen, Osama Alsmadi, Saleh Khudirat, Maysa Al-Hussaini This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7664929/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 Cytomegalovirus (CMV) infections pose a significant risk to individuals with compromised immune systems, particularly those undergoing stem cell transplantation. This study aimed to evaluate a novel real-time PCR based DNA detection approach in whole blood samples with the aim of helping high-risk groups improve CMV diagnosis and therapy. The objective of the study was to validate the efficacy of the RealStar® CMV PCR quantification assay kit 1.0 to diagnose CMV infection. The assay identified CMV in 21 of the 35 blood samples and other infections in 14 samples. The reference Qiagen Artus CMV PCR test exhibited inferior accuracy compared to the RealStar® CMV PCR Kit 1.0. The assay demonstrated a repeatability of 2.14% and reproducibility of 1.29%. Blood components and non-CMV infections did not influence the analytical specificity, which remained at 100%. The test exhibited a detection limit of 265 IU/mL (95% CI: 255–372), which correlated with the reference method (r = 0.93). The RealStar® CMV PCR Kit 1.0 demonstrated strong linearity (R² = 0.95) and achieved 100% predictive value for detecting CMV DNA in blood samples from immunocompromised individuals. Cytomegalovirus Real-time PCR DNA detection immunocompromised patients Stem cell transplant Figures Figure 1 Figure 2 Introduction Patients undergoing organ transplantation are at increased risk of infection with cytomegalovirus (CMV), a member of the Herpesviridae family and subfamily Betaherpesvirinae, which can cause serious illness and even death [ 1 ]. Both the transplanted organ and the recipient's prior (latent) CMV infection can be the source of infection [ 2 ]. However, in immunocompromised patients, primary infection or latent CMV reactivation can lead to fatal diseases [ 3 ]. In a so-called "preemptive" approach, diagnostic tests designed to detect CMV infection early are monitored in the majority individuals at risk of infection or disease. In the early years of CMV antigenemia with Pp65, laboratory surveillance was performed as part of preemptive therapy. Molecular testing has replaced antigenemia as the primary method for the diagnosis and monitoring of CMV. No universal consensus has been achieved on the threshold for starting medication against CMV; however, this is due to considerable differences between assays [ 4 , 5 ]. In recent years, CMV detection techniques have advanced. Rawlinson et al. [ 6 ] state that saliva or urine samples collected within two to three weeks of life are the most effective method to detect CMV in newborns. In the majority of laboratories, culture-based approaches have been replaced by high-throughput molecular techniques such as quantitative polymerase chain reaction (qPCR) [ 5 , 7 ], which has made it easier to conduct CMV screening initiatives all over the world. In this study, a commercially available standardized polymerase chain reaction (PCR) test will be evaluated to determine whether it is effective in detecting CMV infections in immunocompromised patients or in those who have received stem cell transplantation. The purpose of this study was to validate a real-time PCR-based DNA detection approach in whole blood samples with the aim of helping high-risk groups improve CMV diagnosis and therapy. Materials and Methods Sample collection and target population This study utilized patient samples that were subjected to real-time CMV PCR testing using a Qiagen kit. These patients underwent bone marrow transplants at King Hussein Cancer Center (KHCC) from March 2024 to June 2024 to participate in the study. Samples used in the study were chosen through a random selection process. The College of American Pathologists (CAP) and the Clinical and Laboratory Standards Institute (CLSI) together have set validation requirements and standards for quantitative real-time PCR assays including analytical precision, specificity, sensitivity, linearity, precision, carryover, and reportable range. The 35 DNA samples used were extracted from whole blood samples using the QIAamp DSP Virus Kit on a Qiasymphony instrument (Qiagen). The study samples were collected between March 2024 and June 2024, from immunocompromised adult and pediatric patients, specifically those who have undergone stem cell transplantation at the KHCC. These patients are highly susceptible to CMV infection. Only a few studies have evaluated CMV PCR assays in immunocompetent patients. Patient Samples Group 1 (CMV group) comprised of DNA samples from 21 confirmed CMV-positive cases, verified by a previously validated real-time PCR kit (Qiagen). Group 2, comprising diverse illnesses and healthy controls, comprised of 14 DNA samples. These samples were collected from patients with confirmed infections, including EBV, HBV, and BKV. This control group was selected to assess clinical specificity as all exhibited highly comparable highly comparable outcomes. These infections were confirmed by the presence of the corresponding viral load using real-time PCR. In addition, one sample from this group was icteric, and another was lipemic. These two samples were utilized to assess whether or not false-positive results are observed due to the presence of interfering substances. Whole blood DNA extraction Blood samples were collected in K3 EDTA tubes (minimum 2 mL), stored at 8°C, and processed within 24 h to prevent viral degradation. DNA extraction was performed using the QIAamp DSP Virus Kit on the Qiasymphony platform. A volume of 400 µL of whole blood was used per extraction, with a final elution in 80 µl of elution buffer. All extracted DNA was stored at − 20°C until testing. PCR method This retrospective comparative study aimed to validate the Quantitative CMV-Realstar® PCR kit (Altona Diagnostics GmbH, Mörkenstr. 12, D-22767 Hamburg) using the BioRad CFX-96 real-time PCR instrument by contrasting it with the Qiagen Artus CMV reference assay, which served as the reference method employing the Rotor Gene Q system. The RealStar® CMV PCR Kit 1.0 was utilized to identify and quantify human CMV DNA. The assay was conducted following the manufacturer's guidelines using a BioRad CFX-96 real-time PCR apparatus. All values are shown in IU/ml in whole blood. The kit contained all necessary reagents: Taq polymerase, buffer, dNTPs, CMV-specific primers/probes categorized into Mix A and Mix B, internal control, and quantification standards (QS) at four distinct doses. The MIQE guidelines were followed for the quantitative real-time PCR assay, as previously described by Bustin et al. [ 8 ]. Analytical evaluation We define the limit of detection (LOD) as the lowest virus concentration where viral assays detect the virus in ≥ 95% of sample, we assessed the LOD by serial dilution in a CMV-positive sample DNA, starting at 32,558 IU/mL, and ending at 156 IU/ml, as quantified using the CMV kit 4 standards. The dilution points were 5000, 2500, 1250, 1047, 625, 523, 419, 291, 265, 207, and 156 IU/ml. The PCR test was performed using the RealStar kit for diluted samples with titers of 5000, 2500, and 1250 IU/mL in three replicates each, followed by titers of 1047 and 523 IU/mL ten replicates each, 419, 291, and 265 IU/mL twenty replicates each, and finally 207 IU/mL ten replicates. Next, we compared the obtained values with the anticipated values. The limit of quantification (LOQ) was defined as the lowest concentration in this series of dilutions that could be reliably reproduced within a range of 90–100% with a maximum error of 5%. Eight samples were analyzed using polymerase chain reaction (PCR) with varied quantities in three separate runs conducted on separate days by two separate operators to determine the between-run coefficient of variation (CV). To evaluate the within-run CV, two CMV-positive DNA samples were aliquoted into five fractions and used for repeat measurements within the same run by the same operator, on the same instrument, on the same day. CV was compared using the acceptance criteria of < 5.0, referring to institutional standards, such as those from CLSI and CAP. Accuracy was assessed using 19 whole blood samples from patients and two proficiency testing samples with previously confirmed results obtained using the Qiagen Artus CMV assay. The correlation coefficient was calculated and compared with the reference to institutional standards. The size of the correlation interpretation is as follows: 0.90 to 1.00: Very high positive (negative) correlation. 0.70 to 0.90: High positive (negative) correlation. 0.50 to 0.70: Moderate positive (negative) correlation 0.30 to 0.50: Low positive (negative) correlation. 0.00 to 0.30: negligible correlation. To establish the reference range, 13 extracted samples from healthy donors were tested. These samples served as negative controls to determine the expected values in a normal population. Specificity was assessed using samples that tested positive for other viruses, such as EBV, HBV, and BKV, to evaluate CMV cross-reactivity with other pathogens. In addition, icteric and lipemic samples were used to determine whether these factors could cause false positive results. The reportable range was validated by testing a series of four kit standards with values of 1 × 10 7 , 1 × 10 6 , 1 × 10 5 , and 1 × 10 4 , respectively. A serial dilution of the lowest standard yielded results within the anticipated analytical measurement range (AMR). The dilution points were 5000, 2500, 1250, 625, 265, and 156 IU/ml. The measured values were compared with the assigned values, typically plotting the log₁₀ of the measured values against the log₁₀ of the assigned values, and then calculating a regression coefficient, R², which must be around 1, which indicates that the data perfectly fit the linear model. Calculate the probability that a patient possesses a specific ailment given a positive test result (PPV). The NPV, or probability that a patient is free of the ailment, was assessed and juxtaposed with the acceptance requirements, which must meet a minimum of 95% as per institutional standards such as CLSI and CAP. Carryover was evaluated utilizing high-positive and negative CMV samples, which were examined consecutively in the same run to guarantee the absence of cross-contamination across samples. Results Analytical precision The RealStar® CMV PCR Kit 1.0 demonstrated superior analytical performance compared to the gold standard, the Qiagen Artus CMV PCR test, on all critical validation criteria. The analytical precision of the test was above average, with a repeatability CV of 2.14% and a reproducibility CV of 1.29%, both of which were significantly lower than the generally accepted criterion of 5.0%. To confirm the assay's dependability for quantitative CMV detection, these results showed minimal variability both within and between assays. Analytical specificity, Accuracy, and sensitivity The assay correctly identified all 13 negative samples, demonstrating the complete analytical specificity of the assay. Importantly, potentially interfering substances, such as lipemic, or icteric components, did not affect the assay performance, and the assay showed no inhibitory or cross-reactivity with non-CMV pathogens. This high specificity highlights the robustness of the oligonucleotide design used in the RealStar® assay. The accuracy assessment showed a strong positive correlation (r = 0.93) with the Qiagen Artus assay, indicating excellent agreement between the two methods for CMV quantification (Fig. 1; Table 1). Table 2 summarizes the sensitivity performance of the RealStar® CMV PCR Kit 1.0 using serially diluted Bio-Rad reference standards. The assay demonstrated 100% detection rates at concentrations of 1047, 523, 419, 291, and 265 IU/mL, with all tested replicates (ranging from 9 to 23 per dilution) yielding true positive results. The corresponding 95% confidence intervals (CI) for these concentrations ranged from 255 to 1159 IU/mL, affirming the consistency and reliability of detection within this range. At the lowest tested concentration of 207 IU/mL, none of the 10 replicates were detected, indicating that this value falls below the assay’s limit of detection. These results confirm that the CMV-RealStar® assay exhibits high analytical sensitivity, reliably detecting CMV DNA at concentrations down to approximately 265 IU/ml when using the Bio-Rad real-time instrument, making it a suitable tool for early and accurate diagnosis in immunocompromised patients. With a correlation coefficient of 0.93, our data demonstrated a very high positive correlation between the quantitative results obtained from the RealStar® assay and those from the reference Qiagen Artus assay. This confirms the strong agreement between the two methods and supports the reliability of the RealStar® CMV PCR kit assay for CMV detection in clinical practice. Linearity, Reportable, and reference range Figure 2 compares the expected and measured results in concentrations to test the linearity of the RealStar® CMV PCR Kit 1.0. The coefficient of determination (R² = 0.9517) showed linearity within the dynamic range tested, indicating a strong correlation between estimated and predicted values. This shows that the kit can accurately detect and quantify CMV in clinical samples from immunocompromised patients. Table 3 shows the results of the linearity analysis of the RealStar® CMV PCR Kit 1.0 from standard samples throughout a wide dynamic range. The test results, 10,000,000 to 156 IU/mL, were as expected. Quantification was precise, matching observed and expected values for increased concentrations (QS 1–QS 4). Technological and biological factors caused changes at lower and medium concentrations, although the projected values were within acceptable deviation ranges. All replicates showed 0 IU/ml at the lowest diluted concentration (156 IU/ml), indicating a detection limit near this value which is (265 IU/ml). The regression analysis showed a significant linear association (R² = 0.999) within the reportable range. The RealStar® CMV PCR Kit for monitoring viral load in immunocompromised patients was found to be quantitatively precise and reliable for the identification and quantification of CMV DNA in clinical samples. External quality assessment using CAP samples The RealStar® CMV PCR Kit 1.0 was evaluated for external quality assurance using reference samples from the College of American Pathologists (CAP). Four CAP samples were tested: VLS04_2024, VLS13_2023, VLS_03_2023, and VLS_04_2023. The values obtained from the RealStar® kit showed strong agreement with the CAP-assigned ranges for both logarithmic transformed and absolute IU/ml values. For VLS04_2024, the measured viral load was 4.62 log₁₀ IU/mL (41,686), which falls within the CAP-assigned range of 4.35 to 4.93 log₁₀ (22,387 to 85,113), closely matching the CAP mean of 4.60 log₁₀ (39,810). VLS13_2023 demonstrated a measured result of 3.96 log₁₀ (9,135), which was within the CAP-assigned range of 3.35 to 4.07 log₁₀ IU/mL (2,238.7–11,748) and aligned well with the CAP mean of 3.77 log₁₀ (5,919). VLS_03_2023 showed a result of 3.58 log₁₀ (3,801), which is within the CAP range of 3.33 to 3.76 log₁₀ (2,137 to 5,754), with a CAP mean of 3.52 log₁₀ IU/ml (3,311). For VLS_04_2023, the RealStar® kit yielded 4.04 log₁₀ IU/ml (10,964), which corresponded well to the CAP-defined range of 3.62 to 4.13 log₁₀ IU/mL (4,168 to 13,489) and the CAP mean of 3.91 log₁₀ IU/ml (8,128) (Table 4). These results confirm the high precision and consistency of the RealStar® CMV PCR Kit 1.0 in external quality assessments, further supporting its reliability for clinical use in CMV quantification. Establishment of the reference range Control samples from CMV-free individuals were utilized to establish the reference range. All control samples yielded negative results, which validates that the approach identifies the absence of CMV in healthy individuals. This indicates that the assay performs effectively in the reference group, yielding true negative results for those without illness. Sequential testing of high-positive and negative CMV samples within the same run evaluated carryover contamination. The negative results of the sample were juxtaposed with the quantities of the reference method. All negative samples yielded negative results, demonstrating that the assay does not cross-contaminate high-positive and negative samples. In the validation of CMV diagnostic tests, positive predictive value (PPV) and negative predictive value (NPV) are essential to evaluate the test's efficacy in identifying CMV infection, particularly in transplant recipients or immunocompromised patients. Positive Predictive Value (PPV) = 100xTP/ (TP+FP) = 100% (TP true positive; FP false positive) Negative Predictive Value (NPV) = 100xTN/ (FN+TN) = 100% (FN false negative; TN true negative) Discussion Infection with cytomegalovirus (CMV) is common in those with impaired immune systems, particularly those who have recently received a transplant [ 9 ]. This population has been tested with the RealStar® CMV PCR Kit 1.0 for the presence of CMV DNA in plasma and blood samples [ 10 ]. For rapid therapeutic action to be taken, diagnostic sensitivity is crucial. According to Caurie et al. [ 5 ], the assay achieved a positive detection rate of 95% for plasma samples and 265 IU/mL for whole blood specimens. The RealStar® kit outperformed competing commercial tests, demonstrating outstanding performance across extraction methods [ 11 ]. Aptima transcription-mediated amplification test was found to be more effective than other methods in identifying CMV in transplant patient samples, according to a comparative analysis [ 12 ]. The importance of accurate diagnostic methods for the treatment of CMV infections in patients with impaired immune systems is highlighted in this study. The high incidence of co-infections in patients with impaired immune systems highlights the critical need for analytical specificity. The RealStar® CMV PCR Kit 1.0 demonstrated extremely high specificity. More than 100 herpesviruses and diseases, including HIV, hepatitis, BK and JC viruses, were found to have no cross-reactivity in the 2019 research by Yip et al [ 13 ]. This selection supports positive outcomes for CMV infections while reducing unnecessary interventions. Because CMV infection is more common in cancer patients, a correct diagnosis is crucial. Making well-informed decisions about starting antiviral medication is easier with prompt detection. Quantitative PCR technology provides better diagnostic information than traditional approaches [ 14 , 15 ]. The sensitivity, specificity, and reproducibility of the PCR kit were assessed. Whole blood samples are ideal for viremia surveillance because they are easy to collect and the results show that the kit has increased specificity and can effectively identify CMV DNA from these samples. With repeatability and reproducibility variation coefficients of 2.14% and 1.29%, respectively, the assay proved to be precise. It is essential to closely monitor the viral load when caring for transplant recipients. The RealStar® CMV PCR Kit 1.0 showed a high level of agreement with the Abbott RealTime CMV test, with a sensitivity of 99.04% and specificity of 100% in plasma analysis [ 13 ]. In a meta-analysis of dried blood spot PCR testing, Wang et al. [ 16 ] found consistent diagnostic odds ratios across different populations, validating the diagnostic reliability of PCR. The inability to pinpoint certain sensitivity traits was a shortcoming of this comprehensive meta-analysis [ 16 ]. While the increased specificity reduces the likelihood of incorrect treatments caused by false positives, our data show a sensitivity that exceeds 95%. The results of the CAP test show that the RealStar® CMV PCR Kit 1.0 is reliable and accurate compared to the standardized specimens from the COBAS AmpliPrep CMV test. There was a strong correlation with reference standards, as the measured CMV viral loads were in agreement with the CAP-provided averages and were within acceptable CAP limits. This agreement shows that the kit accurately detects CMV DNA in clinical settings. The results show that it works well with different viral loads; therefore, it is useful for keeping an eye on CMV infections in those with weakened immune systems. Hirsch et al. [ 17 ] supported multicenter study to improve infection outcomes in high-risk populations, reporting the precision of the CAP CMV assay in five laboratories. Payandeh et al. developed a real-time PCR method for CMV diagnosis in transplant patients. [ 18 ]. To better manage this at-risk group, their study compared PCR results to those of conventional diagnostic tools, drawing attention to the former's superior sensitivity and specificity. The results are encouraging, but more research is needed to confirm the use of PCR in different groups of patients, with consideration of other real-time platforms other than the 96-CFX Bio-Rad used in this study. This strategy can help clinical professionals improve the precision of their diagnoses. We test the efficacy of the RealStar® CMV assay in a variety of patient populations and push for its integration into clinical procedures. This study had some limitations that must be acknowledged when evaluating the results. The sample size was comparatively limited, perhaps constraining the generalizability of the findings to broader populations. Secondly, the research was performed in a particular geographic area, which may restrict its relevance to other settings. The study duration was rather brief, potentially failing to account for long-term impacts or seasonal fluctuations. The methodological restrictions encompass possible biases in the data gathering and analytical processes. Fifth, technological limitations and ethical issues may have restricted the breadth and profundity of our research. Subsequent research must confront these constraints to enhance and corroborate the findings of this study. Conclusions During CMV detection using the RealStar® CMV PCR Kit 1.0, immunocompromised stem cell transplant patients demonstrated remarkable results. Compared to Qiagen Artus CMV PCR, the assay not only achieved 100% predictive values, but it also displayed improved accuracy, sensitivity, and specificity. The results of this study provide evidence that CMV can be accurately diagnosed in high-risk monitoring and provide justification for clinical integration in the management of CMV in immunocompromised individuals. It is possible that future research with bigger patient cohorts will prove its usefulness across all patient types. Abbreviations AMR Anticipated Measurement Range BKV BK virus CMV Cytomegalovirus EBV Epstein-Barr virus HIV Human immunodeficiency virus HPV Human polyomavirus KHCC King Hussein Cancer Center LOD Limit of Detection LOQ Limit of Quantification NPV Negative Predictive Value PCR Polymerase chain reaction PPV Positive Predictive Value qPCR quantitative polymerase chain reaction Declarations Ethics approval and consent to participate The study was approved by the Institutional Review Board (IRB). The IRB at KHCC works in accordance with the Declaration of Helsinki. Patients’ informed consent was waived as these samples were left over from previously processed samples. No samples were collected for the purpose of validation. Conflict of interest The authors declare that they have no conflict of interest. Funding The study did not receive funding from any sources. Author Contribution Inception of idea: MAH. Collection of data: ZS. Analysis: ZS, OAA, SKH. Writing first draft: ZS, MAH. Review of draft: OAA, SKH. Approval of final draft: All authors. Acknowledgments The authors thank the Office of Scientific Affairs and Research at the King Hussein Cancer Center for their help and support, which improved the quality of this manuscript. Data Availability The datasets generated and/or analysed during the current study are available in the Supplementary files References Stern M, Hirsch H, Cusini A, van Delden C, Manuel O, Meylan P, et al. Cytomegalovirus Serology and Replication Remain Associated With Solid Organ Graft Rejection and Graft Loss in the Era of Prophylactic Treatment. Transplantation. 2014;98:1013–8. https://doi.org/10.1097/tp.0000000000000160 . Chou S. Acquisition of Donor Strains of Cytomegalovirus by Renal-Transplant Recipients. N Engl J Med. 1986;314:1418–23. https://doi.org/10.1056/nejm198605293142205 . Breda G, Almeida B, Carstensen S, Bonfim CM, Nogueira MB, Vidal LR, et al. Human cytomegalovirus detection by real-time PCR and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries. Pathog Glob Health. 2013;107:312–9. https://doi.org/10.1179/2047773213y.0000000114 . Preiksaitis JK, Hayden RT, Tong Y, Pang XL, Fryer JF, Heath AB, et al. Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples. 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Bottino P, Pastrone L, Zanotto E, Sidoti F, Cavallo R, Costa C. Molecular diagnosis of Cytomegalovirus infection: clinical performance of the Aptima transcription-mediated amplification assay toward conventional qPCR chemistry on whole blood samples. J Clin Microbiol. 2024;62. https://doi.org/10.1128/jcm.00906-23 . Yip CCY, Sridhar S, Leung K-H, Cheng AKW, Chan K-H, Chan JFW, et al. Evaluation of RealStar® Alpha Herpesvirus PCR Kit for Detection of HSV-1, HSV-2, and VZV in Clinical Specimens. Biomed Res Int. 2019;2019:1–6. https://doi.org/10.1155/2019/5715180 . De Vries JJC, Van Der Eijk AA, Wolthers KC, Rusman LG, Pas SD, Molenkamp R, et al. Real-time PCR versus viral culture on urine as a gold standard in the diagnosis of congenital cytomegalovirus infection. J Clin Virol. 2012;53. https://doi.org/10.1016/j.jcv.2011.11.006 . Lee H, Oh EJ. Laboratory diagnostic testing for cytomegalovirus infection in solid organ transplant patients. 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Tables Table 1: Samples Real star kit IU/ml QIAGEN kit IU/ml Outcomes S_23 Negative Negative BK positive S_24 Negative Negative HBV positive S_25 Negative Negative HBV positive S_26 Negative Negative EBV positive S_27 Negative Negative EBV positive S_28 Negative Negative EBV positive S_29 Negative Negative HBV positive S_30 Negative Negative Entero positive S_31 Negative Negative EBV positive S_32 Negative Negative LIPMIC S_33 Negative Negative ICTERIC S_34 Negative Negative EBV positive S_35 Negative Negative EBV positive Table 2: BIORAD (IU/ml) Detected Results (Replicates) %Detected samples 95% CI DILUTION WITH 1047 10 / 10 100% 749-1159 DILUTION WITH 523 9 / 9 100% 573-800 DILUTION WITH 419 23 / 23 100% 389_583 DILUTION WITH 291 20 / 20 100% 246-421 DILUTION WITH 265 20 / 20 100% 255-372 DILUTION WITH 207 0 / 10 0% _ Table 3: SAMPLE ID Calculated results Real Star kit IU/ml Expected result IU/ml QS 1 10000000 10000000 QS 2 1000000 1000000 QS 3 100000 100000 QS 4 10000 10000 CMV S4 1/2 _1 3767 5000 CMV S4 1/2 _1 2959 5000 CMV S4 1/2 _1 3517 5000 CMV S4 1/2 _2 1244 2500 CMV S4 1/2 _2 1820 2500 CMV S4 1/2 _2 1948 2500 CMV S4 1/2 _3 692 1250 CMV S4 1/2 _3 587 1250 CMV S4 1/2 _3 674 1250 CMV S4 1/2 _4 595 625 CMV S4 1/2 _4 680 625 CMV S4 1/2 _4 524 625 CMV S4 1/2.3_5 270 265 CMV S4 1/2.3_5 387 265 CMV S4 1/2.3_5 236 265 CMV S4 ½_6 0 156 CMV S4 ½_6 0 156 CMV S4 ½_6 0 156 Regression Coefficient, R² 0.999 Table 4: Sample CAP Log 10 (IU/mL) CAP Range (IU/mL) CAP range log 10 (IU/mL) CAP (mean value) log 10 (IU/mL) CAP Range (IU/mL) CAP (mean value) (IU/mL) VLS04_2024 4.62 41685 4.35-4.93 4.60 22387-85113 39810 VLS13_2023 3.96 9135 3.35- 4.07 3.77 2238.7-11748 5919 VLS03_2023 3.58 3801 3.33- 3.76 3.52 2137-5754 3311 VLS04_2023 4.04 10964 3.62- 4.13 3.91 4168-13489 8128 Additional Declarations No competing interests reported. Supplementary Files AnalyticalVerificationValidationPlanCMVREALSTARBIORADIUPERML.xlsx 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. 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Center","correspondingAuthor":false,"prefix":"","firstName":"Osama","middleName":"","lastName":"Alsmadi","suffix":""},{"id":526310423,"identity":"3a7ff136-8d1f-4109-9350-d931143925be","order_by":2,"name":"Saleh Khudirat","email":"","orcid":"","institution":"King Hussein Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Saleh","middleName":"","lastName":"Khudirat","suffix":""},{"id":526310424,"identity":"b0e8d575-7e67-4210-be32-ff95bc7651d4","order_by":3,"name":"Maysa Al-Hussaini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYDACCSjJxt7GBmIxk6CF5xhpWkCMNDbi3KU7u8f4448aizw+yWdpD37UMLDzE9JidueMmTTPMYliNum044Y9xxiYJRsIabmRY8bMwCaR2Cad3ibN2MDAbHCAsBagw/4BtUgeJ16LgQRvG1CLBNsxYrWklUnz9gG18KSlSfYckyDGL8mbP/74Vpc4v/2YmcSPGptkgiGGDiSSSdXBwGBHupZRMApGwSgY7gAAp2Q3DTk0bq4AAAAASUVORK5CYII=","orcid":"","institution":"King Hussein Cancer 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14:51:27","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92671,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7664929/v1/b5f68cf5af8d4218785cf042.html"},{"id":93241330,"identity":"53d453ef-b572-4df0-ae5c-0a71b7cfa446","added_by":"auto","created_at":"2025-10-10 14:51:27","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":353042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSensitivity performance of CMV PCR Kit\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7664929/v1/e59e11b1eafb22ad07b5a496.jpeg"},{"id":93244517,"identity":"29e0a3b8-441e-4a5a-a323-f3164e94a70b","added_by":"auto","created_at":"2025-10-10 15:07:27","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":255673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinearity and reportable range performance of CMV PCR Kit\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7664929/v1/92be3a43245ef42421ffd560.jpeg"},{"id":93669816,"identity":"fb5a177f-b331-4ddf-a79b-839726985ccd","added_by":"auto","created_at":"2025-10-16 09:47:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1469056,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7664929/v1/52ece40b-a654-4e20-a3ea-7cf3245bc704.pdf"},{"id":93241328,"identity":"3c6e67d6-cc2d-466f-9eab-bb41cab6a21a","added_by":"auto","created_at":"2025-10-10 14:51:27","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":269658,"visible":true,"origin":"","legend":"","description":"","filename":"AnalyticalVerificationValidationPlanCMVREALSTARBIORADIUPERML.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7664929/v1/79585790024edcc0acd00256.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Validation of Real Star® CMV PCR Kit 1.0 for the detection of cytomegalovirus in Immunocompromised Patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePatients undergoing organ transplantation are at increased risk of infection with cytomegalovirus (CMV), a member of the Herpesviridae family and subfamily Betaherpesvirinae, which can cause serious illness and even death [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Both the transplanted organ and the recipient's prior (latent) CMV infection can be the source of infection [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, in immunocompromised patients, primary infection or latent CMV reactivation can lead to fatal diseases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In a so-called \"preemptive\" approach, diagnostic tests designed to detect CMV infection early are monitored in the majority individuals at risk of infection or disease. In the early years of CMV antigenemia with Pp65, laboratory surveillance was performed as part of preemptive therapy. Molecular testing has replaced antigenemia as the primary method for the diagnosis and monitoring of CMV. No universal consensus has been achieved on the threshold for starting medication against CMV; however, this is due to considerable differences between assays [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn recent years, CMV detection techniques have advanced. Rawlinson et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] state that saliva or urine samples collected within two to three weeks of life are the most effective method to detect CMV in newborns. In the majority of laboratories, culture-based approaches have been replaced by high-throughput molecular techniques such as quantitative polymerase chain reaction (qPCR) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which has made it easier to conduct CMV screening initiatives all over the world. In this study, a commercially available standardized polymerase chain reaction (PCR) test will be evaluated to determine whether it is effective in detecting CMV infections in immunocompromised patients or in those who have received stem cell transplantation. The purpose of this study was to validate a real-time PCR-based DNA detection approach in whole blood samples with the aim of helping high-risk groups improve CMV diagnosis and therapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample collection and target population\u003c/h2\u003e\u003cp\u003eThis study utilized patient samples that were subjected to real-time CMV PCR testing using a Qiagen kit. These patients underwent bone marrow transplants at King Hussein Cancer Center (KHCC) from March 2024 to June 2024 to participate in the study. Samples used in the study were chosen through a random selection process. The College of American Pathologists (CAP) and the Clinical and Laboratory Standards Institute (CLSI) together have set validation requirements and standards for quantitative real-time PCR assays including analytical precision, specificity, sensitivity, linearity, precision, carryover, and reportable range. The 35 DNA samples used were extracted from whole blood samples using the QIAamp DSP Virus Kit on a Qiasymphony instrument (Qiagen). The study samples were collected between March 2024 and June 2024, from immunocompromised adult and pediatric patients, specifically those who have undergone stem cell transplantation at the KHCC. These patients are highly susceptible to CMV infection. Only a few studies have evaluated CMV PCR assays in immunocompetent patients.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePatient Samples\u003c/h3\u003e\n\u003cp\u003eGroup 1 (CMV group) comprised of DNA samples from 21 confirmed CMV-positive cases, verified by a previously validated real-time PCR kit (Qiagen). Group 2, comprising diverse illnesses and healthy controls, comprised of 14 DNA samples. These samples were collected from patients with confirmed infections, including EBV, HBV, and BKV. This control group was selected to assess clinical specificity as all exhibited highly comparable highly comparable outcomes. These infections were confirmed by the presence of the corresponding viral load using real-time PCR. In addition, one sample from this group was icteric, and another was lipemic. These two samples were utilized to assess whether or not false-positive results are observed due to the presence of interfering substances.\u003c/p\u003e\n\u003ch3\u003eWhole blood DNA extraction\u003c/h3\u003e\n\u003cp\u003eBlood samples were collected in K3 EDTA tubes (minimum 2 mL), stored at 8\u0026deg;C, and processed within 24 h to prevent viral degradation. DNA extraction was performed using the QIAamp DSP Virus Kit on the Qiasymphony platform. A volume of 400 \u0026micro;L of whole blood was used per extraction, with a final elution in 80 \u0026micro;l of elution buffer. All extracted DNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until testing.\u003c/p\u003e\n\u003ch3\u003ePCR method\u003c/h3\u003e\n\u003cp\u003eThis retrospective comparative study aimed to validate the Quantitative CMV-Realstar\u0026reg; PCR kit (Altona Diagnostics GmbH, M\u0026ouml;rkenstr. 12, D-22767 Hamburg) using the BioRad CFX-96 real-time PCR instrument by contrasting it with the Qiagen Artus CMV reference assay, which served as the reference method employing the Rotor Gene Q system. The RealStar\u0026reg; CMV PCR Kit 1.0 was utilized to identify and quantify human CMV DNA. The assay was conducted following the manufacturer's guidelines using a BioRad CFX-96 real-time PCR apparatus. All values are shown in IU/ml in whole blood. The kit contained all necessary reagents: Taq polymerase, buffer, dNTPs, CMV-specific primers/probes categorized into Mix A and Mix B, internal control, and quantification standards (QS) at four distinct doses. The MIQE guidelines were followed for the quantitative real-time PCR assay, as previously described by Bustin et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnalytical evaluation\u003c/h3\u003e\n\u003cp\u003eWe define the limit of detection (LOD) as the lowest virus concentration where viral assays detect the virus in \u0026ge;\u0026thinsp;95% of sample, we assessed the LOD by serial dilution in a CMV-positive sample DNA, starting at 32,558 IU/mL, and ending at 156 IU/ml, as quantified using the CMV kit 4 standards. The dilution points were 5000, 2500, 1250, 1047, 625, 523, 419, 291, 265, 207, and 156 IU/ml. The PCR test was performed using the RealStar kit for diluted samples with titers of 5000, 2500, and 1250 IU/mL in three replicates each, followed by titers of 1047 and 523 IU/mL ten replicates each, 419, 291, and 265 IU/mL twenty replicates each, and finally 207 IU/mL ten replicates. Next, we compared the obtained values with the anticipated values.\u003c/p\u003e\u003cp\u003eThe limit of quantification (LOQ) was defined as the lowest concentration in this series of dilutions that could be reliably reproduced within a range of 90\u0026ndash;100% with a maximum error of 5%. Eight samples were analyzed using polymerase chain reaction (PCR) with varied quantities in three separate runs conducted on separate days by two separate operators to determine the between-run coefficient of variation (CV). To evaluate the within-run CV, two CMV-positive DNA samples were aliquoted into five fractions and used for repeat measurements within the same run by the same operator, on the same instrument, on the same day. CV was compared using the acceptance criteria of \u0026lt;\u0026thinsp;5.0, referring to institutional standards, such as those from CLSI and CAP.\u003c/p\u003e\u003cp\u003e Accuracy was assessed using 19 whole blood samples from patients and two proficiency testing samples with previously confirmed results obtained using the Qiagen Artus CMV assay. The correlation coefficient was calculated and compared with the reference to institutional standards.\u003c/p\u003e\u003cp\u003eThe size of the correlation interpretation is as follows:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e0.90 to 1.00: Very high positive (negative) correlation.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e0.70 to 0.90: High positive (negative) correlation.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e0.50 to 0.70: Moderate positive (negative) correlation\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e0.30 to 0.50: Low positive (negative) correlation.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e0.00 to 0.30: negligible correlation.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eTo establish the reference range, 13 extracted samples from healthy donors were tested. These samples served as negative controls to determine the expected values in a normal population. Specificity was assessed using samples that tested positive for other viruses, such as EBV, HBV, and BKV, to evaluate CMV cross-reactivity with other pathogens. In addition, icteric and lipemic samples were used to determine whether these factors could cause false positive results. The reportable range was validated by testing a series of four kit standards with values of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, and 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, respectively. A serial dilution of the lowest standard yielded results within the anticipated analytical measurement range (AMR). The dilution points were 5000, 2500, 1250, 625, 265, and 156 IU/ml. The measured values were compared with the assigned values, typically plotting the log₁₀ of the measured values against the log₁₀ of the assigned values, and then calculating a regression coefficient, R\u0026sup2;, which must be around 1, which indicates that the data perfectly fit the linear model.\u003c/p\u003e\u003cp\u003eCalculate the probability that a patient possesses a specific ailment given a positive test result (PPV). The NPV, or probability that a patient is free of the ailment, was assessed and juxtaposed with the acceptance requirements, which must meet a minimum of 95% as per institutional standards such as CLSI and CAP. Carryover was evaluated utilizing high-positive and negative CMV samples, which were examined consecutively in the same run to guarantee the absence of cross-contamination across samples.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAnalytical precision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RealStar® CMV PCR Kit 1.0 demonstrated superior analytical performance compared to the gold standard, the Qiagen Artus CMV PCR test, on all critical validation criteria. The analytical precision of the test was above average, with a repeatability CV of 2.14% and a reproducibility CV of 1.29%, both of which were significantly lower than the generally accepted criterion of 5.0%. To confirm the assay's dependability for quantitative CMV detection, these results showed minimal variability both within and between assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalytical specificity, Accuracy, and sensitivity \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assay correctly identified all 13 negative samples, demonstrating the complete analytical specificity of the assay. Importantly, potentially interfering substances, such as lipemic, or icteric components, did not affect the assay performance, and the assay showed no inhibitory or cross-reactivity with non-CMV pathogens. This high specificity highlights the robustness of the oligonucleotide design used in the RealStar® assay. The accuracy assessment showed a strong positive correlation (r = 0.93) with the Qiagen Artus assay, indicating excellent agreement between the two methods for CMV quantification (Fig. 1; Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 summarizes the sensitivity performance of the RealStar® CMV PCR Kit 1.0 using serially diluted Bio-Rad reference standards. The assay demonstrated 100% detection rates at concentrations of 1047, 523, 419, 291, and 265 IU/mL, with all tested replicates (ranging from 9 to 23 per dilution) yielding true positive results. The corresponding 95% confidence intervals (CI) for these concentrations ranged from 255 to 1159 IU/mL, affirming the consistency and reliability of detection within this range. At the lowest tested concentration of 207 IU/mL, none of the 10 replicates were detected, indicating that this value falls below the assay’s limit of detection. These results confirm that the CMV-RealStar® assay exhibits high analytical sensitivity, reliably detecting CMV DNA at concentrations down to approximately 265 IU/ml when using the Bio-Rad real-time instrument, making it a suitable tool for early and accurate diagnosis in immunocompromised patients.\u0026nbsp;With a correlation coefficient of 0.93, our data demonstrated a very high positive correlation between the quantitative results obtained from the RealStar® assay and those from the reference Qiagen Artus assay. This confirms the strong agreement between the two methods and supports the reliability of the RealStar® CMV PCR kit assay for CMV detection in clinical practice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity, Reportable, and reference range\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 2 compares the expected and measured results in concentrations to test the linearity of the RealStar® CMV PCR Kit 1.0. The coefficient of determination (R² = 0.9517) showed linearity within the dynamic range tested, indicating a strong correlation between estimated and predicted values. This shows that the kit can accurately detect and quantify CMV in clinical samples from immunocompromised patients. Table 3 shows the results of the linearity analysis of the RealStar® CMV PCR Kit 1.0 from standard samples throughout a wide dynamic range. The test results, 10,000,000 to 156 IU/mL, were as expected. Quantification was precise, matching observed and expected values for increased concentrations (QS 1–QS 4). Technological and biological factors caused changes at lower and medium concentrations, although the projected values were within acceptable deviation ranges. All replicates showed 0 IU/ml at the lowest diluted concentration (156 IU/ml), indicating a detection limit near this value which is (265 IU/ml). The regression analysis showed a significant linear association (R² = 0.999) within the reportable range. The RealStar® CMV PCR Kit for monitoring viral load in immunocompromised patients was found to be quantitatively precise and reliable for the identification and quantification of CMV DNA in clinical samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExternal quality assessment using CAP samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RealStar® CMV PCR Kit 1.0 was evaluated for external quality assurance using reference samples from the College of American Pathologists (CAP). Four CAP samples were tested: VLS04_2024, VLS13_2023, VLS_03_2023, and VLS_04_2023. The values obtained from the RealStar® kit showed strong agreement with the CAP-assigned ranges for both logarithmic transformed and absolute IU/ml values.\u003c/p\u003e\n\u003cp\u003eFor VLS04_2024, the measured viral load was 4.62 log₁₀ IU/mL (41,686), which falls within the CAP-assigned range of 4.35 to 4.93 log₁₀ (22,387 to 85,113), closely matching the CAP mean of 4.60 log₁₀ (39,810). VLS13_2023 demonstrated a measured result of 3.96 log₁₀ (9,135), which was within the CAP-assigned range of 3.35 to 4.07 log₁₀ IU/mL (2,238.7–11,748) and aligned well with the CAP mean of 3.77 log₁₀ (5,919). VLS_03_2023 showed a result of 3.58 log₁₀ (3,801), which is within the CAP range of 3.33 to 3.76 log₁₀ (2,137 to 5,754), with a CAP mean of 3.52 log₁₀ IU/ml (3,311). For VLS_04_2023, the RealStar® kit yielded 4.04 log₁₀ IU/ml (10,964), which corresponded well to the CAP-defined range of 3.62 to 4.13 log₁₀ IU/mL (4,168 to 13,489) and the CAP mean of 3.91 log₁₀ IU/ml (8,128) (Table 4). These results confirm the high precision and consistency of the RealStar® CMV PCR Kit 1.0 in external quality assessments, further supporting its reliability for clinical use in CMV quantification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of the reference range\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eControl samples from CMV-free individuals were utilized to establish the reference range. All control samples yielded negative results, which validates that the approach identifies the absence of CMV in healthy individuals. This indicates that the assay performs effectively in the reference group, yielding true negative results for those without illness. Sequential testing of high-positive and negative CMV samples within the same run evaluated carryover contamination. The negative results of the sample were juxtaposed with the quantities of the reference method. All negative samples yielded negative results, demonstrating that the assay does not cross-contaminate high-positive and negative samples. In the validation of CMV diagnostic tests, positive predictive value (PPV) and negative predictive value (NPV) are essential to evaluate the test's efficacy in identifying CMV infection, particularly in transplant recipients or immunocompromised patients.\u003c/p\u003e\n\u003cp\u003ePositive Predictive Value (PPV) = 100xTP/ (TP+FP)\u0026nbsp;= 100% (TP true positive; FP false positive)\u003c/p\u003e\n\u003cp\u003eNegative Predictive Value (NPV) = 100xTN/ (FN+TN) = 100% (FN false negative; TN true negative)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInfection with cytomegalovirus (CMV) is common in those with impaired immune systems, particularly those who have recently received a transplant [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This population has been tested with the RealStar\u0026reg; CMV PCR Kit 1.0 for the presence of CMV DNA in plasma and blood samples [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For rapid therapeutic action to be taken, diagnostic sensitivity is crucial. According to Caurie et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], the assay achieved a positive detection rate of 95% for plasma samples and 265 IU/mL for whole blood specimens. The RealStar\u0026reg; kit outperformed competing commercial tests, demonstrating outstanding performance across extraction methods [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Aptima transcription-mediated amplification test was found to be more effective than other methods in identifying CMV in transplant patient samples, according to a comparative analysis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The importance of accurate diagnostic methods for the treatment of CMV infections in patients with impaired immune systems is highlighted in this study.\u003c/p\u003e\u003cp\u003eThe high incidence of co-infections in patients with impaired immune systems highlights the critical need for analytical specificity. The RealStar\u0026reg; CMV PCR Kit 1.0 demonstrated extremely high specificity. More than 100 herpesviruses and diseases, including HIV, hepatitis, BK and JC viruses, were found to have no cross-reactivity in the 2019 research by Yip et al [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This selection supports positive outcomes for CMV infections while reducing unnecessary interventions. Because CMV infection is more common in cancer patients, a correct diagnosis is crucial. Making well-informed decisions about starting antiviral medication is easier with prompt detection. Quantitative PCR technology provides better diagnostic information than traditional approaches [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The sensitivity, specificity, and reproducibility of the PCR kit were assessed. Whole blood samples are ideal for viremia surveillance because they are easy to collect and the results show that the kit has increased specificity and can effectively identify CMV DNA from these samples.\u003c/p\u003e\u003cp\u003eWith repeatability and reproducibility variation coefficients of 2.14% and 1.29%, respectively, the assay proved to be precise. It is essential to closely monitor the viral load when caring for transplant recipients. The RealStar\u0026reg; CMV PCR Kit 1.0 showed a high level of agreement with the Abbott RealTime CMV test, with a sensitivity of 99.04% and specificity of 100% in plasma analysis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In a meta-analysis of dried blood spot PCR testing, Wang et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] found consistent diagnostic odds ratios across different populations, validating the diagnostic reliability of PCR. The inability to pinpoint certain sensitivity traits was a shortcoming of this comprehensive meta-analysis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. While the increased specificity reduces the likelihood of incorrect treatments caused by false positives, our data show a sensitivity that exceeds 95%.\u003c/p\u003e\u003cp\u003eThe results of the CAP test show that the RealStar\u0026reg; CMV PCR Kit 1.0 is reliable and accurate compared to the standardized specimens from the COBAS AmpliPrep CMV test. There was a strong correlation with reference standards, as the measured CMV viral loads were in agreement with the CAP-provided averages and were within acceptable CAP limits. This agreement shows that the kit accurately detects CMV DNA in clinical settings. The results show that it works well with different viral loads; therefore, it is useful for keeping an eye on CMV infections in those with weakened immune systems. Hirsch et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] supported multicenter study to improve infection outcomes in high-risk populations, reporting the precision of the CAP CMV assay in five laboratories.\u003c/p\u003e\u003cp\u003ePayandeh et al. developed a real-time PCR method for CMV diagnosis in transplant patients. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To better manage this at-risk group, their study compared PCR results to those of conventional diagnostic tools, drawing attention to the former's superior sensitivity and specificity. The results are encouraging, but more research is needed to confirm the use of PCR in different groups of patients, with consideration of other real-time platforms other than the 96-CFX Bio-Rad used in this study. This strategy can help clinical professionals improve the precision of their diagnoses. We test the efficacy of the RealStar\u0026reg; CMV assay in a variety of patient populations and push for its integration into clinical procedures.\u003c/p\u003e\u003cp\u003eThis study had some limitations that must be acknowledged when evaluating the results. The sample size was comparatively limited, perhaps constraining the generalizability of the findings to broader populations. Secondly, the research was performed in a particular geographic area, which may restrict its relevance to other settings. The study duration was rather brief, potentially failing to account for long-term impacts or seasonal fluctuations. The methodological restrictions encompass possible biases in the data gathering and analytical processes. Fifth, technological limitations and ethical issues may have restricted the breadth and profundity of our research. Subsequent research must confront these constraints to enhance and corroborate the findings of this study.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eDuring CMV detection using the RealStar\u0026reg; CMV PCR Kit 1.0, immunocompromised stem cell transplant patients demonstrated remarkable results. Compared to Qiagen Artus CMV PCR, the assay not only achieved 100% predictive values, but it also displayed improved accuracy, sensitivity, and specificity. The results of this study provide evidence that CMV can be accurately diagnosed in high-risk monitoring and provide justification for clinical integration in the management of CMV in immunocompromised individuals. It is possible that future research with bigger patient cohorts will prove its usefulness across all patient types.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAMR Anticipated Measurement Range\u003c/p\u003e\u003cp\u003eBKV BK virus\u003c/p\u003e\u003cp\u003eCMV Cytomegalovirus\u003c/p\u003e\u003cp\u003eEBV Epstein-Barr virus\u003c/p\u003e\u003cp\u003eHIV Human immunodeficiency virus\u003c/p\u003e\u003cp\u003eHPV Human polyomavirus\u003c/p\u003e\u003cp\u003eKHCC King Hussein Cancer Center\u003c/p\u003e\u003cp\u003eLOD Limit of Detection\u003c/p\u003e\u003cp\u003eLOQ Limit of Quantification\u003c/p\u003e\u003cp\u003eNPV Negative Predictive Value\u003c/p\u003e\u003cp\u003ePCR Polymerase chain reaction\u003c/p\u003e\u003cp\u003ePPV Positive Predictive Value\u003c/p\u003e\u003cp\u003eqPCR quantitative polymerase chain reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e The study was approved by the Institutional Review Board (IRB). The IRB at KHCC works in accordance with the Declaration of Helsinki. Patients\u0026rsquo; informed consent was waived as these samples were left over from previously processed samples. No samples were collected for the purpose of validation.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe study did not receive funding from any sources.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eInception of idea: MAH. Collection of data: ZS. Analysis: ZS, OAA, SKH. Writing first draft: ZS, MAH. Review of draft: OAA, SKH. Approval of final draft: All authors.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors thank the Office of Scientific Affairs and Research at the King Hussein Cancer Center for their help and support, which improved the quality of this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the Supplementary files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStern M, Hirsch H, Cusini A, van Delden C, Manuel O, Meylan P, et al. Cytomegalovirus Serology and Replication Remain Associated With Solid Organ Graft Rejection and Graft Loss in the Era of Prophylactic Treatment. 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Infect Agent Cancer. 2021;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13027-021-00349-4\u003c/span\u003e\u003cspan address=\"10.1186/s13027-021-00349-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"512\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSamples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eReal star kit IU/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eQIAGEN kit IU/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOutcomes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBK positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEntero positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLIPMIC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_33\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eICTERIC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS_35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEBV positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"565\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBIORAD (IU/ml) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDetected Results (Replicates)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%Detected samples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDILUTION WITH \u0026nbsp;1047\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10 / 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100% \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e749-1159\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDILUTION WITH\u0026nbsp;523\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9 / 9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e573-800\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDILUTION WITH 419\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e23 / 23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e389_583\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDILUTION WITH 291\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20 / 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e246-421\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDILUTION WITH 265\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20 / 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e255-372\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDILUTION WITH 207\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0 / 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;_\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"361\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSAMPLE ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCalculated results\u0026nbsp;\u003cbr\u003e\u0026nbsp;Real Star kit IU/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eExpected result IU/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQS 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10000000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10000000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQS 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1000000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1000000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQS 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQS 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3767\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2959\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2500\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1820\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2500\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1948\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2500\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e692\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e587\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e674\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e595\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2 _4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e524\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2.3_5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e270\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2.3_5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 1/2.3_5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 ½_6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 ½_6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCMV S4 ½_6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRegression Coefficient, R²\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.999\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4:\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"732\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample CAP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLog 10 (IU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAP\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRange\u003c/strong\u003e\u003cstrong\u003e(IU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAP range log 10 (IU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mean value)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003elog 10 (IU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;CAP\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRange\u003c/strong\u003e\u003cstrong\u003e(IU/mL)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mean value)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(IU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVLS04_2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e41685\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.35-4.93\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22387-85113\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e39810\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVLS13_2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.35- 4.07\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 3.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2238.7-11748\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5919\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVLS03_2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.33- 3.76\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2137-5754\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3311\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVLS04_2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.62- 4.13\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 3.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4168-13489\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"Cytomegalovirus, Real-time PCR, DNA detection, immunocompromised patients, Stem cell transplant","lastPublishedDoi":"10.21203/rs.3.rs-7664929/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7664929/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCytomegalovirus (CMV) infections pose a significant risk to individuals with compromised immune systems, particularly those undergoing stem cell transplantation. This study aimed to evaluate a novel real-time PCR based DNA detection approach in whole blood samples with the aim of helping high-risk groups improve CMV diagnosis and therapy. The objective of the study was to validate the efficacy of the RealStar\u0026reg; CMV PCR quantification assay kit 1.0 to diagnose CMV infection. The assay identified CMV in 21 of the 35 blood samples and other infections in 14 samples. The reference Qiagen Artus CMV PCR test exhibited inferior accuracy compared to the RealStar\u0026reg; CMV PCR Kit 1.0. The assay demonstrated a repeatability of 2.14% and reproducibility of 1.29%. Blood components and non-CMV infections did not influence the analytical specificity, which remained at 100%. The test exhibited a detection limit of 265 IU/mL (95% CI: 255\u0026ndash;372), which correlated with the reference method (r\u0026thinsp;=\u0026thinsp;0.93). The RealStar\u0026reg; CMV PCR Kit 1.0 demonstrated strong linearity (R\u0026sup2; = 0.95) and achieved 100% predictive value for detecting CMV DNA in blood samples from immunocompromised individuals.\u003c/p\u003e","manuscriptTitle":"Validation of Real Star® CMV PCR Kit 1.0 for the detection of cytomegalovirus in Immunocompromised Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-10 14:51:22","doi":"10.21203/rs.3.rs-7664929/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":"fd359d5a-b00f-4fcc-887d-a4edde9ecc5a","owner":[],"postedDate":"October 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-16T09:39:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-10 14:51:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7664929","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7664929","identity":"rs-7664929","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}