Diagnostic Accuracy of ARK Checker® C/G – DyLight® 488: Simultaneous Detection of Giardia and Cryptosporidium by Fluorescent Antibody Microscopy

Diagnostic Accuracy of ARK Checker® C/G – DyLight® 488: Simultaneous Detection of Giardia and Cryptosporidium by Fluorescent Antibody Microscopy | 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 Diagnostic Accuracy of ARK Checker® C/G – DyLight® 488: Simultaneous Detection of Giardia and Cryptosporidium by Fluorescent Antibody Microscopy Yusuke Oshiro, Akira Kawashima, Megumi Akashi, Yasuaki Yanagawa, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8244780/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jan, 2026 Read the published version in Tropical Medicine and Health → Version 1 posted 11 You are reading this latest preprint version Abstract Background Giardiasis and cryptosporidiosis are often misdiagnosed by stool ova and parasite test (classical O&P). Multiplex PCR and rapid antigen immunochromatography (rapid-IC) could offer high diagnostic accuracy; however, their cost and restricted coverage, especially for parasites, limit routine use. This study evaluated the efficacy of adapted fluorescent antibody microscopy using ARK Checker® C/G – DyLight® 488 (FAM-TEST). The reagent is a liquid-form conjugated antibody preparation that directly reacts with Giardia cysts and Cryptosporidium oocysts. Methods Stool samples were incubated with DyLight-488–labeled antibodies as indicated in our original protocol and examined by fluorescence microscopy. Diagnostic accuracy of FAM-TEST was assessed with multiplex PCR results as reference and compared with that of rapid-IC. Samples with discordant results among these methods were additionally tested by conventional PCR with Sanger sequencing to verify infection status. Results A total of 694 stool samples were submitted for microbiological examination. FAM-TEST identified Giardia and/or Cryptosporidium in 35 samples. In addition, 49 FAM-TEST- negative samples from a randomly selected month were included as negative controls. For Giardia , all FAM-TEST results were identical to those of rapid-IC, including three false negatives [88.0% sensitivity, 100% specificity, 100% positive predictive value (PPV), and 95.2% negative predictive value (NPV)]. For Cryptosporidium , FAM-TEST showed 93.8% sensitivity, 100% specificity, 100% PPV, and 98.6% NPV, which were comparable with those of rapid-IC. Notably, nested PCR for Giardia produced negative results for three samples considered to yield FAM-TEST false negatives, raising another possibility of multiplex PCR false positives or extremely low pathogen burden, whereas conventional PCR for Cryptosporidium showed results fully consistent with those of multiplex PCR. Conclusion Concurrent use of FAM-TEST with classical O&P offers a cost-effective, practical diagnostic approach for enteric parasites, which especially strengthens diagnostic accuracy for Giardiasis/Cryptosporidiosis. Giardia duodenalis Cryptosporidium DyLight-488 antibody Fluorescent antibody microscopy Immunochromatography Polymerase chain reaction Figures Figure 1 Figure 2 Figure 3 Introduction Diarrheal diseases affect approximately 4 billion people worldwide annually, with waterborne protozoan infections being a major cause [ 1 – 4 ]. Protozoan infections, such as giardiasis and cryptosporidiosis, are primarily transmitted via the fecal–oral route in situations of poor sanitation, inadequate water supply, and environmental contamination [ 5 – 7 ]. Although these infections are mainly prevalent in developing countries, they can occur in developed countries as sporadic cases or because of an outbreak [ 4 , 6 , 8 ]. In Japan, giardiasis and cryptosporidiosis are classified as “Category V infectious diseases” under the Act on the Prevention of Infectious Diseases and Medical Care for Patients with Infectious Diseases mandating nationwide reporting of all confirmed cases [ 9 , 10 ]. Surveillance data indicate international travelers and HIV-infected individuals to be at high risk for these protozoan infections [ 6 , 11 ]. In addition, notable outbreaks of Cryptosporidium infection have been reported since its classification as a notifiable disease [ 10 ]. Moreover, diarrheal symptoms caused by these protozoa infections are indistinguishable from viral or bacterial gastroenteritis; therefore, these protozoa infections are frequently overlooked in common clinical settings [ 4 , 5 ]. Their diagnosis should be improved to enhance patient care and therefore, enable prompt epidemiological actions to protect public health. The diagnosis of Giardia and Cryptosporidium currently relies on three main methods: morphological identification by classical ova and parasite bright-field microscopy (classical O&P), antigen detection, and polymerase chain reaction (PCR) [ 3 – 5 , 7 ]. This is because intestinal protozoa cannot be cultivated in the microbiology laboratories of medical facilities [ 3 , 4 ]. Classical O&P is highly affected by the skill of the examiner and is rarely used in most developed countries because of low diagnostic accuracy [ 4 , 5 ]. Conversely, multiplex PCR has been increasingly used for diagnosis and research purposes because it offers high sensitivity and specificity [ 11 – 16 ]. It can also detect numerous enteric pathogens, including bacteria, viruses, and protozoa in the same assay. However, multiplex PCR is expensive ( $ 100 or over per test); therefore, its use is highly limited to specialized laboratories. Another common approach is rapid antigen detection by immunochromatography (rapid-IC), which provides rapid and user-friendly results without requiring specialized expertise [ 8 , 17 ]. However, its relatively high cost (~ $ 30 per test for the detection of only a few pathogens) remains a concern for routine testing of enteric infectious diseases. Moreover, multiplex PCR and rapid-IC only target pre-defined pathogens, which is a crucial limitation in the diagnosis of intestinal parasites. Diagnosis of many protozoa as well as most helminths depend on classical O&P in clinical settings, although nucleic acid identification of each parasite can be applied for research purposes. Furthermore, identification by classical O&P is not difficult for most intestinal parasites except for small protozoa (< 10 µm). Thus, classical O&P has the advantage of being usable to examine various intestinal parasites within a routine protocol without incurring additional cost. Improving the diagnostic accuracy of O&P, for example by enabling easier identification of frequently overlooked common but small-sized protozoa, such as Giardia and Cryptosporidium , is a possible solution for enhancing the clinical diagnosis of intestinal parasites. Immunofluorescent antibody testing (IFAT) has been widely used for decades to detect Giardia and Cryptosporidium cysts and oocysts under fluorescence microscopy [ 20 , 21 ]. Commercial IFAT kits, such as Merifluor®, have long been considered reference methods for morphological identification. However, the method is provided as antigen-precoated slides that must be stored in a cold, dark environment and are relatively expensive, making them less suitable for routine diagnostic use. In addition, fluorescein isothiocyanate (FITC) fluorophores used in conventional IFAT are prone to photobleaching, which limits fluorescence stability and signal intensity. To overcome these drawbacks, we have developed a simple fluorescent antibody microscopy method using ARK Checker® C/G – DyLight® 488 (FAM-TEST). The FAM-TEST has been originally used in water quality monitoring and was adapted here for clinical stool examination as a simple and cost-effective diagnostic protocol [ 22 ]. The newly developed FAM-TEST assay enables morphological identification of Giardia and Cryptosporidium in stool specimen by fluorescence microscopy after staining with DyLight🄬488-conjugated antibodies. In the present study, diagnostic performance of FAM-TEST was evaluated relative to multiplex PCR results and compared with rapid-IC results. Methods Sample collections and study approval Stool samples submitted for microbiological examination from patients with suspected infectious gastroenteritis were collected between January 2022 and April 2025 (Fig. 1). Samples of sufficient volume for FAM-TEST, rapid-IC, and multiplex PCR were selected. These diagnostic tests, as well as DNA extraction using the QIAamp PowerFecal Pro DNA Kit© (Qiagen, Hilden, Germany), were performed immediately after sampling. Extracted DNA was stored at −80°C. Microbiology results were the only patient clinical information used. Informed consent was not obtained because all submitted specimens were anonymized; however, an opt-out was employed in accordance with ethical guidelines. This study was approved by the Institutional Review Board of the National Center for Global Health and Medicine (NCGM-S-004414). All procedures were performed in accordance with the Declaration of Helsinki and the Ethical Guidelines for Medical and Biological Research Involving Human Subjects in Japan. The ethics committee waived the requirement for individual informed consent because the study used only anonymized residual clinical specimens. Fluorescent Antibody Microscopy method using ARK Checker® C/G – DyLight® 488 (FAM-TEST) for Giardia and Cryptosporidium Specimens were prepared using the formalin-ether sedimentation method. The sedimented fraction (10 μL) was mixed with 10 μL of ARK Checker C/G - DyLight 488 and mounted on a glass slide under a coverslip. Fluorescence images were obtained through a 510–550-nm wavelength filter after B-excitation (490 nm) (Fig. 2). Four clinical laboratory technologists, who had no experience of fluorescence microscopy, performed FAM-TEST diagnosis. They underwent a 1-hour training session using positive control samples of G. duodenalis and Cryptosporidium spp . prior to the study. Each diagnosis was made by a single examiner randomly selected from 4 technologists. Rapid Immunochromatographic (rapid-IC) Assays, and Multiplex PCR Rapid-IC was performed using a GIARDIA/CRYPTOSPORIDIUM QUIK CHEK test (TECH LAB, USA), and multiplex PCR was performed using a Multiplex PCR FilmArray Gastrointestinal Panel (BioMérieux, France) according to the manufacturer’s instructions. Sequence analysis of discordant cases For cases with discrepancies among FAM-TEST, rapid-IC assay, and multiplex PCR results, we performed PCR followed by Sanger sequencing using DNA extracted from stool samples. For Giardia duodenalis , nested PCR targeting the glutamate dehydrogenase ( gdh ) gene was performed. Outer primers Ghd1 (5′-TTCCGTRTYCAGTACAACTC-3′) and Gdh2 (5′-ACCTCGTTCTGRGTGGCGCA-3′), and nested primers Gdh3 (5′-ATGACYGAGCTYCAGAGGCACGT-3′) and Gdh4 (5′-GTGGCGCARGGCATGATGCA-3′) were used [23]. For Cryptosporidium spp., conventional PCR targeting the 18S rRNA gene was conducted using forward (5′-AAGCTCGTAGTTGGATTTCTG-3′) and reverse (5′-TAAGGTGCTGAAGGAGTAAGG-3′) primers [24]. The cycling conditions were as follows: an initial denaturation at 94°C for 3 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The nested Giardia PCR used an annealing temperature of 67°C and 25 cycles. Sequence analysis of each amplicon was performed by Sanger sequencing using appropriate forward and reverse primers (Eurofins Genomics, Tokyo, Japan). Statistical analysis Statistical analyses were performed using GraphPad Prism (v10.2.3). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) with 95% confidence intervals (Cis) were calculated for FAM-TEST and rapid-IC using multiplex PCR as a reference. Fisher’s exact test was used to compare FAM-TEST and rapid-IC results, with statistical significance set at p < 0.05. Results Sample collection During the 3 years of the study period, 696 stool samples collected from patients with suspected infectious colitis were examined (Fig. 1). Of these, two samples were excluded from the analysis because of insufficient sample volume. Therefore, FAM-TEST was performed on 694 samples. We identified 35 stool samples positive for Giardia and/or Cryptosporidium , which we classified as FAM-TEST-positive stool samples. These protozoa were not identified in the other 659 samples. As an unbiased collection of FAM-TEST-negative stool samples for use as control samples, we chose all the FAM-TEST-negative samples examined in 1 month, October 2024. Overall, we therefore selected 84 samples, 35 FAM-TEST-positive and 49 FAM-TEST-negative samples, for evaluating the diagnostic accuracy of three methods (Supplementary data 1). Diagnostic value of FAM-TEST Diagnostic accuracy of FAM-TEST for Giardia and Cryptosporidium was evaluated by comparison with multiplex PCR (FilmArray Gastrointestinal Panel©) as a reference. Thereafter, it was compared with rapid-IC tests (GIARDIA/CRYPTOSPORIDIUM QUIK CHEK © tests). Performance of FAM-TEST for Giardia The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FAM-TEST are shown in Table 1A. For all FAM-TEST-positive giardiasis cases, Giardia lamblia (syn. G. duodenalis ) was detected by multiplex PCR (100% PPV). G. lamblia was also detected by multiplex PCR in three FAM-TEST-negative samples, suggesting false negative results of FAM-TEST (95.2% NPV). The diagnostic performance of rapid-IC was similarly assessed (Table 1B). PPV, NPV, sensitivity, and specificity of rapid-IC were all the same as those of FAM-TEST. More surprisingly, the same three samples showed false negative results for FAM-TEST and rapid-IC. These results indicate that diagnostic accuracy of FAM-TEST is comparable to that of rapid-IC. They also indicate that the false negative results for FAM-TEST and rapid-IC probably occurred because the pathogen burden was beneath the level of detection by these tests. Performance of FAM-TEST for Cryptosporidium The performance of FAM-TEST for detecting Cryptosporidium is shown in Table 2A. Cryptosporidium spp . was detected by multiplex PCR in all FAM-TEST-positive samples, and in one sample among 64 FAM-TEST-negative samples (100% PPV, 98.6% NPV) (Table 2). In addition, rapid-IC produced no false positive results for Cryptosporidium . However, rapid-IC produced false negative results in two cases (100% PPV, 97.1% NPV), one of which was the sample that gave a false negative result by FAM-TEST. These results indicate the diagnostic accuracy of FAM-TEST for Cryptosporidium to be highly comparable to that of rapid-IC. Evaluation of Discordant Cases Using Nested and Conventional PCR As described above, discordant (false positive/negative) results produced by the different testing methods were seen in five cases, three discordant for Giardia and two discordant for Cryptosporidium (Table 3). As already mentioned, the cases discordant for Giardia yielded the same results by three diagnostic measures; they were FAM-TEST/rapid-IC-negative but multiplex PCR-positive. To confirm the presence of these pathogens in these samples, we performed conventional or nested PCR with sequencing analysis. Notably, in all samples discordant for Giardia , G. duodenalis could not be identified by nested PCR (Table 3A, Case 1-3), whereas G. duodenalis was clearly identified in the Giardia -positive sample (Table 3A, Case 4&5). These results raised two possibilities for the discordant Giardia results; one is an extremely low pathogen burden of G. duodenalis , giving a positive result only by multiplex PCR, and the other is a false positive multiplex PCR result. For cases with discordant Cryptosporidium results, we performed conventional PCR, which produced the same results as multiplex PCR (Table 3B). These findings confirm that multiplex PCR reliably identified Cryptosporidium spp . in the present analysis. Discussion Improved treatment of intestinal parasitic diseases requires a low-cost, rapid, and technically simple diagnostic measure covering various parasites. In addition, the method should be developed as highly sensitive to small-sized common protozoa, such as Giardia and Cryptosporidium , which are frequently overlooked by classical O&P. Multiplex PCR and antigen testing show high sensitivity to these protozoa [ 8 , 14 , 17 , 25 ]; however, these tests are generally expensive. Moreover, both tests can detect only pre-defined pathogens; therefore, other “untargeted” parasites need to be diagnosed separately. Currently, some intestinal protozoa and most helminths are not recognized by multiplex PCR or rapid-IC, and need to be identified morphologically by classical O&P in clinical settings. In the present study, we examined FAM-TEST using fluorescence-conjugated specific antibodies against G. duodenalis and Cryptosporidium spp. for increased sensitivity to these protozoa, as an auxiliary diagnosis method to classical O&P. The FAM-TEST system differs from conventional immunofluorescence assays (IFAT) primarily in the use of DyLight 488–conjugated monoclonal antibodies. This modification improves optical brightness and photostability compared to traditional fluorescein isothiocyanate (FITC)-labeled antibodies, allowing more consistent visualization. First, FAM-TEST exhibited high specificity for Giardia , comparable to that of rapid-IC. Additionally, the sensitivity of FAM-TEST was the same as that of rapid-IC. However, FAM-TEST and rapid-IC produced false negative results for Giardia in the same samples relative to the reference results of multiplex PCR. G. duodenalis was not identified by nested PCR targeting gdh . These results indicate that the pathogen burden was too low to be detected by these methods but not by multiplex PCR, or that multiplex PCR produced a false positive result. The manufacturer of the Multiplex PCR FilmArray Gastrointestinal Panel notes cross-reactivity between Giardia and other gut microbiota, such as Bifidobacterium spp. and Ruminococcus spp. [ 16 ]. In addition, multiplex PCR frequently reports multiple pathogens in one stool specimen, some of which do not match the results of other diagnostic methods, such as bacterial culture [ 26 ]. In fact, in the present study, half of FAM-TEST-positive “ Giardia and/or Cryptosporidium containing” samples (18 out of 35) showed multiple pathogens by multiplex PCR ( G. lamblia / Cryptosporidium spp. plus one or more other microorganisms were positive simultaneously. Supplementary data 1). Such multiplex PCR results often make it difficult for physicians to determine the causative agent of intestinal disease. Considered together, FAM-TEST has good diagnostic value for giardiasis and is comparable to that of rapid-IC. We emphasize that FAM-TEST produced only one false negative case for Cryptosporidium , whereas rapid-IC produced an additional false negative case. Additionally, FAM-TEST did not produce any false positives. In general, identification of Cryptosporidium oocysts is extremely difficult because of their relatively small size compared with other protozoa. We predict that FAM-TEST can decrease the number of overlooked or misdiagnosed cases of cryptosporidiosis that frequently occur by classical O&P examination. Fluorescence-based diagnostic techniques have also been successfully applied to several pathogens [ 27 ]. For example, auramine staining has been used not only for tuberculosis screening (excitation: 460 nm, emission: 550 nm) [ 28 , 29 ], but only the diagnosis of Cryptosporidium in low-resource settings, providing a fluorescent option that does not require antibodies or a cold chain [ 30 ]. The running cost of FAM-TEST is extremely low (up to $ 2 per test) compared with that of multiplex PCR ( $ 100 or over per test) or rapid-IC assays (~ $ 30 per test). In addition, FAM-TEST can be simultaneously performed with classical O&P. In fact, tested stool samples contained some enteric parasites (e.g. Sarcocystis spp. and Taenia saginata ), which were uncovered by multiplex PCR (OAK-40 & -48 in Supplementary data 1). They were morphologically diagnosed by classical O&P. Furthermore, Cyclospora cayetanensis can be identified by fluorescent microscopy through its autofluorescence by ultraviolet excitation (supplementary data 2). Species-level discrimination (e.g., C. parvum vs. C. hominis) or differentiation of Giardia assemblages, however, still requires PCR or sequencing. Based on these findings, we suggest a FAM-TEST-containing diagnostic algorithm for enteric parasites (Fig. 3 ). First, FAM-TEST with classical O&P is employed to screen for enteric parasites when infectious gastroenteritis is suspected. Its implementation could enhance diagnostic accuracy while minimizing the risk of overlooking common protozoa and maintaining cost-effectiveness. Rapid-IC and/or multiplex PCR should be performed in limited cases to narrow down differential diagnoses (e.g. ruling out of infectious etiology before diagnosing non-infectious inflammatory bowel diseases). This study has some limitations. First, it was conducted as a single-center study at a general hospital in Tokyo, which serves as a national reference center for travel and HIV-related infections. The number of intestinal parasite cases seen is relatively high compared with other facilities in Japan; however, positive cases of Giardia and/or Cryptosporidium are still limited. In addition, the small number of examiners from the center involved in the study were more experienced in classical O&P than general practitioners. Therefore, diagnostic accuracy could be overestimated. Considered together, a larger scale study conducted at multiple facilities in resource limited areas is warranted for more reliable assessment of the diagnostic utility of FAM-TEST. In addition, negative control samples were selected from a single month during the study period, which may introduce spectrum bias. Although this approach minimized storage-related degradation, random sampling across the entire collection period would provide more robust validation. In conclusion, we demonstrated that FAM-TEST has comparable diagnostic accuracy for Giardia and Cryptosporidium compared with rapid-IC while having the advantages of cost-effectiveness, examiner independence, and practical feasibility. Continuous efforts to improve the diagnostic flow for enteric parasites are needed for better clinical practice. Abbreviations FAM-TEST: Fluorescent antibody microscopy using DyLight 488 PCR: Polymerase chain reaction O&P: Ova and parasite test rapid-IC: rapid antigen detection by immunochromatography IFAT: Immunofluorescent antibody testing (IFAT) FITC: Fluorescein isothiocyanate (FITC) PPV: Positive predictive value NPV: Negative predictive value HIV: Human Immunodeficiency Virus Declarations Acknowledgments We thank Jeremy Allen, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. Author contributons All authors contributed significantly to the study. Y.O., K.Yagita, and K.W. were responsible for the study overall and for study design and conceptualisation. Y.O., A.K., and K.W. wrote the manuscript. Y.O., E.A., K.S., and T.K. performed the fluorescent antibody microscopy and rapid immunochromatographic assay. A.K., R.S., M.K., and K.Yagita performed the PCR analysis. A.K., Y.Y., N.A., T.A., and D.M. recruited participants and managed clinical data collection. H.U., K.Yamamoto, J.A., and H.G. supervised the study. Figures and tables were prepared by A.K. and Y.O. Funding was acquired by A.K. and K.W. All authors approved the final version of the manuscript. Funding This work was supported by the Emerging/Re-emerging Infectious Diseases Project of Japan from the Japan Agency for Medical Research and Development (grant numbers JP24jk0210050h0001 and JP23fk0108681h0701), and by a grant from the National Center for Global Health and Medicine (23A2017). Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Ethical approval statement This study was approved by the Institutional Review Board of the National Center for Global Health and Medicine (approval number: NCGM-S-004414). 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J Clin Microbiol. 2003 Feb;41(2):623-626. doi: 10.1128/JCM.41.2.623-626.2003. Kutsuna S, Hayakawa K, Mezaki K, Yamamoto K, Ohmagari N. Spectrum of enteropathogens in cases of traveler's diarrhea that were detected using the FilmArray GI panel: New epidemiology in Japan. J Infect Chemother. 2021 Jan;27(1):49-54. doi: 10.1016/j.jiac.2020.08.009. Coulibaly G, Georges Togo AC, Somboro AM, Kone M, Traore FG, Diallo F, et al. Use of light-emitting diode fluorescence microscopy to detect acid-fast bacilli in sputum as proficient alternative tool in the diagnosis of pulmonary tuberculosis in countries with limited resource settings. Int J Mycobacteriol. 2023 Apr-Jun;12(2):144-150. doi: 10.4103/ijmy.ijmy_13_23. Hooja S, Pal N, Malhotra B, Goyal S, Kumar V, Vyas L. Comparison of Ziehl Neelsen & Auramine O staining methods on direct and concentrated smears in clinical specimens. Indian J Tuberc. 2011 Apr;58(2):72-6. Sharma M, Broor S, Maheshwari M, Sudan DPS. Comparison of conventional diagnostic methods with molecular method for the diagnosis of pulmonary tuberculosis. Indian J Tuberc. 2003 Apr;70(2):182-189. doi: 10.1016/j.ijtb.2022.04.006. Johansen ØH, Abdissa A, Zangenberg M, Mekonnen Z, Eshetu B, Bjørang O, et al. Performance and operational feasibility of two diagnostic tests for cryptosporidiosis in children (CRYPTO-POC): a clinical, prospective, diagnostic accuracy study. Lancet Infect Dis. 2021 May;21(5):722-730. doi: 10.1016/S1473-3099(20)30556-9. Tables TABLE 1 Sensitivity and specificity of each method for Giardia detection with reference to multiplex PCR A. Performance of fluorescent antibody microscopy examination Giardiasis diagnosis relative to multiplex PCR a Performance relative to PCR (no. of samples) PPV (% [95% CI a ]) NPV (% [95% CI]) Positive Negative Total Fluorescent antibody microscopy examination Positive 22 0 22 100 (85.1–100) Negative 3 59 62 95.2 (86.7–98.7) Total 25 59 Sensitivity (% [95% CI a ]) 88.0 (70.0–95.8) Specificity (% [95% CI a ]) 100 (93.9–100) B. Performance of rapid-IC test Giardiasis diagnosis relative to multiplex PCR a Performance relative to PCR (no. of samples) PPV (% [95% CI a ]) NPV (% [95% CI]) Positive Negative Total Rapid-IC test b Positive 22 0 22 100 (85.1–100) Negative 3 59 62 95.2 (86.7–98.7) Total 25 59 Sensitivity (% [95% CI a ]) 88.0 (70.0–95.8) Specificity (% [95% CI a ]) 100 (93.9–100) a Performed using a FilmArray Intestinal Panel b CI, confidence interval. c Immunochromatography by GIARDIA/CRYPTOSPORIDIUM QUIK CHEK test. TABLE 2 Sensitivity and specificity of each method for Cryptosporidium detection with reference to multiplex PCR A. Performance of fluorescent antibody microscopy examination Cryptosporidiosis diagnosis relative to multiplex PCR a Performance relative to PCR (no. of samples) PPV (% [95% CI a ]) NPV (% [95% CI]) Positive Negative Total Fluorescent antibody microscopy examination Positive 15 0 15 100 (79.6–100) Negative 1 68 69 98.6 (92.2–99.9) Total 16 68 Sensitivity (% [95% CI a ]) 93.8 (71.7–99.7) Specificity (% [95% CI a ]) 100 (94.7–100) B.Performance of rapid-IC test Cryptosporidiosis diagnosis relative to multiplex PCR a Performance relative to PCR (no. of samples) PPV (% [95% CI a ]) NPV (% [95% CI]) Positive Negative Total Rapid-IC test b Positive 14 0 14 100 (78.5–100) Negative 2 68 70 97.1 (90.2–99.5) Total 16 68 Sensitivity (% [95% CI a ]) 87.5 (64.0–97.8) Specificity (% [95% CI a ]) 100 (94.7–100) a Performed using a FilmArray Intestinal Panel b CI, confidence interval. c Immunochromatography by GIARDIA/CRYPTOSPORIDIUM QUIK CHEK test. TABLE 3 Analysis of discordant cases among different diagnostic methods. FAM-TEST Rapid-IC FilmArray Nested PCR with sequencing for Giardia gdh Interpretation Case 1 (OAK-41) Negative Negative Positive Negative FilmArray false positive or FAM-TEST, Rapid-IC, and Nested PCR false negative Case 2 (OAK-48) Negative Negative Positive Negative FilmArray false positive or FAM-TEST, Rapid-IC, and Nested PCR false negative Case 3 (OAK-32) Negative Negative Positive Negative FilmArray false positive or FAM-TEST, Rapid-IC, and Nested PCR false negative Case 4 (OAK-21) Positive Positive Positive Positive Giardia positive result Case 5 (OAK-17) Positive Positive Positive Positive Giardia positive result A. Giardia test results for discordant cases B. Cryptosporidium test results for discordant cases FAM-TEST Rapid-IC FilmArray Conventional PCR with sequencing for Cryptosporidium 18S rRNA Interpretation Case 1 (OAK-41) Negative Negative Negative Negative Cryptosporidium negative result Case 2 (OAK-48) Negative Negative Negative Negative Cryptosporidium negative result Case 3 (OAK-32) Positive Positive Positive Positive Cryptosporidium positive result Case 4 (OAK-21) Positive Negative Positive Positive Rapid-IC false negative result Case 5 (OAK-17) Negative Negative Positive Positive FAM-TEST and Rapid-IC false negative result Additional Declarations No competing interests reported. Supplementary Files Supplementarydata1.xlsx Supplementarydata2.tiff Cite Share Download PDF Status: Published Journal Publication published 27 Jan, 2026 Read the published version in Tropical Medicine and Health → Version 1 posted Editorial decision: Revision requested 27 Dec, 2025 Reviews received at journal 22 Dec, 2025 Reviews received at journal 16 Dec, 2025 Reviewers agreed at journal 13 Dec, 2025 Reviewers agreed at journal 12 Dec, 2025 Reviewers agreed at journal 12 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviewers invited by journal 07 Dec, 2025 Editor assigned by journal 07 Dec, 2025 Submission checks completed at journal 07 Dec, 2025 First submitted to journal 30 Nov, 2025 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. 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Between January 2022 and April 2025, 696 stool samples were submitted for microbiological examination. Among them, 694 samples were included for analysis, with two excluded. Thirty-five samples tested positive for \u003cem\u003eGiardia\u003c/em\u003e and/or \u003cem\u003eCryptosporidium\u003c/em\u003eby fluorescent antibody microscopy (FAM-TEST-positive samples), and an additional 49 FAM-TEST-negative samples submitted in October 2024 were selected as controls. These 84 samples underwent full analysis, including multiplex PCR and rapid immunochromatographic (IC) testing.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8244780/v1/f0c71f0f4d9ea0bfd370bd11.png"},{"id":97944389,"identity":"2e522b09-d12d-45a6-91f5-7512a983983b","added_by":"auto","created_at":"2025-12-11 05:11:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6782159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBright-field microscopy and fluorescent antibody microscopy (FAM-TEST) images of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGiardia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCryptosporidium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e Bright-field microscopy image (A), and FAM-TEST image (B) of \u003cem\u003eGiardia\u003c/em\u003e cysts, indicated by red arrows. Bright-field microscopy image (C), and FAM-TEST image (D) of \u003cem\u003eCryptosporidium\u003c/em\u003eoocysts, indicated by yellow arrows. FAM-TEST images were obtained through an absorption filter (510–550 nm) after excitation with a blue laser (490 nm).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8244780/v1/166a24458335818e6495e038.png"},{"id":98423150,"identity":"fc770671-53ff-4c01-8091-5653a6804564","added_by":"auto","created_at":"2025-12-17 16:31:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":631482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed diagnostic flowchart for infectious enteritis including protozoa detection by FAM-TEST.\u003c/strong\u003eThis diagram illustrates a suggested diagnostic algorithm for infectious colitis. Initial screening tests include Gram staining and culture for bacterial pathogens, as well as fluorescent antibody microscopy (FAM-TEST) combined with classical ova and parasite bright-field microscopy (classical O\u0026amp;P) for intestinal protozoa and helminths. If no causative agents are identified despite persistence of clinical symptoms, rapid immunochromatographic (IC) assays or multiplex PCR testing are recommended as secondary tests. This approach aims to optimize diagnostic accuracy while maintaining cost-effectiveness and minimizing overlooked protozoan infections, such as giardiasis and cryptosporidiosis.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8244780/v1/9457162423f28071d22fe994.png"},{"id":101690488,"identity":"1c6132ad-47b5-4e84-98c3-805e9517e5cc","added_by":"auto","created_at":"2026-02-02 16:03:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10989893,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8244780/v1/469aeec8-ce90-4a3e-a4b2-9b04559b7a53.pdf"},{"id":98422123,"identity":"998bc950-9fe4-440b-a7db-d2db4aa2fca2","added_by":"auto","created_at":"2025-12-17 16:30:29","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14071,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8244780/v1/3ade9ddfd21126e45e20c9d5.xlsx"},{"id":97944404,"identity":"c3324b5f-6fff-430b-ad04-5d8f284d0f6e","added_by":"auto","created_at":"2025-12-11 05:11:57","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32656378,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8244780/v1/529e2c3e31b58151179ad874.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDiagnostic Accuracy of ARK Checker® C/G – DyLight® 488: Simultaneous Detection of \u003cem\u003eGiardia\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e by Fluorescent Antibody Microscopy\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiarrheal diseases affect approximately 4\u0026nbsp;billion people worldwide annually, with waterborne protozoan infections being a major cause [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Protozoan infections, such as giardiasis and cryptosporidiosis, are primarily transmitted via the fecal\u0026ndash;oral route in situations of poor sanitation, inadequate water supply, and environmental contamination [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although these infections are mainly prevalent in developing countries, they can occur in developed countries as sporadic cases or because of an outbreak [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn Japan, giardiasis and cryptosporidiosis are classified as \u0026ldquo;Category V infectious diseases\u0026rdquo; under the Act on the Prevention of Infectious Diseases and Medical Care for Patients with Infectious Diseases mandating nationwide reporting of all confirmed cases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Surveillance data indicate international travelers and HIV-infected individuals to be at high risk for these protozoan infections [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, notable outbreaks of \u003cem\u003eCryptosporidium\u003c/em\u003e infection have been reported since its classification as a notifiable disease [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, diarrheal symptoms caused by these protozoa infections are indistinguishable from viral or bacterial gastroenteritis; therefore, these protozoa infections are frequently overlooked in common clinical settings [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Their diagnosis should be improved to enhance patient care and therefore, enable prompt epidemiological actions to protect public health.\u003c/p\u003e\u003cp\u003eThe diagnosis of \u003cem\u003eGiardia\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e currently relies on three main methods: morphological identification by classical ova and parasite bright-field microscopy (classical O\u0026amp;P), antigen detection, and polymerase chain reaction (PCR) [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This is because intestinal protozoa cannot be cultivated in the microbiology laboratories of medical facilities [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Classical O\u0026amp;P is highly affected by the skill of the examiner and is rarely used in most developed countries because of low diagnostic accuracy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Conversely, multiplex PCR has been increasingly used for diagnosis and research purposes because it offers high sensitivity and specificity [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It can also detect numerous enteric pathogens, including bacteria, viruses, and protozoa in the same assay. However, multiplex PCR is expensive (\u003cspan\u003e$\u003c/span\u003e100 or over per test); therefore, its use is highly limited to specialized laboratories. Another common approach is rapid antigen detection by immunochromatography (rapid-IC), which provides rapid and user-friendly results without requiring specialized expertise [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, its relatively high cost (~\u003cspan\u003e$\u003c/span\u003e30 per test for the detection of only a few pathogens) remains a concern for routine testing of enteric infectious diseases. Moreover, multiplex PCR and rapid-IC only target pre-defined pathogens, which is a crucial limitation in the diagnosis of intestinal parasites. Diagnosis of many protozoa as well as most helminths depend on classical O\u0026amp;P in clinical settings, although nucleic acid identification of each parasite can be applied for research purposes. Furthermore, identification by classical O\u0026amp;P is not difficult for most intestinal parasites except for small protozoa (\u0026lt;\u0026thinsp;10 \u0026micro;m). Thus, classical O\u0026amp;P has the advantage of being usable to examine various intestinal parasites within a routine protocol without incurring additional cost. Improving the diagnostic accuracy of O\u0026amp;P, for example by enabling easier identification of frequently overlooked common but small-sized protozoa, such as \u003cem\u003eGiardia\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e, is a possible solution for enhancing the clinical diagnosis of intestinal parasites.\u003c/p\u003e\u003cp\u003eImmunofluorescent antibody testing (IFAT) has been widely used for decades to detect \u003cem\u003eGiardia\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e cysts and oocysts under fluorescence microscopy [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Commercial IFAT kits, such as Merifluor\u0026reg;, have long been considered reference methods for morphological identification. However, the method is provided as antigen-precoated slides that must be stored in a cold, dark environment and are relatively expensive, making them less suitable for routine diagnostic use. In addition, fluorescein isothiocyanate (FITC) fluorophores used in conventional IFAT are prone to photobleaching, which limits fluorescence stability and signal intensity. To overcome these drawbacks, we have developed a simple fluorescent antibody microscopy method using ARK Checker\u0026reg; C/G \u0026ndash; DyLight\u0026reg; 488 (FAM-TEST). The FAM-TEST has been originally used in water quality monitoring and was adapted here for clinical stool examination as a simple and cost-effective diagnostic protocol [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The newly developed FAM-TEST assay enables morphological identification of \u003cem\u003eGiardia\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e in stool specimen by fluorescence microscopy after staining with DyLight\u0026#127276;488-conjugated antibodies.\u003c/p\u003e\u003cp\u003eIn the present study, diagnostic performance of FAM-TEST was evaluated relative to multiplex PCR results and compared with rapid-IC results.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSample collections and study approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStool samples submitted for microbiological examination from patients with suspected infectious gastroenteritis were collected between January 2022 and April 2025 (Fig. 1). Samples of sufficient volume for FAM-TEST, rapid-IC, and multiplex PCR were selected. These diagnostic tests, as well as DNA extraction\u0026nbsp;using the QIAamp PowerFecal Pro DNA Kit\u0026copy; (Qiagen, Hilden, Germany), were performed immediately after sampling. Extracted DNA was stored at \u0026minus;80\u0026deg;C. Microbiology results were the only patient clinical information used. Informed consent was not obtained because all submitted specimens were anonymized; however, an opt-out was employed in accordance with ethical guidelines. This study was approved by the Institutional Review Board of the National Center for Global Health and Medicine (NCGM-S-004414). All procedures were performed in accordance with the Declaration of Helsinki and the Ethical Guidelines for Medical and Biological Research Involving Human Subjects in Japan. The ethics committee waived the requirement for individual informed consent because the study used only anonymized residual clinical specimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFluorescent Antibody Microscopy method using ARK Checker\u0026reg; C/G \u0026ndash; DyLight\u0026reg; 488 (FAM-TEST) for Giardia and Cryptosporidium\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecimens were prepared using the formalin-ether sedimentation method. The sedimented fraction (10 \u0026mu;L) was mixed with 10 \u0026mu;L of ARK Checker C/G - DyLight 488 and mounted on a glass slide under a coverslip. Fluorescence images were obtained through a 510\u0026ndash;550-nm wavelength filter after B-excitation (490 nm) (Fig. 2). Four clinical laboratory technologists, who had no experience of fluorescence microscopy, performed FAM-TEST diagnosis. They underwent a 1-hour training session using positive control samples of \u003cem\u003eG. duodenalis\u003c/em\u003e and \u003cem\u003eCryptosporidium\u0026nbsp;\u003c/em\u003espp\u003cem\u003e.\u003c/em\u003e prior to the study. Each diagnosis was made by a single examiner randomly selected from 4 technologists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRapid Immunochromatographic (rapid-IC) Assays, and Multiplex PCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRapid-IC was performed using a GIARDIA/CRYPTOSPORIDIUM QUIK CHEK test (TECH LAB, USA), and multiplex PCR was performed using a Multiplex PCR FilmArray Gastrointestinal Panel (BioM\u0026eacute;rieux, France) according to the manufacturer\u0026rsquo;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSequence analysis of discordant cases\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cases with discrepancies among FAM-TEST, rapid-IC assay, and multiplex PCR results, we performed PCR followed by Sanger sequencing using DNA extracted from stool samples. For \u003cem\u003eGiardia duodenalis\u003c/em\u003e, nested PCR targeting the glutamate dehydrogenase (\u003cem\u003egdh\u003c/em\u003e) gene was performed. Outer primers Ghd1 (5\u0026prime;-TTCCGTRTYCAGTACAACTC-3\u0026prime;) and Gdh2 (5\u0026prime;-ACCTCGTTCTGRGTGGCGCA-3\u0026prime;), and nested primers Gdh3 (5\u0026prime;-ATGACYGAGCTYCAGAGGCACGT-3\u0026prime;) and Gdh4 (5\u0026prime;-GTGGCGCARGGCATGATGCA-3\u0026prime;) were used [23]. For \u003cem\u003eCryptosporidium\u003c/em\u003e spp., conventional PCR targeting the 18S rRNA gene was conducted using forward (5\u0026prime;-AAGCTCGTAGTTGGATTTCTG-3\u0026prime;) and reverse (5\u0026prime;-TAAGGTGCTGAAGGAGTAAGG-3\u0026prime;) primers [24]. The cycling conditions were as follows: an initial denaturation at 94\u0026deg;C for 3 minutes, followed by 40 cycles of denaturation at 94\u0026deg;C for 30 seconds, annealing at 60\u0026deg;C for 30 seconds, and extension at 72\u0026deg;C for 30 seconds. The nested \u003cem\u003eGiardia\u003c/em\u003e PCR used an annealing temperature of 67\u0026deg;C and 25 cycles.\u0026nbsp;Sequence analysis of each amplicon was performed by Sanger sequencing using appropriate forward and reverse primers (Eurofins Genomics, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism (v10.2.3). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) with 95% confidence intervals (Cis) were calculated for FAM-TEST and rapid-IC using multiplex PCR as a reference. Fisher\u0026rsquo;s exact test was used to compare FAM-TEST and rapid-IC results, with statistical significance set at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSample collection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the 3 years of the study period, 696 stool samples collected from patients with suspected infectious colitis were examined (Fig. 1). Of these, two samples were excluded from the analysis because of insufficient sample volume. Therefore, FAM-TEST was performed on 694 samples. We identified 35 stool samples positive for \u003cem\u003eGiardia\u003c/em\u003e and/or \u003cem\u003eCryptosporidium\u003c/em\u003e, which we classified as FAM-TEST-positive stool samples. These protozoa were not identified in the other 659 samples. As an unbiased collection of FAM-TEST-negative stool samples for use as control samples, we chose all the FAM-TEST-negative samples examined in 1 month, October 2024. Overall, we therefore selected 84 samples, 35 FAM-TEST-positive and 49 FAM-TEST-negative samples, for evaluating the diagnostic accuracy of three methods (Supplementary data 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDiagnostic value of FAM-TEST\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiagnostic accuracy of FAM-TEST for \u003cem\u003eGiardia\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e was evaluated by comparison with multiplex PCR (FilmArray Gastrointestinal Panel\u0026copy;) as a reference. Thereafter, it was compared with rapid-IC tests (GIARDIA/CRYPTOSPORIDIUM QUIK CHEK\u003csup\u003e\u0026copy;\u003c/sup\u003e tests).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePerformance of FAM-TEST for Giardia\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FAM-TEST are shown in Table 1A. For all FAM-TEST-positive giardiasis cases, \u003cem\u003eGiardia lamblia\u003c/em\u003e (syn.\u003cem\u003e\u0026nbsp;G. duodenalis\u003c/em\u003e) was detected by multiplex PCR (100% PPV). \u003cem\u003eG. lamblia\u003c/em\u003e was also detected by multiplex PCR in three FAM-TEST-negative samples, suggesting false negative results of FAM-TEST (95.2% NPV). The diagnostic performance of rapid-IC was similarly assessed (Table 1B). PPV, NPV, sensitivity, and specificity of rapid-IC were all the same as those of FAM-TEST. More surprisingly, the same three samples showed false negative results for FAM-TEST and rapid-IC. These results indicate that diagnostic accuracy of FAM-TEST is comparable to that of rapid-IC. They also indicate that the false negative results for FAM-TEST and rapid-IC probably occurred because the pathogen burden was beneath the level of detection by these tests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePerformance of FAM-TEST for Cryptosporidium\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe performance of FAM-TEST for detecting \u003cem\u003eCryptosporidium\u003c/em\u003e is shown in Table 2A. \u003cem\u003eCryptosporidium\u0026nbsp;\u003c/em\u003espp\u003cem\u003e.\u003c/em\u003e was detected by multiplex PCR in all FAM-TEST-positive samples, and in one sample among 64 FAM-TEST-negative samples (100% PPV, 98.6% NPV) (Table 2). In addition, rapid-IC produced no false positive results for \u003cem\u003eCryptosporidium\u003c/em\u003e. However, rapid-IC produced false negative results in two cases (100% PPV, 97.1% NPV), one of which was the sample that gave a false negative result by FAM-TEST. These results indicate the diagnostic accuracy of FAM-TEST for \u003cem\u003eCryptosporidium\u003c/em\u003e to be highly comparable to that of rapid-IC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEvaluation of Discordant Cases Using Nested and Conventional PCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs described above, discordant (false positive/negative) results produced by the different testing methods were seen in five cases, three discordant for \u003cem\u003eGiardia\u003c/em\u003e and two discordant for \u003cem\u003eCryptosporidium\u003c/em\u003e (Table 3). As already mentioned, the cases discordant for \u003cem\u003eGiardia\u003c/em\u003e yielded the same results by three diagnostic measures; they were FAM-TEST/rapid-IC-negative but multiplex PCR-positive. To confirm the presence of these pathogens in these samples, we performed conventional or nested PCR with sequencing analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotably, in all samples discordant for \u003cem\u003eGiardia\u003c/em\u003e, \u003cem\u003eG. duodenalis\u003c/em\u003e could not be identified by nested PCR (Table 3A, Case 1-3), whereas \u003cem\u003eG. duodenalis\u003c/em\u003e was clearly identified in the \u003cem\u003eGiardia\u003c/em\u003e-positive sample (Table 3A, Case 4\u0026amp;5). These results raised two possibilities for the discordant \u003cem\u003eGiardia\u003c/em\u003e results; one is an extremely low pathogen burden of\u003cem\u003e\u0026nbsp;G. duodenalis\u003c/em\u003e, giving a positive result only by multiplex PCR, and the other is a false positive multiplex PCR result.\u003c/p\u003e\n\u003cp\u003eFor cases with discordant \u003cem\u003eCryptosporidium\u003c/em\u003e results, we performed conventional PCR, which produced the same results as multiplex PCR (Table 3B). These findings confirm that multiplex PCR reliably identified \u003cem\u003eCryptosporidium\u0026nbsp;\u003c/em\u003espp\u003cem\u003e.\u003c/em\u003e in the present analysis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImproved treatment of intestinal parasitic diseases requires a low-cost, rapid, and technically simple diagnostic measure covering various parasites. In addition, the method should be developed as highly sensitive to small-sized common protozoa, such as \u003cem\u003eGiardia\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e, which are frequently overlooked by classical O\u0026amp;P. Multiplex PCR and antigen testing show high sensitivity to these protozoa [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]; however, these tests are generally expensive. Moreover, both tests can detect only pre-defined pathogens; therefore, other \u0026ldquo;untargeted\u0026rdquo; parasites need to be diagnosed separately. Currently, some intestinal protozoa and most helminths are not recognized by multiplex PCR or rapid-IC, and need to be identified morphologically by classical O\u0026amp;P in clinical settings. In the present study, we examined FAM-TEST using fluorescence-conjugated specific antibodies against \u003cem\u003eG. duodenalis\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e spp. for increased sensitivity to these protozoa, as an auxiliary diagnosis method to classical O\u0026amp;P. The FAM-TEST system differs from conventional immunofluorescence assays (IFAT) primarily in the use of DyLight 488\u0026ndash;conjugated monoclonal antibodies. This modification improves optical brightness and photostability compared to traditional fluorescein isothiocyanate (FITC)-labeled antibodies, allowing more consistent visualization.\u003c/p\u003e\u003cp\u003eFirst, FAM-TEST exhibited high specificity for \u003cem\u003eGiardia\u003c/em\u003e, comparable to that of rapid-IC. Additionally, the sensitivity of FAM-TEST was the same as that of rapid-IC. However, FAM-TEST and rapid-IC produced false negative results for \u003cem\u003eGiardia\u003c/em\u003e in the same samples relative to the reference results of multiplex PCR. \u003cem\u003eG. duodenalis\u003c/em\u003e was not identified by nested PCR targeting \u003cem\u003egdh\u003c/em\u003e. These results indicate that the pathogen burden was too low to be detected by these methods but not by multiplex PCR, or that multiplex PCR produced a false positive result. The manufacturer of the Multiplex PCR FilmArray Gastrointestinal Panel notes cross-reactivity between \u003cem\u003eGiardia\u003c/em\u003e and other gut microbiota, such as \u003cem\u003eBifidobacterium\u003c/em\u003e spp. and \u003cem\u003eRuminococcus\u003c/em\u003e spp. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, multiplex PCR frequently reports multiple pathogens in one stool specimen, some of which do not match the results of other diagnostic methods, such as bacterial culture [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In fact, in the present study, half of FAM-TEST-positive \u0026ldquo;\u003cem\u003eGiardia\u003c/em\u003e and/or \u003cem\u003eCryptosporidium\u003c/em\u003e containing\u0026rdquo; samples (18 out of 35) showed multiple pathogens by multiplex PCR (\u003cem\u003eG. lamblia\u003c/em\u003e/\u003cem\u003eCryptosporidium\u003c/em\u003e spp. plus one or more other microorganisms were positive simultaneously. Supplementary data 1). Such multiplex PCR results often make it difficult for physicians to determine the causative agent of intestinal disease. Considered together, FAM-TEST has good diagnostic value for giardiasis and is comparable to that of rapid-IC.\u003c/p\u003e\u003cp\u003eWe emphasize that FAM-TEST produced only one false negative case for \u003cem\u003eCryptosporidium\u003c/em\u003e, whereas rapid-IC produced an additional false negative case. Additionally, FAM-TEST did not produce any false positives. In general, identification of \u003cem\u003eCryptosporidium\u003c/em\u003e oocysts is extremely difficult because of their relatively small size compared with other protozoa. We predict that FAM-TEST can decrease the number of overlooked or misdiagnosed cases of cryptosporidiosis that frequently occur by classical O\u0026amp;P examination.\u003c/p\u003e\u003cp\u003eFluorescence-based diagnostic techniques have also been successfully applied to several pathogens [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For example, auramine staining has been used not only for tuberculosis screening (excitation: 460 nm, emission: 550 nm) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], but only the diagnosis of Cryptosporidium in low-resource settings, providing a fluorescent option that does not require antibodies or a cold chain [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe running cost of FAM-TEST is extremely low (up to \u003cspan\u003e$\u003c/span\u003e2 per test) compared with that of multiplex PCR (\u003cspan\u003e$\u003c/span\u003e100 or over per test) or rapid-IC assays (~\u003cspan\u003e$\u003c/span\u003e30 per test). In addition, FAM-TEST can be simultaneously performed with classical O\u0026amp;P. In fact, tested stool samples contained some enteric parasites (e.g. \u003cem\u003eSarcocystis\u003c/em\u003e spp. and \u003cem\u003eTaenia saginata\u003c/em\u003e), which were uncovered by multiplex PCR (OAK-40 \u0026amp; -48 in Supplementary data 1). They were morphologically diagnosed by classical O\u0026amp;P. Furthermore, \u003cem\u003eCyclospora cayetanensis\u003c/em\u003e can be identified by fluorescent microscopy through its autofluorescence by ultraviolet excitation (supplementary data 2). Species-level discrimination (e.g., C. parvum vs. C. hominis) or differentiation of Giardia assemblages, however, still requires PCR or sequencing.\u003c/p\u003e\u003cp\u003eBased on these findings, we suggest a FAM-TEST-containing diagnostic algorithm for enteric parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). First, FAM-TEST with classical O\u0026amp;P is employed to screen for enteric parasites when infectious gastroenteritis is suspected. Its implementation could enhance diagnostic accuracy while minimizing the risk of overlooking common protozoa and maintaining cost-effectiveness. Rapid-IC and/or multiplex PCR should be performed in limited cases to narrow down differential diagnoses (e.g. ruling out of infectious etiology before diagnosing non-infectious inflammatory bowel diseases).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis study has some limitations. First, it was conducted as a single-center study at a general hospital in Tokyo, which serves as a national reference center for travel and HIV-related infections. The number of intestinal parasite cases seen is relatively high compared with other facilities in Japan; however, positive cases of \u003cem\u003eGiardia\u003c/em\u003e and/or \u003cem\u003eCryptosporidium\u003c/em\u003e are still limited. In addition, the small number of examiners from the center involved in the study were more experienced in classical O\u0026amp;P than general practitioners. Therefore, diagnostic accuracy could be overestimated. Considered together, a larger scale study conducted at multiple facilities in resource limited areas is warranted for more reliable assessment of the diagnostic utility of FAM-TEST. In addition, negative control samples were selected from a single month during the study period, which may introduce spectrum bias. Although this approach minimized storage-related degradation, random sampling across the entire collection period would provide more robust validation.\u003c/p\u003e\u003cp\u003eIn conclusion, we demonstrated that FAM-TEST has comparable diagnostic accuracy for \u003cem\u003eGiardia\u003c/em\u003e and \u003cem\u003eCryptosporidium\u003c/em\u003e compared with rapid-IC while having the advantages of cost-effectiveness, examiner independence, and practical feasibility. Continuous efforts to improve the diagnostic flow for enteric parasites are needed for better clinical practice.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFAM-TEST: Fluorescent antibody microscopy using DyLight 488\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eO\u0026amp;P: Ova and parasite test\u003c/p\u003e\n\u003cp\u003erapid-IC: rapid antigen detection by immunochromatography\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIFAT: Immunofluorescent antibody testing (IFAT)\u003c/p\u003e\n\u003cp\u003eFITC: Fluorescein isothiocyanate (FITC)\u003c/p\u003e\n\u003cp\u003ePPV: Positive predictive value\u003c/p\u003e\n\u003cp\u003eNPV: Negative predictive value\u003c/p\u003e\n\u003cp\u003eHIV: Human Immunodeficiency Virus\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Jeremy Allen, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed significantly to the study. Y.O., K.Yagita, and K.W. were responsible for the study overall and for study design and conceptualisation. Y.O., A.K., and K.W. wrote the manuscript. Y.O., E.A., K.S., and T.K. performed the fluorescent antibody microscopy and rapid immunochromatographic assay. A.K., R.S., M.K., and K.Yagita performed the PCR analysis. A.K., Y.Y., N.A., T.A., and D.M. recruited participants and managed clinical data collection. H.U., K.Yamamoto, J.A., and H.G. supervised the study. Figures and tables were prepared by A.K. and Y.O. Funding was acquired by A.K. and K.W. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Emerging/Re-emerging Infectious Diseases Project of Japan from the Japan Agency for Medical Research and Development (grant numbers JP24jk0210050h0001 and JP23fk0108681h0701), and by a grant from the National Center for Global Health and Medicine (23A2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of the National Center for Global Health and Medicine (approval number: NCGM-S-004414). Informed consent was not obtained because all submitted specimens were anonymized and used as residual samples originally collected for routine diagnostic purposes; however, an opt-out approach was employed in accordance with institutional ethical guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCenters for Disease Control and Prevention (CDC). Stool Specimens - Detection of Parasite Antigens (DPDx).CDC; Last reviewed Nov 2020cdc.govcdc.gov.\u003c/li\u003e\n \u003cli\u003eShane AL, Mody RK, Crump JA, Tarr PI, Steiner TS, Kotloff K, et al. 2017 Infectious Diseases Society of America clinical practice guidelines for the diagnosis and management of infectious diarrhea. Clin Infect Dis. 2017 Nov 29;65(12):e45-e80. doi: 10.1093/cid/cix669.\u003c/li\u003e\n \u003cli\u003eMomčilović M, Cantacessi C, Arsić-Arsenijević V, Otranto D, Tasić-Ota\u0026scaron;ević S. Rapid diagnosis of parasitic diseases: current scenario and future needs. Clin Microbiol Infect. 2019 Mar;25(3):290-309. doi: 10.1016/j.cmi.2018.04.028.\u003c/li\u003e\n \u003cli\u003eGarcia LS, Arrowood MJ, Kokoskin E, Paltridge GP, Pillai DR, Procop GW, et al. Practical Guidance for Clinical Microbiology Laboratories: Laboratory diagnosis of parasites from the gastrointestinal tract. Clin Microbiol Rev. 2017 Nov 15;31(1):e00025-17. doi: 10.1128/CMR.00025-17.\u003c/li\u003e\n \u003cli\u003eSoares R, Tasca T. 2016. Giardiasis: an update review on sensitivity and specificity of methods for laboratory diagnosis. J Microbiol Methods. 2016 Oct;129:98-102.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSquire SA, Ryan U. Cryptosporidium and Giardia in Africa: current and future challenges. Parasitol Vectors. 2017 Apr 20;10(1):195. doi: 10.1016/j.mimet.2016.08.017.\u003c/li\u003e\n \u003cli\u003eVicente B, Freitas A, Freitas M, Midlej V. Systematic Review of Diagnostic Approaches for Human Giardiasis: Unveiling Optimal Strategies. Diagnostics (Basel). 2024 Feb 7;14(4):364. doi: 10.3390/diagnostics14040364.\u003c/li\u003e\n \u003cli\u003eKabir M, Ahmed E, Hossain B, Alam M, Ahmed S, Taniuchi M, et al. Giardia/Cryptosporidium QUIK CHEK Assay Is More Specific Than Quantitative Polymerase Chain Reaction for Rapid Point-of-care Diagnosis of Cryptosporidiosis in Infants in Bangladesh. Clin Infect Dis. 2018 Nov 28;67(12):1897-1903. doi: 10.1093/cid/ciy372.\u003c/li\u003e\n \u003cli\u003eEl-Alfy ES, Nishikawa Y. Cryptosporidium species and cryptosporidiosis in Japan: a literature review and insights into the role played by animals in its transmission. J Vet Med Sci. 2020 Aug 19;82(8):1051-1067. doi: 10.1292/jvms.20-0151.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKabir MHB, Kato K. Examining the molecular epidemiology of Giardia and Eimeria species in Japan: a comprehensive review. J Vet Med Sci. 2024 May 25;86(5):563-574. doi: 10.1292/jvms.23-0525.\u003c/li\u003e\n \u003cli\u003eNooshadokht M, Kalantari-Khandani B, Sharifi I, Kamyabi H, Liyanage NP, Lagenaur LA, et al. Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer. J Microbiol Methods. 2017 Oct;141:35-41. doi: 10.1016/j.mimet.2017.07.004.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNazeer JT, El Sayed Khalifa K, von Thien H, El-Sibaei MM, Abdel-Hamid MY, Tawfik RA, Tannich E. Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt. Parasitol Res. 2013 Feb;112(2):595-601. doi: 10.1007/s00436-012-3171-8.\u003c/li\u003e\n \u003cli\u003eElsafi SH, Al-Maqati TN, Hussein MI, Adam AA, Hassan MM, Al Zahrani EM. Comparison of microscopy, rapid immunoassay, and molecular techniques for the detection of Giardia lamblia and Cryptosporidium parvum. Parasitol Res. 2013 Apr;112(4):1641-1646. doi: 10.1007/s00436-013-3319-1.\u003c/li\u003e\n \u003cli\u003eAutier B, Belaz S, Razakandrainibe R, Gangneux JP, Robert-Gangneux F. Comparison of three commercial multiplex PCR kits for the diagnosis of intestinal protozoa. Parasite. 2018;25:48. doi: 10.1051/parasite/2018049. Epub 2018 Sep 18.\u003c/li\u003e\n \u003cli\u003eBasmaciyan L, Fran\u0026ccedil;ois A, Vincent A, Valot S, Bonnin A, Costa D, et al. Commercial simplex and multiplex PCR assays for the detection of Giardia intestinalis, Entamoeba spp., and Cryptosporidium spp.: comparative evaluation of seven commercial kits with in-house PCR. Microorganisms. 2021 Nov 10;9(11):2325. doi: 10.3390/microorganisms9112325.\u003c/li\u003e\n \u003cli\u003eBuss SN, Leber A, Chapin K, Fey PD, Bankowski MJ, Jones MK, et al. Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J Clin Microbiol. 2015 Mar;53(3):915-25. doi: 10.1128/JCM.02674-14.\u003c/li\u003e\n \u003cli\u003eMinak J, Kabir M, Mahmud I, Liu Y, Liu L, Haque R, et al. Evaluation of rapid antigen point-of-care tests for detection of Giardia and Cryptosporidium species in human fecal specimens. J Clin Microbiol. 2012 Jan;50(1):154-156. doi: 10.1128/JCM.01194-11.\u003c/li\u003e\n \u003cli\u003eCampbell SM, Pettersen FO, Brekke H, Hanevik K, Robertson LJ. Transition to PCR diagnosis of cryptosporidiosis and giardiasis in the Norwegian healthcare system: could the increase in reported cases be due to higher sensitivity or a change in the testing algorithm? Eur J Clin Microbiol Infect Dis. 2022 May;41(5):835-839. doi: 10.1007/s10096-022-04426-3.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLarsen TG, Ethelberg S, Nielsen HL, Hartmeyer GN, Nielsen L, Zangenberg M, et al. From rare to recognized: enhanced detection uncovers Cryptosporidium endemicity and species diversity in Denmark. Emerg Microbes Infect. 2025 Dec;14(1):2529893. doi: 10.1080/22221751.2025.2529893.\u003c/li\u003e\n \u003cli\u003eGarcia LS, Shum AC, Bruckner DA. Evaluation of a new monoclonal antibody combination reagent for direct fluorescence detection of Giardia cysts and Cryptosporidium oocysts in human fecal specimens. J Clin Microbiol. 1992 Dec;30(12):3255-7. doi: 10.1128/jcm.30.12.3255-3257.1992.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChalmers RM, Campbell BM, Crouch N, Charlett A, Davies AP. Comparison of diagnostic sensitivity and specificity of seven Cryptosporidium assays used in the UK. J Med Microbiol. 2011 Nov;60(Pt 11):1598-1604. doi: 10.1099/jmm.0.034181-0.\u003c/li\u003e\n \u003cli\u003eIzumiyama S, Yagita K. Laboratory diagnosis of Cryptosporidiosis and Giardiasis. Infectious Agents Surveillance Report. 2014 Aug 14;35(8):197-200. Japanese.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCacci\u0026ograve; SM, Beck R, Lalle M, Marinculic A, Pozio E. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int J Parasitol. 2008 Nov;38(13):1523-31. doi: 10.1016/j.ijpara.2008.04.008.\u003c/li\u003e\n \u003cli\u003eJohnson DW, Pieniazek NJ, Griffin DW, Misener L, Rose JB. Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. Appl Environ Microbiol. 1995 Nov;61(11):3849-55. doi: 10.1128/aem.61.11.3849-3855.1995.\u003c/li\u003e\n \u003cli\u003eJohnston SP, Ballard MM, Beach MJ, Causer L, Wilkins PP. Evaluation of three commercial assays for detection of Giardia and Cryptosporidium organisms in fecal specimens. J Clin Microbiol. 2003 Feb;41(2):623-626. doi: 10.1128/JCM.41.2.623-626.2003.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKutsuna S, Hayakawa K, Mezaki K, Yamamoto K, Ohmagari N. Spectrum of enteropathogens in cases of traveler\u0026apos;s diarrhea that were detected using the FilmArray GI panel: New epidemiology in Japan. J Infect Chemother. 2021 Jan;27(1):49-54. doi: 10.1016/j.jiac.2020.08.009.\u003c/li\u003e\n \u003cli\u003eCoulibaly G, Georges Togo AC, Somboro AM, Kone M, Traore FG, Diallo F, et al. Use of light-emitting diode fluorescence microscopy to detect acid-fast bacilli in sputum as proficient alternative tool in the diagnosis of pulmonary tuberculosis in countries with limited resource settings. Int J Mycobacteriol. 2023\u0026nbsp;Apr-Jun;12(2):144-150. doi: 10.4103/ijmy.ijmy_13_23.\u003c/li\u003e\n \u003cli\u003eHooja S, Pal N, Malhotra B, Goyal S, Kumar V, Vyas L. Comparison of Ziehl Neelsen \u0026amp; Auramine O staining methods on direct and concentrated smears in clinical specimens. Indian J Tuberc. 2011 Apr;58(2):72-6.\u003c/li\u003e\n \u003cli\u003eSharma M, Broor S, Maheshwari M, Sudan DPS. Comparison of conventional diagnostic methods with molecular method for the diagnosis of pulmonary tuberculosis. Indian J Tuberc. 2003 Apr;70(2):182-189. doi: 10.1016/j.ijtb.2022.04.006.\u003c/li\u003e\n \u003cli\u003eJohansen \u0026Oslash;H, Abdissa A, Zangenberg M, Mekonnen Z, Eshetu B, Bj\u0026oslash;rang O, et al. Performance and operational feasibility of two diagnostic tests for cryptosporidiosis in children (CRYPTO-POC): a clinical, prospective, diagnostic accuracy study. Lancet Infect Dis. 2021 May;21(5):722-730. doi: 10.1016/S1473-3099(20)30556-9.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTABLE 1 Sensitivity and specificity of each method for \u003cem\u003eGiardia\u003c/em\u003e detection with reference to multiplex PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Performance of fluorescent antibody microscopy examination\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" align=\"\" width=\"973\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 347px;\"\u003e\n \u003cp\u003eGiardiasis diagnosis relative to multiplex PCR\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 334px;\"\u003e\n \u003cp\u003ePerformance relative to PCR (no. of samples)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 142px;\"\u003e\n \u003cp\u003ePPV (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 141px;\"\u003e\n \u003cp\u003eNPV (% [95% CI])\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eFluorescent antibody microscopy examination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e100 (85.1\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e95.2 (86.7\u0026ndash;98.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Total\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eSensitivity (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e88.0 (70.0\u0026ndash;95.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eSpecificity (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e100 (93.9\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\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\u0026nbsp;B. Performance of rapid-IC test\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" align=\"\" width=\"973\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 347px;\"\u003e\n \u003cp\u003eGiardiasis diagnosis relative to multiplex PCR\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 334px;\"\u003e\n \u003cp\u003ePerformance relative to PCR (no. of samples)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 142px;\"\u003e\n \u003cp\u003ePPV (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 141px;\"\u003e\n \u003cp\u003eNPV (% [95% CI])\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eRapid-IC test \u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e100 (85.1\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e95.2 (86.7\u0026ndash;98.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Total\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eSensitivity (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e88.0 (70.0\u0026ndash;95.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eSpecificity (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e100 (93.9\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\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\u0026nbsp;\u003csup\u003ea\u003c/sup\u003e Performed using a FilmArray Intestinal Panel \u003csup\u003eb\u003c/sup\u003e CI, confidence interval. \u003csup\u003ec\u0026nbsp;\u003c/sup\u003eImmunochromatography by GIARDIA/CRYPTOSPORIDIUM QUIK CHEK test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTABLE 2 Sensitivity and specificity of each method for \u003cem\u003eCryptosporidium\u003c/em\u003e detection with reference to multiplex PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Performance of fluorescent antibody microscopy examination\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" align=\"\" width=\"973\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 347px;\"\u003e\n \u003cp\u003eCryptosporidiosis diagnosis relative to multiplex PCR\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 334px;\"\u003e\n \u003cp\u003ePerformance relative to PCR (no. of samples)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 142px;\"\u003e\n \u003cp\u003ePPV (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 141px;\"\u003e\n \u003cp\u003eNPV (% [95% CI])\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eFluorescent antibody microscopy examination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e100 (79.6\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e98.6 (92.2\u0026ndash;99.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Total\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eSensitivity (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e93.8 (71.7\u0026ndash;99.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eSpecificity (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e100 (94.7\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\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\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB.Performance of rapid-IC test\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" align=\"\" width=\"973\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 347px;\"\u003e\n \u003cp\u003eCryptosporidiosis diagnosis relative to multiplex PCR\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 334px;\"\u003e\n \u003cp\u003ePerformance relative to PCR (no. of samples)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 142px;\"\u003e\n \u003cp\u003ePPV (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 141px;\"\u003e\n \u003cp\u003eNPV (% [95% CI])\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eRapid-IC test \u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e100 (78.5\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e97.1 (90.2\u0026ndash;99.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u0026emsp;\u0026emsp;\u0026emsp;\u0026emsp;Total\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eSensitivity (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e87.5 (64.0\u0026ndash;97.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eSpecificity (% [95% CI\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e])\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e100 (94.7\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\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\u003csup\u003ea\u003c/sup\u003e Performed using a FilmArray Intestinal Panel \u003csup\u003eb\u0026nbsp;\u003c/sup\u003eCI, confidence interval. \u003csup\u003ec\u0026nbsp;\u003c/sup\u003eImmunochromatography by GIARDIA/CRYPTOSPORIDIUM QUIK CHEK test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTABLE 3 Analysis of discordant cases among different diagnostic methods.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" align=\"\" width=\"1058\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eFAM-TEST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eRapid-IC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eFilmArray\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eNested PCR with sequencing for \u003cem\u003eGiardia\u003c/em\u003e \u003cem\u003egdh\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003eInterpretation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 1 (OAK-41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003eFilmArray false positive or FAM-TEST, Rapid-IC, and Nested PCR false negative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 2 (OAK-48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003eFilmArray false positive or FAM-TEST, Rapid-IC, and Nested PCR false negative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 3 (OAK-32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003eFilmArray false positive or FAM-TEST, Rapid-IC, and Nested PCR false negative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 4 (OAK-21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003e\u003cem\u003eGiardia\u003c/em\u003e positive result\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 5 (OAK-17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003e\u003cem\u003eGiardia\u003c/em\u003e positive result\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;A. \u003cem\u003eGiardia\u003c/em\u003e test results for discordant cases\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB. \u003cem\u003eCryptosporidium\u003c/em\u003e test results for discordant cases\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" align=\"\" width=\"1058\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eFAM-TEST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eRapid-IC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eFilmArray\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eConventional PCR with sequencing for \u003cem\u003eCryptosporidium\u003c/em\u003e 18S rRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003eInterpretation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 1 (OAK-41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003e\u003cem\u003eCryptosporidium\u003c/em\u003e negative result\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 2 (OAK-48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003e\u003cem\u003eCryptosporidium\u003c/em\u003e negative result\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 3 (OAK-32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003e\u003cem\u003eCryptosporidium\u003c/em\u003e positive result\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 4 (OAK-21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003eRapid-IC false negative result\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eCase 5 (OAK-17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePositive\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 498px;\"\u003e\n \u003cp\u003eFAM-TEST and Rapid-IC false negative result\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-medicine-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tmah","sideBox":"Learn more about [Tropical Medicine and Health](https://tropmedhealth.biomedcentral.com/)","snPcode":"41182","submissionUrl":"https://submission.springernature.com/new-submission/41182/3","title":"Tropical Medicine and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Giardia duodenalis, Cryptosporidium, DyLight-488 antibody, Fluorescent antibody microscopy, Immunochromatography, Polymerase chain reaction","lastPublishedDoi":"10.21203/rs.3.rs-8244780/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8244780/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eGiardiasis and cryptosporidiosis are often misdiagnosed by stool ova and parasite test (classical O\u0026amp;P). Multiplex PCR and rapid antigen immunochromatography (rapid-IC) could offer high diagnostic accuracy; however, their cost and restricted coverage, especially for parasites, limit routine use. This study evaluated the efficacy of adapted fluorescent antibody microscopy using ARK Checker\u0026reg; C/G \u0026ndash; DyLight\u0026reg; 488 (FAM-TEST). The reagent is a liquid-form conjugated antibody preparation that directly reacts with \u003cem\u003eGiardia\u003c/em\u003e cysts and \u003cem\u003eCryptosporidium\u003c/em\u003e oocysts.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eStool samples were incubated with DyLight-488\u0026ndash;labeled antibodies as indicated in our original protocol and examined by fluorescence microscopy. Diagnostic accuracy of FAM-TEST was assessed with multiplex PCR results as reference and compared with that of rapid-IC. Samples with discordant results among these methods were additionally tested by conventional PCR with Sanger sequencing to verify infection status.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eA total of 694 stool samples were submitted for microbiological examination. FAM-TEST identified \u003cem\u003eGiardia\u003c/em\u003e and/or \u003cem\u003eCryptosporidium\u003c/em\u003e in 35 samples. In addition, 49 FAM-TEST- negative samples from a randomly selected month were included as negative controls. For \u003cem\u003eGiardia\u003c/em\u003e, all FAM-TEST results were identical to those of rapid-IC, including three false negatives [88.0% sensitivity, 100% specificity, 100% positive predictive value (PPV), and 95.2% negative predictive value (NPV)]. For \u003cem\u003eCryptosporidium\u003c/em\u003e, FAM-TEST showed 93.8% sensitivity, 100% specificity, 100% PPV, and 98.6% NPV, which were comparable with those of rapid-IC. Notably, nested PCR for \u003cem\u003eGiardia\u003c/em\u003e produced negative results for three samples considered to yield FAM-TEST false negatives, raising another possibility of multiplex PCR false positives or extremely low pathogen burden, whereas conventional PCR for \u003cem\u003eCryptosporidium\u003c/em\u003e showed results fully consistent with those of multiplex PCR.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eConcurrent use of FAM-TEST with classical O\u0026amp;P offers a cost-effective, practical diagnostic approach for enteric parasites, which especially strengthens diagnostic accuracy for Giardiasis/Cryptosporidiosis.\u003c/p\u003e","manuscriptTitle":"Diagnostic Accuracy of ARK Checker® C/G – DyLight® 488: Simultaneous Detection of Giardia and Cryptosporidium by Fluorescent Antibody Microscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 05:11:51","doi":"10.21203/rs.3.rs-8244780/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-27T09:05:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T12:34:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T05:45:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227828044072449957882208051566486541804","date":"2025-12-13T14:12:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146106042877216980047451904276629184448","date":"2025-12-12T18:37:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294098113742561896624703060497997680411","date":"2025-12-12T13:01:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236683850529882702770765142708915631809","date":"2025-12-10T08:52:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-08T02:03:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-08T01:41:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-08T01:40:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Medicine and Health","date":"2025-11-30T23:22:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-medicine-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tmah","sideBox":"Learn more about [Tropical Medicine and Health](https://tropmedhealth.biomedcentral.com/)","snPcode":"41182","submissionUrl":"https://submission.springernature.com/new-submission/41182/3","title":"Tropical Medicine and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91b2183a-55c0-437d-8d96-640ff438af6a","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:01:00+00:00","versionOfRecord":{"articleIdentity":"rs-8244780","link":"https://doi.org/10.1186/s41182-026-00907-9","journal":{"identity":"tropical-medicine-and-health","isVorOnly":false,"title":"Tropical Medicine and Health"},"publishedOn":"2026-01-27 15:58:31","publishedOnDateReadable":"January 27th, 2026"},"versionCreatedAt":"2025-12-11 05:11:51","video":"","vorDoi":"10.1186/s41182-026-00907-9","vorDoiUrl":"https://doi.org/10.1186/s41182-026-00907-9","workflowStages":[]},"version":"v1","identity":"rs-8244780","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8244780","identity":"rs-8244780","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}