F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.17362.1 Research Note Articles Comparative evaluation of sensitivity and specificity of immunochromatography kit for the rapid detection of norovirus and rotavirus in Bangladesh

[version 1; peer review: 2 approved with reservations]

 Shaha Modhusudon Formal Analysis Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4245-0830 1 Sifat Sadia Farzana Formal Analysis Methodology Writing – Review & Editing 2 Mamun Md. Al Investigation Methodology Writing – Review & Editing 2 Billah Md. Baki Methodology Writing – Review & Editing 3 Sharif Nadim Formal Analysis Investigation Writing – Review & Editing 2 Nobel Nasir Uddin Formal Analysis Investigation Writing – Review & Editing 2 Parvez Anowar Khasru Investigation Supervision Writing – Review & Editing 2 Talukder Ali Azam Investigation Validation Writing – Review & Editing 2 Nomura Akiko Conceptualization Supervision Writing – Review & Editing 4 Ushijima Hiroshi Supervision Writing – Review & Editing 4 Dey Shuvra Kanti Conceptualization Funding Acquisition Supervision Writing – Review & Editing https://orcid.org/0000-0002-2440-5673 a 2 Microbial Biotechnology Division, National Institute of Biotechnology, Dhaka, 1349, Bangladesh Department of Microbiology, Jahangirnagar University, Dhaka, 1342, Bangladesh Department of Zoology, Jahangirnagar University, Dhaka, 1342, Bangladesh Division of Microbiology, Department of Pathology and Microbiology, Nihon University, Tokyo, Japan shuvradey@yahoo.com

No competing interests were disclosed.

 11 2 2019 2019 8 173 24 1 2019 Copyright: © 2019 Shaha M et al. 2019 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

We report a comprehensive analysis of sensitivity and specificity of immunochromatography kit (IC Kit) for the rapid detection of norovirus and rotavirus in Bangladesh. The IC kit (IP-Noro/Rota) provides highest sensitivity (100%) to both viruses compared to the reference method reverse transcription- polymerase chain reaction (RT- PCR) for diagnosis. Furthermore, the test provides a high specificity of 98.9% and 96.1% to diagnose norovirus and rotavirus, respectively, as well as good agreement with the reference method. We also found high prevalence of rotavirus infection (74%) among Bangladeshi pediatric population, of which most of the patients were less than five years old, suffering from severe dehydration, abdominal pain and vomiting. This study is the first to report the ease and rapid detection of norovirus and rotavirus by IC kits in Bangladesh. Therefore, IP-Noro/Rota kit is recommended for the rapid detection of these viruses in routine diagnosis as well as during outbreaks.

 Immunochromatography kit Norovirus Rotavirus Rapid detection Bangladesh. Ministry of Science and Technology, Government of the People’s Republic of Bangladesh This research was supported by Grants-in-Aid from the Ministry of Education, The People’s Republic of Bangladesh. Introduction

Diarrheal diseases represent a major worldwide public health problem, particularly in developing countries 1 . Acute gastroenteritis is a very common disease in young children 2 . It has been reported that about 3–5 billion cases of acute gastroenteritis occur each year in children less than 5 years old and 1.5 to 2.5million children of that group die from severe diarrhoea 3– 5 . Of that, about half amillion death is caused by rotavirus infections 6 . On the other hand, norovirus is responsible for almost half of the foodborne gastroenteritis outbreaks and 75–90% of non-bacterial gastroenteritis outbreaks 7 .

When outbreaks of gastroenteritis occur in communities, rapid identification of pathogens is essential to ensure the administration of the appropriate treatment and control. Furthermore, definite diagnosis plays an important role to decrease the unnecessary use of antibiotics. In the case of emergency, there is no rapid detection method available in Bangladesh. In this regard, a rapid diagnosis kit with good sensitivity and specificity is essential. Developing such a kit may raise the reliability for rapid diagnosis in developing countries, where the prevalence of norovirus and rotavirus is increased. Herein, we report a comprehensive analysis of the sensitivity and specificity of an immunochromatography kit (IC Kit) for the rapid detection of norovirus and rotavirus in Bangladesh.

 Methods Participants

In this study, we evaluate the newly developed IC test kit for norovirus and rotavirus detection (IP-Noro/Rota; ImmunoProbe Co., Ltd., Saitama, Japan) in 100 stool samples collected from pediatric patients with acute gastroenteritis (severe dehydration, abdominal pain and vomiting) in Bangladesh during January to June 2015. The study was ethically approved by the ethical review committee of Jahangirnagar University, Bangladesh.

 Test methods

Reverse transcription PCR (RT-PCR) was used as the reference test for both norovirus and rotavirus detection 3 . The PCR is a molecular biology technique that allows for nucleic acid fragment from a complex pool of DNA. Faecal specimens were thawed, diluted with distilled water to 10% suspensions, and centrifuged at 10,000xg for 10 min. Viral RNA was extracted from 140µl of the supernatant using a spin-column technique (QIAamp Viral RNA kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For reverse transcription, 3µl of extracted RNA was mixed with a reaction mixture consisting of 1µl of oligo dT primer (Promega, Madison, USA) and 1µl of nuclease free water in microcentrifuge tube, then kept at 70°c for 5 mins and then chill for 5 mins. After that 4µl of 5X reaction buffer (Promega, Madison, USA), 2µl of MgCl 2, 1µl of PCR Nucleotide Mix (Promega, Madison, USA), 0.5µl of Ribonuclease Inhibitor (Promega, Madison, USA), 1µl of Reverse Transcriptase (Promega, Madison, USA), 6.5µl of nuclease free water were mixed with the same microcentrifuge tube. Then the solution was heated at 25°C for 5 mins, 42°C for 60 min and 70°C for 15 mins. Norovirus and rotavirus were detected by PCR analysis of cDNA with specific primers previously published 8– 9 . The amplification was carried out in a thermal cycler (2720 Thermal Cycler, Applied Biosystems, USA). The PCR was performed at 94°C for 3 mins followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 60 s and a final extension at 72°C and then held at 4°C. The PCR products were electrophoresed in a 1.5% agarose gel, followed by staining with ethidium bromide (0.5 g/ml) for 20 min and then visualized under ultraviolet (UV) light. The bands were recorded by photography.

To evaluate the sensitivity and specificity of this IP-Noro/Rota test kits, all the 100 samples were tested for norovirus and rotavirus antigens by this kit following manufacturer’s instructions (IP-Noro/Rota; ImmunoProbe Co., Ltd., Saitama, Japan). It took only 10-15 min to obtain the result. A positive result for both pathogens is two lines; the left control line (C) and the right test-positive line (T), whereas, a negative result consisted of a single left control line (C) ( Figure 1).

Detection method of the IP-Rota/Noro kit.

The test is positive if two lines appear in the membrane ( a). The test is negative when only one line appears in the control area ( b).

 Analysis

The sensitivity and specificity of IP-Rota/Noro test kit were calculated mathematically as described below:

Sensitivity for IC kit = Both IC kit and RT-PCR Positive × 100/ RT-PCR positive

Specificity for IC kit = Both IC kit and RT-PCR negative × 100 / RT-PCR negative

 Results

The working plan for evaluation of sensitivity and specificity of immunochromatography methods for rapid detection of rotavirus and norovirus associated with paediatric diarrhoea in Bangladesh is described in Figure 2.

Methods for rapid detection of rotavirus and norovirus associated with paediatric diarrhoea in Bangladesh.

By the RT-PCR method, 10 and 74 samples were confirmed as norovirus and rotavirus, respectively. It was found that all the isolated norovirus belongs to the genogroup II (data not shown). On the other hand, G1P 8 rotavirus strain was found the most prevalent among the Bangladeshi pediatric population after characterization of G-types (VP-7) and P-types (VP-4) of rotavirus-positive samples. The youngest patient was 21 days and the oldest 56 months; the average age was 14 months. The most common clinical symptoms of rotavirus and norovirus infected patients were dehydration, vomiting, fever and abdominal pain.

Of the 10 and 74 samples positive for norovirus and rotavirus, respectively, by RT-PCR, IP- Noro/Rota kit recognized all positive samples with 100% sensitivity. However, the kit gave one false positives for norovirus and three false positives for rotavirus detection, resulting in a specificity of 98.9% and 96.1%, respectively ( Table 1 and Table 2).

Comparison of norovirus detection in stool samples by IP-Noro/Rota kit and RT-PCR method (n=100).
Test Result RT- PCR Total Sensitivity Specificity
+ -
IP-Noro/Rota kit: 10 1 9 100% 98.9%
+
- 0 91 91
Total 10 90 100
Comparison of rotavirus detection in stool samples by IP-Rota/Noro kit and RT-PCR method (n=100).
Test Result RT- PCR Total Sensitivity Specificity
+ -
IP-Rota/Noro kit: 74 3 71 100% 96.1%
+
- 0 29 29
Total 74 26 100
Conclusions

The clinical symptoms of the patients with acute gastroenteritis are generally not indicative of a specific pathogen. In Bangladesh, the outbreak of norovirus and rotavirus diarrhea occurs mainly in the winter season 3 , when the IC kits could be used for rapid screening, as other existing diagnosis methods are time consuming. The rapid IC kit test is easy to perform at a low cost and it takes only 10–15 min to diagnose with a simple procedure and does not require special equipment or a skilled technician.

Our findings clearly indicate that rotavirus and norovirus are the most important enteropathogen responsible for acute viral gastroenteritis among infants and children in Bangladesh, where 74% of the cases were caused by rotavirus only. The IC kit provides a high specificity and sensitivity as well as good agreement with the reference method, RT-PCR, for the detection of rotavirus and norovirus. Therefore, IC-Noro/Rota kit will be easy and useful assay for the rapid detection of these viruses in routine diagnosis as well as during the outbreaks. This is the first report about the rapid detection of rotavirus by IC kits in Bangladesh. Finally, it is strongly recommended to use the IC kit as an alternative method for rapid diagnosis of norovirus and rotavirus infections, especially in developing countries like Bangladesh.

 Data availability Underlying data

Figshare: Raw Data- IC kit.csv, https://doi.org/10.6084/m9.figshare.7616630.v1 10 .

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgements

We thank the ImmunoProbe Co., Ltd. (Saitama, Japan) for kindly providing the IP-Rota/Noro kit.

 Cheng AC McDonald JR Thielman NM : Infectious diarrhea in developed and developing countries. J Clin Gastroenterol. 2005;39(9):757–773. 16145337 10.1097/01.mcg.0000177231.13770.07 Howidi M Al Kaabi N El Khoury AC : Burden of acute gastroenteritis among children younger than 5 years of age--a survey among parents in the United Arab Emirates. BMC Pediatr. 2012;12:74. 22708988 10.1186/1471-2431-12-74 3407526 Dey SK Nguyen TA Phan TG : Molecular and epidemiological trend of norovirus associated gastroenteritis in Dhaka City, Bangladesh. J Clin Virol. 2007;40(3):218–223. 17881286 10.1016/j.jcv.2007.08.005 Dey SK Phan TG Mizuguchi M : Genetic diversity and emergence of norovirus GII/4-2006b in Japan during 2006-2007. Clin Lab. 2011;57(3–4):193–199. 21500726 Chow CM Leung AK Hon KL : Acute gastroenteritis: from guidelines to real life. Clin Exp Gastroenterol. 2010;3:97–112. 21694853 10.2147/CEG.S6554 3108653 Tagbo BN Mwenda JM Armah G : Epidemiology of rotavirus diarrhea among children younger than 5 years in Enugu, South East, Nigeria. Pediatr Infect Dis J. 2014;33 Suppl 1:S19–22. 24343608 10.1097/INF.0000000000000047 Barreira DMPG Fumian TM Tonini MAL : Detection and molecular characterization of the novel recombinant norovirus GII.P16-GII.4 Sydney in southeastern Brazil in 2016. PLoS One. 2017;12(12):e0189504. 29236779 10.1371/journal.pone.0189504 5728567 Yan H Yagyu F Okitsu S : Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR. J Virol Methods. 2003;114(1):37–44. 14599677 10.1016/j.jviromet.2003.08.009 Dey SK Thongprachum A Ota Y : Molecular and epidemiological trend of rotavirus infection among infants and children in Japan. Infect Genet Evol. 2009;9(5):955–61. 19560560 10.1016/j.meegid.2009.06.017 Shaha M Sifat SF Al Mamun Md. : Raw Data- IC kit.csv. figshare. Dataset.2019. http://www.doi.org/10.6084/m9.figshare.7616630.v1 10.5256/f1000research.18987.r45947 Reviewer response for version 1 Page Nicola A. 1 2 Referee https://orcid.org/0000-0001-5845-4417 National Institute for Communicable Diseases (NICD), Johannesburg, South Africa Department of Medical Virology, University of Pretoria, Pretoria, South Africa

Competing interests: No competing interests were disclosed.

 26 3 2019 Copyright: © 2019 Page NA 2019 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The paper reports the performance of an immunochromatography cartridge for the rapid detection of norovirus and rotavirus against an RT-PCR reference method. The specimens selected (n=100) were collected between January and June 2015 in Bangladesh and had been frozen prior to use. The authors also calculated the sensitivity and specificity of the kit and made recommendations regarding the potential use. However, the paper has a few problems:

1. The calculations in Table 1 and 2 are incorrect. The sum of the rows and columns do not total 100. Consider correcting the tables an indicated below. 

Table 1. Comparison of norovirus detection in stool samples by IP-Noro/Rota kit and RT-PCR method (n=100)

Test result                             RT-PCR

IP-Noro/Rota kit        Positive          Negative      Total

Positive                         10                      1               11 

Negative                         0                     89               89

Total                              10                    90              100  

Table 2. Comparison of rotavirus detection in stool samples by IP-Noro/Rota kit and RT-PCR method (n=100)

Test result                             RT-PCR

IP-Noro/Rota kit     Positive           Negative       Total

Positive                        74                     3                77

Negative                        0                    23               23

Total                             74                   26              100

The calculations for specificity are, therefore, incorrect. This should be correct in the next version of the paper.

2. The authors briefly mention the broad typing results for norovirus (genogroup II) and rotavirus (G1P[8]) but fail to give any indication of the methods used to obtain these results. It makes it difficult for the reviewers and readers to assess the results presented. In addition, no detailed genotyping results were presented for the viral strains detected. Most current papers evaluating diagnostic assays for enteric viruses include genotyping data for the virus strains detected - see references below.

3. The authors failed to report the manufacturers’ sensitivity and specificity for the kit or to compare their results to other studies that evaluated the same or similar kits – see articles below. Alternatively, if this is a new formulation of the kit being evaluated the authors should indicate this fact.

4. The authors utilized stool specimens that were frozen for an unspecified period (but approximately three years if the study was completed in 2018). The prolonged storage or repeated freeze/thawing could have contributed to a degree of degradation of the viruses in the stool material and should have been listed as a potential limitation of the study. However, the screening of the frozen stool specimens is a previously utilized approach for evaluating diagnostic kits.

5. If the kit is introduced according to the author’s recommendations, it is more likely that fresh stool specimens will be screened. Therefore, the authors should consider evaluating the kit using fresh stool specimens from routine surveillance or during an outbreak which would mimic real world use and provide better estimates of sensitivity and specificity.      

6. Based on the detection rate of rotavirus in the stool specimens selected for the evaluation (74%), these specimens were collected during the rotavirus season in Bangladesh. While the results indicate that the kit would be adequate for use during the rotavirus season (barring a few false positive results), it does not give any indication of the kits performance when the rotavirus prevalence declines, outside of the rotavirus season.

Additional comments:

The term “developing country” is imprecise and inaccurate with no standard definition of what constitutes a developing country. The World Bank has defined standards for classifying countries so a better definition or term is a low-income country. Please correct in paragraphs 1 and 2 of the Introduction and in the last paragraph of the conclusions.  

Please check your nomenclature for rotavirus genotypes – G1P[8] and not G1P 8.

The statement “ Our findings clearly indicate that rotavirus and norovirus are the most important enteropathogen responsible for acute viral gastroenteritis among infants and children in Bangladesh, where 74% of the cases were caused by rotavirus only” is not valid. The authors only screened for rotavirus and norovirus. Other enteric viruses may also be important in gastroenteritis in children in Bangladesh but because the authors failed to screen for these pathogens, these observations would have been missed and, therefore, conclusions regarding viral enteropathogen importance are incorrect.     

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

No

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Viruses associated with diarrhoeal diseases, rotavirus vaccines, molecular characterization and phylogenetics of enteric viruses, epidemiology and public health

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References : Evaluation of immunochromatography tests for detection of rotavirus and norovirus among Vietnamese children with acute gastroenteritis and the emergence of a novel norovirus GII.4 variant. J Trop Pediatr .2007;53(4) : 10.1093/tropej/fmm021 264-9 17496324 10.1093/tropej/fmm021 : Evaluation of 4 immunochromatographic tests for rapid detection of norovirus in faecal samples. J Clin Virol .2013;56(3) : 10.1016/j.jcv.2012.11.001 194-8 23177165 10.1016/j.jcv.2012.11.001 : Comparison of the rapid methods for screening of group a rotavirus in stool samples. J Trop Pediatr .2011;57(5) : 10.1093/tropej/fmq101 375-7 21030457 10.1093/tropej/fmq101 Shaha Modhusudon National Institute of Biotechnology, Bangladesh

Competing interests: Authors have no competing interests

 24 2 2020

I would like to thank the respectable reviewer for the constructive comments and valuable suggestions. The revised manuscript is corrected based on the reviewer’s comments.

The point-by-point response is given below-

The corrected tables are given in page 5 and 6 of this revised manuscript.

This is a short research note. The genotyping was obtained by sequencing and the phylogenetic tree is not shown here in this manuscript.

The results of this study are compared with the existing reports and the results part is updated in page 5.

The laboratory analysis was conducted at the time of collection of the samples. After analysis of the samples, the leftover is stored for long time and is still available in the lab.

The evaluation of the kit was tested using the fresh samples.

The kit gave a specificity of 100% as compared to the RT-PCR analysis, though some false positive results may have been given. The statement is updated in the revised manuscript.

 Additional corrections-

-In all the paragraphs, the term “developing countries” is replaced by “low-income countries” in the revised manuscript.

-The nomenclature of the rotavirus genotypes is corrected.

-The statement is updated in the revised manuscript.

 10.5256/f1000research.18987.r44319 Reviewer response for version 1 Rahman Mustafizur 1 Referee https://orcid.org/0000-0002-6876-0191 International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh

Competing interests: No competing interests were disclosed.

 20 2 2019 Copyright: © 2019 Rahman M 2019 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

In this manuscript, the authors evaluate a newly developed immunochromatography (IC) kit for detecting norovirus and rotavirus by comparing the results with Reverse transcription PCR method as gold standard. They conducted comprehensive analysis of the sensitivity and specificity of the IC kit and concluded that the kit could be performed for rapid detection of norovirus and rotavirus with low cost and recommended the kits for routine use in low-income countries like Bangladesh.

Major comments:

1. Lack of information about sample quality: Authors claimed that the samples were collected from January to June 2015 and stored in -20⁰C, but did not mention the duration of storage in the freezer before testing, which could affect the evaluation. The reviewer suggests to use freshly collected samples for this type of comprehensive analysis.

2. Selection of inappropriate gold-standard for comparison: Authors selected Reverse transcription PCR (RT-PCR) for comparison. This PCR depends on primers which were designed more than 15 years ago (published in 2003); however, according to recent data and considering high diversity of the viruses, these primers may fail to detect the viruses correctly in many samples. Thus, this assay cannot be used as gold standard. Using more reliable and latest detection methods such as real time RT-PCR can be used as a gold standard.

3. Miscalculation: Analysis for sensitivity and specificity was incorrectly performed. For example, specificity of the kit for rotavirus detection (in Table 2) should be 89.6%.

4. Concluding remarks: The statement “Our findings clearly indicate that rotavirus and norovirus are the most important enteropathogen responsible for acute viral gastroenteritis among infants and children in Bangladesh, where 74% of the cases were caused by rotavirus only.” cannot be made from the data presented in this paper. This study was particularly designed to evaluate a kit; not for describing rotavirus and norovirus prevalence.

The authors strongly recommended to use the IC kit as an alternative method for rapid and low-cost diagnosis without the cost analysis by comparing with other available detection kits. Mentioning the unit cost for this IC test could be helpful to support this statement.

One of the major limitations of this study is the lack of information on the capability of the IC kit to detect different genotypes of viruses by this IC kit. All norovirus strains detected in this study belonged to norovirus genogroup II and other norovirus genogroups were ignored. For example, in Bangladesh, about 15-30% of noroviruses belong to genogroups other than genogroup II 1 , 2 . Similarly, the author did not mention genetic variation of rotaviruses detected in this study except G1P 3 . A well-designed study by including different genotypes with appropriate sample size is recommended for this type of kit evaluation.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Virology; Infectious diseases; Rotavirus vaccine; norovirus epidemiology; viral hepatitis, HIV

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References : Epidemiologic and Genotypic distribution of Noroviruses among children with Acute Diarrhea and Healthy Controls in a Low-income Rural Setting. Clin Infect Dis .2018; 10.1093/cid/ciy915 30351379 10.1093/cid/ciy915 : Norovirus diarrhea in Bangladesh, 2010-2014: prevalence, clinical features, and genotypes. J Med Virol .88(10) : 10.1002/jmv.24530 1742-50 27003679 10.1002/jmv.24530 : Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR. J Virol Methods .2003;114(1) :37-44 14599677 Shaha Modhusudon National Institute of Biotechnology, Bangladesh

Competing interests: Authors have no competing interests

 24 2 2020

I would like to thank the respectable reviewer for his valuable comments. The point-by-point response is given below-

The laboratory analysis was conducted at the time of collection of the samples. After analysis of the samples, the leftover is stored for long time and is still available in the lab.

Before selecting the primer pairs, a quality check was performed using the existing Genbank sequences of respective viruses. The primer oligo location in the viral genomes seems to be conserved.

The corrected calculations are given in the revised manuscript.

The statement is updated in the revised manuscript.

 The cost analysis was not performed, but the price of the kit would be much lower compared to the current diagnostic cost.

The kit is developed to detect all genotypes of underlying viruses, not a specific genotype. This is a short research note to report the sensitivity and specificity of the kit against rotavirus and norovirus. However, reviewer’s suggestion will be considered in the next study.