Comparative evaluation of sensitivity and specificity of immunochromatography kit for the rapid detection of norovirus and rotavirus in Bangladesh

Introduction

Diarrheal diseases represent a major worldwide public health problem, particularly in developing countries¹. Acute gastroenteritis is a very common disease in young children². 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 infections⁶. On the other hand, norovirus is responsible for almost half of the foodborne gastroenteritis outbreaks and 75–90% of non-bacterial gastroenteritis outbreaks⁷.

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

Test methods

Reverse transcription PCR (RT-PCR) was used as the reference test for both norovirus and rotavirus detection³. 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 MgCl₂, 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).

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).

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.

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, G1P⁸ 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).

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 season³, 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