Evaluation of a New Norovirus Genogroups GI and GII In Vitro Molecular Diagnostic Assay Using Clinical Specimens Collected from Acute Diarrheal Outbreaks

Abstract

Norovirus is a leading cause of acute gastroenteritis (AGE) in Taiwan. To improve diagnosis as part of laboratory surveillance, AGE surveillance was conducted using a new fluorescent probe hydrolysis-based insulated isothermal polymerase chain reaction (PCR) method, the POCKIT system, and the results were compared with those obtained from conventional methods. A total of 119 clinical stool samples from reported AGE outbreaks were collected for this study. From 83 real-time reverse transcription PCR (rRT-PCR) norovirus-positive cases, the POCKIT system identified 78 with a sensitivity of 90.3% in GI genogroup and 96.7% in GII genogroup. The specificity for both GI and GII genogroups was 100%. Overall, the POCKIT system is faster and easier to use than the conventional rRT-PCR method, and because of its high sensitivity and specificity, this system is a promising alternative for the detection of norovirus in patients with AGE, and would benefit public health laboratories for near real-time surveillance of AGE epidemic outbreaks.

Introduction

Infectious acute gastroenteritis (AGE) causes high morbidity and mortality in children and is often caused by viruses; identification of the infectious agent is essential for patient management (Marignani et al., 2004; Collaborators, 2018). However, because clinical signs and symptoms of AGE are similar to those of many viruses, making an etiological diagnosis based solely on clinical presentation is difficult and sometimes leads to delays in therapeutic management.

Norovirus is a leading cause of AGE, and it is estimated to cause nearly 20% of all global AGE cases (Ahmed et al., 2014). Once infected, patients may exhibit symptoms and spread the disease to others (Ngabo et al., 2016). In Taiwan, norovirus often causes large outbreaks of AGE within institutions, and an AGE surveillance was conducted by Taiwan Centers for Disease Control (CDC), which is based on the reporting system from schools, populous institutes, and laboratory diagnostics. Increasing the detection sensitivity of norovirus as a cause of AGE is an essential step for defining appropriate treatments and taking effective isolation precautions.

Real-time reverse transcription polymerase chain reaction (rRT-PCR) is considered the gold standard norovirus screening and detection method. However, the procedure is labor-intensive and time-consuming because of the many required techniques and steps; moreover, it also will introduce the possibility of contamination through these procedures and make them vulnerable to false-positive (or false-negative) results.

A fluorescent probe hydrolysis-based insulated isothermal PCR (iiPCR) assay was recently developed and made commercially available (Tsai et al., 2014). Through an automated iiPCR device, the POCKIT™ Central Nucleic Acid Analyzer (GeneReach Biotechnology Corp., Taichung City, Taiwan), norovirus GI and GII genotypes can be simultaneously detected in a closed system that requires <3 min of hands-on time and ∼85 min of instrumentation time. This study determined whether the results provided by the iiPCR assay would benefit public health laboratories for near real-time surveillance of AGE outbreaks.

Materials and Methods

This prospective observational study tested clinical samples for norovirus. All samples were tested with the POCKIT Series Nucleic Acid Analyzer and were compared its performance with an rRT-PCR test serving as the reference method.

Clinical specimens

A total of 301 AGE outbreaks were reported to the Taiwan CDC between September and December 2020, and 119 stool samples with sufficient residual volume for testing (>700 μL) from the reported AGE outbreaks were used in this study. Outbreaks were defined as two or more cases of gastroenteritis linked by place and time. A new outbreak was arbitrarily defined as having occurred at least 7 days after the last case in a previous outbreak or as having occurred in a separate patient care unit, such as a ward or hospital (LeBaron et al., 1990; Lew et al., 1990). Stool samples from AGE outbreaks were collected between September and December 2020 in Taiwan.

Patients with AGE experienced vomiting or diarrhea (have three or more loose or liquid stools per day). The biological materials obtained in this study were used for standard diagnostic procedures as requested by patient's physician; the procedures were conducted in accordance with no specific sampling method and no modification were made to sampling protocols. According to the Communicable Disease Control Act in Taiwan, all suspected gastroenteritis outbreaks must be reported and stool samples must be collected to Taiwan CDC through the Notifiable Diseases Surveillance System. In accordance with local regulations, the procedures did not require specific consent from patients.

rRT-PCR detection of norovirus

The specimens from patients were submitted to the Taiwan CDC for viral tests. A viral nucleic acid extraction from 200 μL fecal suspension was performed according to the manufacturer's recommendations of a kit (QIAamp Viral RNA Mini kit; Qiagen, Hilden, Germany). Viral tests for norovirus were performed by rRT-PCR (Cannon et al., 2017). All norovirus-positive samples from the rRT-PCR were amplified using PCR at the ORF1/ORF2 junction and were sequenced with primers (Kojima et al., 2002). The classification of genogroups and genotypes were through publicly accessible typing tools, the online Human Calicivirus Typing Tool (Chhabra et al., 2019; Tatusov et al., 2021), available at https://norovirus.ng.philab.cdc.gov.

POCKIT norovirus GI and GII detection

Clinical stool sample was resuspended in 1 × phosphate-buffered saline (PBS) (100 mg stool +900 μL PBS) and centrifuged at 13,000 × g for 10 min at 4°C and analyzed by POCKIT system according to the manufacturer's instructions. In brief, a total of 200 μL supernatant was loaded into the sample well of extraction cartridge. Insert the sample-loaded extraction cartridge and the transfer cartridge to POCKIT, press confirm to proceed to start the run, and the reaction will complete in 85 min (Supplementary Appendix Fig. SA1). This system uses iiPCR technology to target the ORF1/ORF2 junction of the norovirus for a qualitative detection of norovirus GI and GII in liquid or soft stool from patients suspected of having a norovirus infection.

QIAstate-Dx gastrointestinal panel testing

The QIAstate-Dx Gastrointestinal Panel (GIP) assay (Qiagen) was performed according to the manufacturer's instructions. After vortexing, 200 μL of the resuspended sample was inserted into a GIP test cartridge. The barcode of the GIP test cartridge and the barcode of the corresponding sample were scanned by the QIAstat-Dx operational module. The GIP test cartridge was then loaded into the QIAstat-Dx analyzer module. Each report contains the results of all 24 viral, bacterial, and parasite targets, as well as those for the internal control.

Statistics

The kappa statistic test (κ) was used to examine the level of consistency between the two assays. Test results from separate laboratories and operators in proficiency studies were analyzed using the chi-square test.

Ethical approval

This study was approved by the Institution Review Board of Taiwan Centers for Disease Control (No. IRB110101). The consent was waived for this study as there was no personal information collected from subjects.

Results

Of the 119 stool samples, 83 laboratory-confirmed norovirus-positive samples and 36 negative samples were performed to evaluate the analytical sensitivity of the POCKIT system. Of the 83 positive samples, 31 were GI, 52 were GII, and no GI/GII combined infections were detected in any sample. The demographic and clinical symptoms data are displayed in Tables 1 and 2 for GI and GII, respectively. This study included samples from 74 (62.2%) male patients and 45 (31.8%) female patients aged 1–60 years. The most common clinical manifestations were vomiting, diarrhea, and fever.

Table 1.

Age Distribution and Symptoms of Patients with Acute Gastroenteritis in GI Testing During 2020–2021 in Taiwan

	Patients (n = 57)		Symptoms
	Male	Female	Fever	Diarrhea	Vomiting	Nausea	Stomachache	Others a
Age group	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
<10	3 (5.3)	4 (7.0)	1 (1.8)	2 (3.5)	7 (12.3)		1 (1.8)
11–20	13 (22.8)	7 (12.3)	2 (3.5)	17 (29.8)	12 (21.0)	4 (7.0)	4 (7.0)
21–30	8 (14.0)	3 (5.3)	2 (3.5)	8 (14.0)	6 (10.5)	3 (5.3)	5 (8.8)	2 (3.5)
31–40	1 (1.8)	2 (3.5)		2 (3.5)	2 (3.5)	1 (1.8)	1 (1.8)
41–50	4 (7.0)	0 (0.0)		4 (7.0)	2 (3.5)	1 (1.8)		1 (1.8)
51–60	2 (3.5)	2 (3.5)		4 (7.0)	1 (1.8)	2 (3.5)	2 (3.5)
>60	1 (1.8)	7 (12.3)	1 (1.8)	4 (7.0)	5 (8.8)	1 (1.8)	1 (1.8)
Total	32 (56.2)	25 (43.8)	6 (10.60)	41 (71.8)	35 (61.4)	12 (21.2)	14 (24.7)	3 (5.3)

a

Others included muscle soreness, chillness, and general body aches.

Table 2.

Age Distribution and Symptoms of Patients with Acute Gastroenteritis in GII Testing During 2020–2021 in Taiwan

	Patients (n = 62)		Symptoms
	Male	Female	Fever	Diarrhea	Vomiting	Nausea	Stomachache	Others a
Age group	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
<10	19 (30.6)	10 (16.1)	12 (19.4)	8 (12.9)	25 (40.3)	1 (1.6)	9 (14.5)
11–20	12 (19.4)	4 (6.5)	4 (6.5)	8 (12.9)	11 (17.7)	3 (4.8)	7 (11.3)	1 (1.6)
21–30	4 (6.5)	0 (0.0)	1 (1.6)	2 (3.2)	2 (3.2)	1 (1.6)	1 (1.6)
31–40	1 (1.6)	0 (0.0)		1 (1.6)	1 (1.6)	1 (1.6)	1 (1.6)
41–50	3 (4.8)	2 (3.2)	1 (1.6)	2 (3.2)	1 (1.6)	1 (1.6)	1 (1.6)	1 (1.6)
51–60	1 (1.6)	3 (4.8)	1 (1.6)	1 (1.6)	2 (3.2)	1 (1.6)	1 (1.6)
>60	2 (3.2)	1 (1.6)	2 (3.2)	2 (3.2)	2 (3.2)
Total	42 (67.7)	20 (32.3)	21 (33.9)	24 (38.6)	44 (70.8)	8 (12.9)	20 (32.2)	2 (3.2)

a

Others included muscle soreness, headache, and cardialgia.

The results of the POCKIT system and the real-time PCR tests are displayed in Table 3. Of the 83 rRT-PCR norovirus-positive cases (GI 31and GII 52), POCKIT identified 78 (GI 28 and GII 50), with a sensitivity 90.3% (28/31) for GI genogroup and 96.1% (50/52) for GII genogroups, the specificity for both GI and GII genogroups was 100%, and κ values of 0.89 and 0.97, respectively (Table 3). The study period coincided with the emergence of the coronavirus disease pandemic, which limited the availability of QIAstat-Dx GIP imports.

Table 3.

Performance of POCKIT System on Clinical Samples Compared with Reference Real-Time Reverse Transcription Polymerase Chain Reaction Assay

	Norovirus rRT-PCR
		GI genogroup		GII genogroup
		Positive	Negative	Positive	Negative
POCKIT	Positive	28	0	50	0
	Negative	3	26	2	10
	Total	31	26	52	10

rRT-PCR, real-time reverse transcription polymerase chain reaction.

A total of 24 samples were used in the QIAstat-Dx GIP assay. Of the 8 rRT-PCR norovirus-positive cases (GI 3 and GII 5), the QIAstat-Dx GIP assay identified 6 (GI 2 and GII 4), with a positive predictive value of 66.7% for GI and 80.0% for GII. Comparing the results of POCKIT system with QIAstat-Dx GIP assay, a better positive and negative agreement was observed between the rRT-PCR assay and the POCKIT iiPCR (Supplementary Appendix Table S1).

Discussion

Diagnostic detection of norovirus is crucial for managing the large scale of diarrheal outbreaks, to enable real-time screening and detection is essential. PCR-based assays are recommended as the gold standard for diagnostic testing for many infectious diseases (Robilotti et al., 2015). Because rRT-PCR detection for norovirus is both sensitive and specific (Cannon et al., 2017), these assays have up to 100-times higher sensitivity of detection than one-step RT-PCR tests (Munir and Kibenge, 2004).

Since norovirus is fecal–oral transmitted and frequently causes large outbreaks, early detection and identification of norovirus-infected individuals and foods remain a key strategy to interrupt transmission. Moreover, possible impact factors analysis of norovirus infection from our other project shows that the burden of norovirus is likely to increase in response to climate change and population density as increasing in kindergarten and nursing home. Longitudinal data from previous study revealed that prevalences of aging population and multimorbidity substantially increased in Taiwan from 2000 to 2013 (Lai et al., 2020).

This study is the first to evaluate the suitability of the POCKIT system for laboratory diagnosing norovirus among patients with diarrhea during outbreaks in Taiwan. The specificity and sensitivity of the POCKIT system exhibited satisfactory performances among the GI and GII genogroups. From our evaluation results, the POCKIT assay had a specificity of 100% for the GI and GII genogroups, and its sensitivity was 90.3% with the GI genogroup and 96.7% with the GII genogroup, this revealed similar diagnostic accuracies with rRT-PCR assay and facilitates urgent testing and laboratory surveillance.

Previous study evaluated several molecular platforms for the detection of norovirus, and showed that the sensitivity of the FilmArray, Luminex xTAG GIP, and TaqMan Array Card were 87.8%, 78.0% and 87.8%, respectively (Chhabra et al., 2017). The POCKIT system showed higher sensitivity than these molecular platforms and is useful to help to detect norovirus when outbreaks occur. The difference in performance of the POCKIT system in detection differed between GI and GII with 9.6% testing negative that were PCR positive for GI and 3.8% for GII, it still suggests that there is the potential to miss a substantial number of positives.

Thus, the POCKIT system is appropriate for use at the outbreak level, and a second detection system would be required if it is applied at the individual case level. In addition, a previous study that evaluated the performance of the QIAstat-Dx GIP in detecting and identifying causative agents of infectious gastroenteritis reported a perfect performance record with a total of 16 norovirus GI and GII positive in 172 patients (Boers et al., 2020).

However, due to limited availability of COVID-19 pandemic, only 24 QIAstat-Dx GIPs tests were performed in this study. The QIAstat-Dx GIP assay exhibited lower sensitivity (GI 66.7% and GII 80.0%) than POCKIT system, and yielded an overall 95.8% consistency results with rRT-PCR. Because the sample size was too small, the data of QIAstat-Dx GIPs shown here are unlikely to be statistically significant or reliable; however, a multicenter comparative study of QIAstat-Dx GIP indicates a good correlation and positive percent agreement of 98.2% with other multiplexed molecular assays (Hannet et al., 2019).

The POCKIT device was specifically designed to provide optimal conditions for fluorescent probe-based iiPCR, and has the capacity to detect 520- and 550-nm signals simultaneously. The iiPCR is highly sensitive and specific for the detection of both RNA and DNA, and because it can be used within a single heating source, it does not require an expensive thermocycler (Chang et al., 2012; Tsai et al., 2012). With its high levels of sensitivity and specificity, the POCKIT system can detect AGE outbreaks in terms of norovirus infection at an early stage and subclinical infections. Given quick turnaround time of the POCKIT system, it could be a useful test in controlling outbreaks and such results could help implement effective public health surveillance and monitoring systems.

A shortcoming of the POCKIT system is limited by flexibility and high-throughput testing, only detects eight samples in each run. If large-scale foodborne outbreaks occur, it is unable to cope with plenty of clinical samples. Nevertheless, POCKIT only requires the addition of an aliquot supernatant of feces into the test cartridge by a single pipetting step and reports results in nearly an hour, its high sensitivity and ease of use make it possible to be applied to the diagnostic of norovirus outbreaks, and can facilitate not only urgent testing but also laboratory surveillance for diverse genogroups and genotypes of norovirus.

This makes the POCKIT system more suitable for being applied at the front line of outbreak level. The quick turnaround time of the system could potentially be a very useful test in controlling outbreaks. POCKIT substantially offers advantages such as accuracy, easy of handling, faster turnaround time, reduce labor and employee training, potential costs, and this assay may be more favorable than other available methods.

Conclusion

This study demonstrated the use of a high-order testing, yet is simple to use and requires minimal hands-on time that is feasible for application worldwide. Its use may lead to an increased diagnosis rate of norovirus infections and improved patient outcomes. Our results indicated that the POCKIT system generates specific, sensitive, and reproducible results, and can therefore serve as an alternative method for laboratory screening and a valuable platform for AGE outbreaks investigation.