1 INTRODUCTION

According to the data in the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO), norovirus is the dominant cause of acute gastroenteritis that led to diarrhea and vomiting worldwide in recent years.^{1, 2} Norovirus causes an estimated 685 million cases and 50,000 child deaths, and costs about $60 billion worldwide due to healthcare costs and lost productivity every year.¹ Diarrheal diseases were the eighth leading cause of death at all ages and the fifth leading cause of death in children younger than 5 years.³ WHO estimated that norovirus was responsible for approximately 1/5 of the cases of diarrheal diseases, leading 35, 000 deaths worldwide in 2010 and constituting the most deaths among the diarrheal diseases.^{2, 4, 5} Norovirus was divided into at least 6 genogroups (GⅠ–GⅥ), and GⅠ and GⅡ were the predominant genogroups that caused acute gastroenteritis in humans.^6-8 Norovirus infection usually caused a self-limiting illness in healthy adults with 2- to 3-day course, while in the elderly, young children, and immunocompromised individuals, it was associated with severe complications such as developing severe disease and mortality.^{6, 7} Human norovirus has a substantially high prevalence and mortality across both healthcare and community settings, particularly in developing countries, and brings humans a severe global burden of disease.^{5, 9, 10}

Since the rapid spread of norovirus is a major public health issue, rapid laboratory diagnosis is essential to facilitate the execution of appropriate control measures to reduce transmission and outbreaks of the virus. The methods for the detection of norovirus can be concluded in terms of electron microscopy, immunology, and molecular biology.^{8, 11} Unfortunately, electron microscopy was not widely available for detecting norovirus in microbiology laboratories because of its low sensitivity, specificity, and expensiveness.¹¹ The development of enzyme immunoassays, such as IDEIA and RIDASCREEN, has been challenging because norovirus genotypes that formed most virus antigens were many (n = 29) and had the antigenic drift in certain strains.⁸ In addition, due to the problem of sample processing, the IDEIA norovirus assay was at low sensitivity and raised serious questions regarding its usefulness in routine screening for norovirus.¹² Currently, reverse transcription-polymerase chain reaction (RT-PCR) is known as the gold standard in the detection of norovirus due to its high sensitivity and specificity in clinical and environmental samples.^{6, 8} However, the disadvantages of RT-PCR–based assays are also apparent, including high cost, time-consuming, and dependence on sophisticated equipment and expertise, leading to the limitation in its use in resource-poor settings without equipped laboratories.^{11, 13}Loop-mediated isothermal amplification (LAMP), created by Notomi et al.,¹⁴ was a remarkable alternative to RT-PCR for the diagnosis of norovirus infection. LAMP allowed RNA detection in an isothermal condition by using Bst DNA Polymerase that had high displacement activity, which was performed with simple and single-temperature incubation sources and thus got rid of the limitation of expensive equipment.^{6, 13, 14} LAMP has many advantages compared with RT-PCR, including high sensitivity, high specificity, rapidity, low cost, and intuitive results, which can be easily read with the naked eye with the help of color-based reporters.¹⁵

While many LAMP assays have been presented to detect norovirus by research groups, it is essential to systematically evaluate and conclude the performance of LAMP and the quality of these studies. So far, no overall analysis of the accuracy of LAMP for norovirus seems to have been researched. Therefore, here, we intend to determine the accuracy of LAMP methods for the diagnosis of norovirus infection, using systematic review and meta-analysis techniques.

2 METHODS

3 RESULTS

3.3 Quality assessment

To ensure scientific rigor and objectivity, two evaluations were conducted at different times. The quality assessment of the eight included articles is catalogued in Table S2. The overall risk of bias and applicability results are presented in Figures 1 and 2. Most studies were only performed when there were a low risk of bias and little concern about the applicability of the results. In the aspect of patient selection, all studies had a low risk of bias due to the inclusion of continuous or randomized samples and avoidance of inappropriate exclusions, while the applicability concerns were little high. In the aspect of the index test, approximately half of the studies were at low risk, while still two studies were classified as having “Unclear” risk of bias and one study as having a high risk of bias, because they were unclear in the elaboration of thresholds or explained the index test under the condition of knowing the results of the reference standards. In terms of the reference standard, all the studies had a low risk of bias and high clinical applicability, because the reference standard could correctly classify the target condition in all studies and the results of the reference standard were interpreted without knowledge of the results of the index test in more studies. In terms of flow and timing, a low risk of bias was existed in all studies, owing to that there was an appropriate interval between the index test and the reference standard and patients received the same reference standard. Also, all patients were included in the analysis except for the study of Iturriza-Gomara et al.

3.6 Merge analysis results

With using the random-effects model, the pooled data to evaluate the diagnostic accuracy of LAMP are shown in Figures 3-7. LAMP technology was utilized to detect norovirus, whose pooled sensitivity, specificity, positive LR, negative LR, and their 95% CI were 0.96 (0.95–0.97), 0.99 (0.99–1.00), 91.14 (31.88–260.56), 0.06 (0.04–0.09), and 1473.68 (562.96–3857.70), respectively. Moreover, the value of the combined diagnostic OR was 1473.68 (562.96–3857.70). Diagnostic OR is the ratio of positive LR to negative LR, reflecting the degree of connection between the results of diagnostic tests and diseases. The larger the diagnostic OR value, the better the discrimination effect of the diagnostic test. The SROC curve is shown in Figure 8, with AUC and Q* of 0.9920 and 0.9636 (SE = 0.0139), respectively. SROC is a comprehensive index reflecting continuous variables of sensitivity and specificity and revealing the relationship between them, and the AUC means the area under the SROC curve. The value of AUC getting closer to 1 indicates a better diagnosis, and Q* point is the intersection of the upper left and lower right diagonal with the SROC curve; the closer it is to the upper left, the better it is. According to the above results, it indicates that LAMP has high accuracy in the area of diagnosis of norovirus infection.

3.7 Publication bias

Deeks’ funnel graph asymmetry was drawn, and the result, p = 0.02 (p < 0.05), is shown in Figure 9, which indicated a low potential for publication bias in the present study. However, the small number of studies included in the analysis resulted in a low capacity to detect bias.

4 DISCUSSION

Human norovirus is the leading cause of foodborne illness globally, which has caused a considerable public health and economic burden.³³ Complex pathogenesis, different susceptibility, and difficult culture methods in vitro limit the research, diagnosis, and prevention of viruses.^{33, 34} A rapid and distinct diagnosis of norovirus infection is of great importance to treat patients early and control disease progression.

Among the detection methods of norovirus, RT-PCR is regarded as the gold standard in the detection of norovirus with inherent advantages. However, the disadvantages of RT-PCR–based assays lead to the limitation in its use in resource-poor settings without equipped laboratories. It is usually only performed in public health laboratory with advanced research equipment.³⁵

LAMP is a novel, rapid, specific, sensitive, and simple nucleic acid amplification technique located in six regions in the target gene using four primers and amplified RNA at a constant temperature. The most significant one is its rapidity; LAMP allows immediate diagnosis in just 30–50 min.³⁶ Recently, LAMP has been utilized for the rapid detection of some pathogens, and their results showed high accuracy and availability in the diagnosis of the corresponding pathogens.^37-39 In the detection of norovirus, LAMP with hydroxynaphthol blue dye could be observed visually through the change of color; therefore, the instrument of molecular electrophoresis is not required.^{27, 28, 31} This can decline costs, reduce procedure, and rapidly make a clear diagnosis. The previous study showed that the relative detection limitation was the concentration of the positive sample after 5⁶ dilution or norovirus RNA load of >10³ copies/tube, which was equivalent to that of the conventional RT-PCR.^{27, 28} Therefore, LAMP has more advantages in detecting norovirus.

In this study, we majorly focused on the importance and applicability of LAMP in detecting norovirus promptly. Based on the strict inclusion and exclusion criteria, we finally included eight articles to form quality evaluation and extracted eleven data sets for meta-analysis. The results in this analysis displayed that the pooled sensitivity and specificity of LAMP were 0.96 (0.95–0.97) and 0.99 (0.99–1.00), respectively, suggesting that LAMP had a lower rate of omission diagnosis and misdiagnosis in detecting norovirus. Furthermore, the negative LR, positive LR, and diagnostic OR were 0.06 (0.04–0.09), 91.14 (31.88–260.56), and 1473.68 (562.96–3857.70), respectively. A greater value contributed to a better distinguishing efficiency of diagnostic experiments if diagnostic OR was >1. Meanwhile, the AUC was 0.9920 and Q* was 0.9636 in SROC; the curve was close to the top left corner. The results proved a higher value, suggesting a greater diagnostic potential of LAMP for norovirus infection.

From the comprehensive analysis, one may conclude that the capacity of LAMP in the early diagnosis of norovirus is considerably high. LAMP may be a practical and reliable method for detecting norovirus.

In addition, Deeks’ funnel plots and the linear regression pattern were made to examine the publication bias of the included studies. The results implied the presence of publication bias, which might be that some negative results had not been published. It reduced the reliability and persuasiveness for the accuracy of LAMP of this analysis.

Further analysis was employed to investigate the source of heterogeneity of the included studies. To minimize the heterogeneity, this study had set rigorous inclusion/exclusion criteria earlier. The value of I² of pooled sensitivity (48.5%), specificity (75.1%), negative LR (60.5%), and positive LR (69.0%) indicated the existence of heterogeneity in this study. The threshold effect and non-threshold effects were analyzed to discover the heterogeneous sources. The correlation index (0.164) and p-value (0.631) of eleven data showed the lack of diagnostic threshold. Moreover, in non-threshold effect analysis, the result of diagnostic OR (I²: 32.9%) indicated the presence of non-threshold effect in the included studies. Considering the few articles included, subgroup analysis was not conducted to investigate its heterogeneity. Through browsing the included articles, possible factors of heterogeneity originating from non-threshold effect might be identified, including the virus conditions in patients, concurrent infection, the sample situation (collection, storage, and transportation), and experiment status (technics, standard tests, and operators).

Nevertheless, this analysis has several limitations. First, although we searched and screened all the relevant documents with the retrieval strategies, it was difficult to ensure that no articles were missing. Second, unpublished studies that might have several negative outcomes were not researched, which led to publication bias. Third, the number of the included articles was comparatively small. There were only eight articles to include and evaluate. Fourth, some articles included were relatively old. Half of the included articles were published before 2012. Finally, the existence of generic quality in the included studies could result in the rise of heterogeneity.

In conclusion, the study illustrates that LAMP has high sensitivity and specificity in the detection of norovirus. LAMP proves feasible and promising in the rapid diagnosis of norovirus infection. The results of this study provided information for finding an efficient method for the clinical detection of norovirus. However, further extension of this approach is encouraged to ensure the accuracy and practicability of this hopeful test in the future.

CONFLICT OF INTEREST

The authors guarantee that there were no competing interests.

AUTHOR CONTRIBUTIONS

XG and XF conceived and designed the experiments. XF and RX searched literature. XF, NX, AL, and RX screened literature, extracted data, and assessed the quality. XF contributed to the production of figures and tables by the analysis tools. YP was mainly responsible for the overall thinking. All authors participated in the writing, reading, and revising of the article and approved of the final version of the article.