Norovirus Diagnostics and Serology in Travelers' Diarrhea—Where Do We Go From Here?
In addition to being the leading cause of gastroenteritis outbreaks in the developed world, and second only to rotavirus as a cause of significant morbidity in the developing world, noroviruses (NoVs) cause a substantial proportion of acute enteric illness among travelers to the developing world (and most recently as a risk to consider among those who may be undergoing fecal microbiota transplantation).[1, 2] However, a number of gaps exist in our current knowledge base with respect to NoV epidemiology in travelers, including disease burden, history of natural infection, acquired immunity, etc. Understanding the epidemiology of such infections can be challenging, especially for a pathogen that is opportunistic (eg, causes asymptomatic infection), antigenically diverse, and genetically unstable.
In this issue of the journal, Ajami and colleagues present data comparing detection of NoV by reverse transcription-polymerase chain reaction (RT-PCR) in stool and serological responses to dominant GI.1 and GII.4 strains, and a more recently developed functional histoblood group antigen (HBGA) antibody blocking assay. To put the results and limitations of this study in context, it is first worthwhile to review the relevant biology of NoV which makes detection by molecular and serological methods a challenge. NoV, like most single stranded RNA viruses, generates a high rate of mutation given the lack of proofreading ability of the viral RNA polymerase. The major viral capsid protein, VP1, includes a shell domain (S) and a protruding domain (P), the latter of which is involved in cellular interactions between the host and the virus. The P domain is subject to significant antigenic variation that exists between genogroups, genotypes, and even within genotypes. Such variation impacts the ability of diagnostic assays to efficiently detect NoVs because of potential epitopic variation of the capture antigen for immune-based assays, and translationally silent primer-binding mutations.
NoV Diagnostics: What Do We Really Know?
In the study by Ajami and colleagues, among 75 students evaluated for acute travelers diarrhea (TD) in the summer of 2004, NoV was detected in 16% with three fourths due to GI.1. These data are intriguing as they exceed the rate of NoV described from previously reported systematic reviews on TD etiology in civilian (6.6%) and military travelers (8.4%).[7, 8] However, it is important to note that these earlier systematic reviews report studies that predominantly used enzyme-linked immunosorbent assay and electron microscopy detection methods which are known to be less sensitive. The rates reported in this study are more consistent with recent reports using the more sensitive RT-PCR method with regional variability.[9, 10] However, identification does not equal causation, and while this study did not include asymptomatic controls to adequately address this issue, the overall copathogen identification rate of 75% [seven of nine being enterotoxigenic Escherichia coli (ETEC)] raises questions about NoV attribution in this setting. The authors point out that coinfections are not uncommon and, specifically, ETEC and NoV as well as other pathogens have been found to be coincident in recent studies.[11, 12] One question to be answered is if there is a unique interaction between these two pathogens which results in this apparent association (eg, common environmental/exposure relationship, NoV asymptomatic infection increases susceptibility to ETEC, or visa-versa), or if highly sensitive genotypic detection methods result in the likelihood of detection of commonly circulating pathogens traditionally known to independently cause TD, or if these data represent a situation of serial infections, particularly given that NoV shedding is known to occur for up to 2 months in symptomatic and asymptomatic infections. Platts-Mills and colleagues recently forwarded the notion of quantitative diagnostics to be used for infectious diarrhea to better define the attribution of infections causing disease in the likely situation where multiple pathogens are identified. Embarking on an integrated and standardized multi-site study design among travelers with cases and controls using an expanded case definition to include acute gastroenteritis not meeting the classical TD case definition, in combination with quantitative PCR and discriminatory methods directly on stool for detection and differentiation of an array of enteropathogens, would go far to answer some of these basic questions of NoV epidemiology in travelers.[15, 16]
NoV Serology: What Might It Tell Us?
Serological diagnostics are used for a number of infectious diseases in the clinical setting, and typically reserved for pathogens that may be difficult to directly isolate. In this study, which matched stool samples with paired pre-travel and post-travel serum, the concordance of seroconversion with positively identified NoV by PCR in the stool was poor with only one individual with NoV-positive stool developing a ≥4-fold increase in serum antibody titers (seroconversion). The authors also attribute NoV to an additional two subjects for whom seroconversion was noted, but stool samples were not available. While such testing was not optimized owing to potential epitopic heterogeneity between the two strain types used and those that actually caused disease, the results call into question the value of serology for NoV diagnosis and more work is needed to better understand the cross-reactivity across strains. The results are particularly confounded by data from a NoV experimental human model where immune responses (as measured by increases in HBGA blocking titers) among those who were asymptomatic when challenged were higher compared with those who were symptomatic.
Not surprisingly, this study describes the presence of baseline levels of antibodies [immunoglobulin G (IgG), IgA, and HBGA blocking] against GI.1 and GII.4 type strains among pre-illness samples, which is consistent with the finding of nearly 100% serological response against NoV among populations in the developed world by the age of 20.[18-22] The higher baseline titers to GII.4 relative to the GI.1 may be due to differences in antigen cross-reactivity/antibody affinity, the predominance of GII.4 globally, or some other assay performance characteristic. The lack of asymptomatic controls precluded the ability to evaluate serum biomarkers for protection against illness or modification of disease severity which are important questions, particularly in light of a putative serum correlate of protection which has been identified in human experimental models and a vaccine which is in clinical development. Studies evaluating these correlates in a setting of natural infection among travelers are needed.
The stated limitations of this study methodology do not provide convincing evidence to completely abandon the potential utility of seroepidemiological methods. Expansion of virus-like particle arrays that might efficiently increase the detection of nonhomologous exposure, or identification of unique NoV biomarkers specific to NoV disease (as opposed to infection) combined with quantitative molecular diagnostics could be of value both in clinical and travel medicine. While the field has come a long way in understanding NoV despite an agent which still cannot be cultured, further development of direct and indirect methods will be worthwhile to better understand the burden of acute NoV-attributed illness, and in light of recent data linking NoV with persistent long-term gastrointestinal health consequences, to further appreciate what may be the iceberg below.[24-26]
The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the US Government. This is a US Government work. There are no restrictions on its use. There were no financial conflicts of interests among any of the author. This work was conducted under support of the Military Infectious Disease Research Program and Department of Defense Global Emerging Infections Surveillance and Response System funding.