Pediatric rotavirus and norovisrus diarrhea in Nicaragua

Abstract: Diarrheal diseases are still one of the major health problems in developing countries with rotavirus (RV) being the most important pathogen of severe diarrhea in young children. Norovirus (NoV), a common cause of gastroenteritis is now recognized as an important cause of sporadic diarrhea and hospitalization in children worldwide. Estimates of the disease burden indicate that every year, RV causes approximately 111 million episodes of gastroenteritis, 2 million of hospitalizations and approximately 600,000 deaths in children <5 years of age, with most of the mortality in developing countries. Likewise, recent estimations indicate that NoV cause 900,000 clinical visits among children in industrialized countries, and up to 200,000 deaths of children <5 years of age in developing countries. Thus viral intestinal pathogens are associated with approximately 800.000 deaths in young children every year predominantly in developing countries. In, this thesis the importance, molecular epidemiology and host genetic factors associated with RV and NoV diarrhea in Nicaraguan children have been investigated. Between February and March 2005 a nationwide outbreak of acute gastroenteritis associated with an exaggerated increase in mortality in children <2 years of age was observed in Nicaragua. A total of 108 stool samples from children and adults of 13 towns or major cities of the country were investigated. RV was detected in 72 (67%) of the 108 samples examined. Surprisingly, most (85%) of the RVpositive samples were typed as P[8]G4, a virus not previously observed in Nicaragua. This viral strain was found to have several amino acid mutations that modified antigenic sites and the secondary structure of VP7. The structural changes observed in this virus may have increased virulence and enable this particular virus strain to escape neutralization. Following the nationwide outbreak of rotavirus, a diarrhea surveillance study was conducted in the city of León between March 2005 and February 2006 to investigate the role of NoVs infections in pediatric diarrhea. NoV was detected in 12% (65/542) of the children; of these, 11% (45/409) were in the community and 15% (20/133) among hospitalized children, with most strains (88%) belonging to genogroup (G) II. A significant proportion (18/31) of NoV-positive children with dehydration required intravenous rehydration. Nucleotide sequence analysis (38/65) of the N-terminal and shell region in the capsid gene revealed that at least six genotypes (GI.4, GII.2, GII.4, GII.7, GII.17, and a potentially novel cluster termed GII.18-Nica ) circulated during the study period, with GII.4 virus being predominant (26/38). GII.4 virus infected predominantly young children (<2 years old) and was the most common strain found among hospitalized cases. Molecular epidemiological analysis revealed circulation of NoV genotypes with significant diversity (GII.2, GII.4, GII.17 and GII.18-Nica) in April followed by decreased diversity (GI.4, GII.4 and GII.18-Nica) in May-June and restriction mainly to GII.4 in July. Our findings suggests that NoV is an important etiological agent of acute diarrhea among children of <2 years of age in Nicaragua. Host genetic resistance to NoV has been observed in challenge and outbreak studies in populations from Europe, Asia and USA. This, thesis also include an study to investigate if histoblood group antigens (HBGA) and secretor status (defined by a nonsense G428A mutation in FUT2 gene) are associated with NoV susceptibility in the Nicaraguan population. A subset of 28 NoV-positive patients and 131 healthy population controls were investigated in relation to blood types, Lewis phenotypes (Lea+b-, Lea-b+ and Lea-b-), secretor status and NoV antibody prevalence and titers. Similar to reports from Europe, none of the nonsecretor or Lea+b- individuals was symptomatically infected. Moreover, only 3% of the Nicaraguan population was nonsecretor in contrast to 20% in Europe. The globally dominating GII.4 virus was found to infect all blood groups except AB, nonsecretors and Lea+b- individuals. AB individuals were found to have significantly lower antibody-prevalence than both A and O individuals (P < 0.05) and also significantly lower antibody-titers than blood group A, B and O (P < 0.05) further suggesting that, AB individuals are highly resistant to NoV infection. The Lewis investigation revealed not only that Lewis status (Lea+b-, Lea-b+ and Lea-b-) is not a predictive marker for NoV infection, an observation consistent with a previous report but also that the Lea-b- individuals can be infected with both GI and GII viruses, an observation not previously made. Furthermore, no significant difference in antibody-prevalence was observed between different Lewis phenotypes. Surprisingly, 25% of the Nicaraguan population was Lea-b- as compared with the 5.7% and 10% observed in Sweden and Spain, respectively. This study extended previous knowledge about the role of HBGAs in NoV disease in a population with different genetic background than North America and Europe. The recognition of NoV as an important cause of gastroenteritis is in part due to recent development of sensitive and specific diagnostic methods. In, this thesis I describe a sensitive and specific LUX real-time PCR assay for detection and quantification of NoV. The LUX system uses a fluorophore attached to one primer having a self-quenching hairpin structure, making it cost-effective and specific. The assay simultaneously detected and distinguished between GI and GII NoV by using genogroup specific primers and melting temperature analysis. Quantification limit per real-time PCR reaction was 10 and 20 gene copies for GII and GI, respectively. The assay correctly identified all (n = 11) coded control specimens in a reference panel containing various NoV genogroups and genotypes. Of the clinical specimens from Nicaragua the LUX real-time PCR assay identified NoV in 29/42 samples which correlated with TaqMan assay, but not with a commercial ELISA (24/42) or a conventional PCR (targeting the RdRp) (25/42). One possible reason why the conventional PCR method failed to detect certain NoV-positive specimens might be that viral RNA concentration was too low. Another reason might be that the sites targeted (RdRp) with the conventional PCR primers are less conserved.