Monitoring of SARS-CoV-2 RNA in wastewater as an epidemiological surveillance tool in Mendoza, Argentina.

Wastewater-based epidemiology (WBE) is an emerging tool that gives temporal and spatial information on a population's health status. Here, we report the epidemiological dynamics of a population of ~1.2 million residents in the metropolitan region of Mendoza province, Argentina, within the period July 2020 to January 2021. We combined the use of WBE of two wastewater treatment plants with epidemiological surveillance of the corresponding populations. We applied two viral concentration methods (polyethylene glycol precipitation and aluminum-based adsorption-flocculation) and RNA isolation methods in each wastewater sample to increase the possibility of detection and quantification of nucleocapsid markers (N1 and N2) of SARS-CoV-2 by RT-qPCR. Overall, our results allowed us to trace the rise, exponential growth, plateau, and fall of SARS-CoV-2 infections for 26 weeks. Individual analysis for each wastewater treatment plant showed a positive correlation between the viral load of SARS-CoV-2 genetic markers and COVID-19 cases that were diagnosed per week. Our findings indicate that WBE is a useful epidemiological indicator to anticipate the increase in COVID-19 cases and monitor the advance of the pandemic and different waves of infections.

Epidemiology of hepatitis B and C virus infection in Central West Argentina.

Little information is available regarding the prevalence of viral hepatitis in Central West Argentina. This study aims to give new information regarding HBV and HCV prevalence, genotypes, and risk factors in Central West Argentina and the suitability of dried blood spot (DBS) sampling for HBV and HCV screening. METHODS: A total of 622 individuals were included; the mean age was 36.6 ± 14.3 years and 55.4% were females. HBV and HCV markers were detected using serological and molecular analysis, and risk factors were evaluated using statistical analysis. RESULTS: Using serum samples, the HBsAg prevalence was 1.8%, the rate of HBV exposure (anti-HBc positivity) was 5.3%, and the rate of HBV immunity was 34.9%. HBV DNA was found in four out of 11 HBsAg+ samples, and the viruses in three of these samples were classified as genotypes A1, A2 and F2a. Multivariate analysis showed that anti-HBs positivity was associated with the level of schooling and history of HBV vaccination. The anti-HCV prevalence was 2.6%, and HCV RNA was found in 11 samples, seven of which contained viruses of genotypes 1a (n = 2), 1b (n = 3) and 2 (n = 2). The sensitivity of the DBS assay for HBsAg, anti-HBc, and anti-HCV was 100%, 66.6%, and 75%, respectively, and the specificity was above 98% for all markers when compared to serum. CONCLUSION: A low rate of HBV immunity was observed, demonstrating the importance of HBV vaccination. High HCV prevalence was found, and HCV 1b was closely related to other Argentinian isolates. Finally, the performance of DBS testing in this population needs more optimization to increase its sensitivity and specificity.

Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma.

Gliomas are the most common intracranial primary tumors, for which very few therapeutic options are available. The most malignant subtype is the glioblastoma, a disease associated with a 5-year survival rate lower than 5%. Given that research in glycobiology continues highlighting the role of glycans in tumor cell biology, it offers an interesting niche for the search of new therapeutic targets. In this study, we characterized aberrant glycosylation and its impact on cell biology over a broad panel of high- and low-grade glioma cell lines. Results show high expression of terminal Lewis glycans, mainly SLex, and overexpression of sialyl- and fucosyltransferases involved in their biosynthesis in high-grade glioma cell lines. Moreover, we report an association of complex multi-antennary N-glycans presenting ß1,6-GlcNAc branches with the high-grade glioma cells, which also overexpressed the gene responsible for these assemblies, MGAT5. In addition, downmodulation of N-glycosylation by treatment with the inhibitors Tunicamycin/Swainsonine or MGAT5 silencing decreased SLex expression, adhesion and migration in high-grade glioma cells. In contrast, no significant changes in these cell capacities were observed in low-grade glioma after treatment with the N-glycosylation inhibitors. Furthermore, inhibition of histone deacetylases by Trichostatin A provoked an increase in the expression of SLex and its biosynthetic related glycosyltransferases in low-grade glioma cells. Our results describe that aggressive glioma cells show high expression of Lewis glycans anchored to complex multi-antennary N-glycans. This glycophenotype plays a key role in malignant cell behavior and is regulated by histone acetylation dependent mechanisms.

Proteomic atlas of organ vasculopathies triggered by Staphylococcus aureus sepsis.

Sepsis is a life-threatening condition triggered by a dysregulated host response to microbial infection resulting in vascular dysfunction, organ failure and death. Here we provide a semi-quantitative atlas of the murine vascular cell-surface proteome at the organ level, and how it changes during sepsis. Using in vivo chemical labeling and high-resolution mass spectrometry, we demonstrate the presence of a vascular proteome that is perfusable and shared across multiple organs. This proteome is enriched in membrane-anchored proteins, including multiple regulators of endothelial barrier functions and innate immunity. Further, we automated our workflows and applied them to a murine model of methicillin-resistant Staphylococcus aureus (MRSA) sepsis to unravel changes during systemic inflammatory responses. We provide an organ-specific atlas of both systemic and local changes of the vascular proteome triggered by sepsis. Collectively, the data indicates that MRSA-sepsis triggers extensive proteome remodeling of the vascular cell surfaces, in a tissue-specific manner.

Viral hepatitis and Treponema pallidum prevalence in persons who underwent premarital blood tests in Argentina.

The objective of this study was to estimate the prevalence of different serological markers of hepatitis A, B and C viruses and Treponema pallidum among the adult population of Argentina. To achieve this, adults who attended health services for premarital exams (which are mandatory and includes screening for syphilis) were recruited. A cross-sectional study was designed with a cluster sampling strategy. Couples who attended selected health services for premarital screening between 2013 and 2014 in Buenos Aires, Cordoba, Mendoza and Santa Fe provinces were included. A total of 3833 individuals were recruited. Anti-HAV prevalence was 63.9%, anti-HCV 0.3%, anti-HBc (without HBsAg) 1.9%, HBsAg 0.3%, and T pallidum 0.8%. Anti-HAV was higher among older participants, foreigners and those from the lower strata. HBV increased with age and was higher among foreigners and those with lower formal educational level. Anti-HCV frequency increased with age. Premarital screening of viral hepatitis could constitute an instance of diagnosis, vaccination and inclusion in care of those in need. Results from this study will allow the national hepatitis programs to design public policies in order to diminish the impact of these infections on the population.

Expression of bladder cancer-associated glycans in murine tumor cell lines.

The characterization of murine cell lines is of great importance in order to identify preclinical models that could resemble human diseases. Aberrant glycosylation includes the loss, excessive or novel expression of glycans and the appearance of truncated structures. MB49 and MB49-I are currently the only two murine cell lines available for the development of preclinical bladder cancer models. The glycans Lewis X (LeX), Sialyl lewis X (SLeX) and Sialyl Tn (STn) have previously been associated with aggressiveness, dissemination and poor prognosis in human bladder cancer, additionally N-glycolyl GM3 (NGcGM3) is a neo-antigen expressed in many types of tumors; however, to the best of our knowledge, its expression has not previously been assessed in this type of cancer. Taking into account the relevance of glycans in tumor biology and considering that they can act as targets of therapies and biomarkers, the present study evaluated the expression of LeX, SLeX, STn and NGcGM3 in MB49 and MB49-I cells, in different growth conditions such as monolayer cultures, three-dimensional multicellular spheroids and mouse heterotopic and orthotopic tumors. The expression of LeX was not detected in either cell line, whereas SLeX was expressed in monolayers, spheroids and orthotopic tumors of both cell lines. STn was only identified in MB49 monolayers and spheroids. There are no reports concerning the expression of NGcGM3 in human or murine bladder cancer. In our hands, MB49 and MB49-I expressed this ganglioside in all the growth conditions evaluated. The assessment of its expression in cancer cell lines and patient tumors is of great importance, considering the relevance of this ganglioside in tumor biology. The data obtained by the present study demonstrates that glycan expression may be substantially altered depending on the growth conditions, highlighting the importance of the characterization of murine cancer models. To the best of our knowledge, the present study is the first to examine the expression of cancer-associated glycans, in the two murine cell lines available for the development of preclinical studies in bladder cancer.

Aberrant O-glycosylation modulates aggressiveness in neuroblastoma.

Neuroblastoma (NB) is the most common pediatric malignancy diagnosed before the first birthday in which MYCN oncogene amplification is associated with poor prognosis. Although aberrant glycosylation is an important actor in cell biology, little is known about its role in pediatric cancers such as NB. In this work we characterized the glycophenotype and the enzyme expression involved in glycans biosynthesis in five established human NB cell lines and in patient-derived primary tumors with different MYCN status. Our results show a high expression of Lewis glycan family both in MYCN-amplified cell lines and patient samples. Additionally, we report that MYCN-amplified cells overexpressed Core 2-initiating glycosyltransferase C2GNT1 in association with specific ST3Gals and FUTs, and showed increased binding to E- and P- selectins. Silencing of C2GNT1 expression in NB cells diminished expression of Lewis glycans, decreased the E- and P-selectin binding, and reduced cell adhesion, migration and proliferation in vitro. Treatment of MYCN-non-amplified cells with Trichostatin A (TSA), an histone deacetylase inhibitor, increased the expression of Lewis glycans and the enzymes involved in their biosynthesis. Our results demonstrate that MYCN-amplified NB cells overexpress Lewis family glycans, which belong to the Core 2 O-glycans group. Their expression plays a key role in the malignant behaviour of the NB cells and it is modulated by epigenetic mechanisms.

Antibody-dependent cell-mediated cytotoxicity induced by active immunotherapy based on racotumomab in non-small cell lung cancer patients.

Antitumor strategies based on positive modulation of the immune system currently represent therapeutic options with prominent acceptance for cancer patients' treatment due to its selectivity and higher tolerance compared to chemotherapy. Racotumomab is an anti-idiotype (anti-Id) monoclonal antibody (mAb) directed to NeuGc-containing gangliosides such as NeuGcGM3, a widely reported tumor-specific neoantigen in many human cancers. Racotumomab has been approved in Latin American countries as an active immunotherapy for advanced non-small cell lung cancer (NSCLC) treatment. In this work, we evaluated the induction of Ab-dependent cell-mediated cytotoxicity (ADCC) in NSCLC patients included in a phase III clinical trial, in response to vaccination with racotumomab. The development of anti-NeuGcGM3 antibodies (Abs) in serum samples of immunized patients was first evaluated using the NeuGcGM3-expressing X63 cells, showing that racotumomab vaccination developed antigen-specific Abs that are able to recognize NeuGcGM3 expressed in tumor cell membranes. ADCC response against NeuGcGM3-expressing X63 (target) was observed in racotumomab-treated- but not in control group patients. When target cells were depleted of gangliosides by treatment with a glucosylceramide synthase inhibitor, we observed a significant reduction of the ADCC activity developed by sera from racotumomab-vaccinated patients, suggesting a target-specific response. Our data demonstrate that anti-NeuGcGM3 Abs induced by racotumomab vaccination are able to mediate an antigen-specific ADCC response against tumor cells in NSCLC patients.

Seven-year follow-up of the immune response after one or 2 doses of inactivated hepatitis A vaccine given at 1 year of age in the Mendoza Province of Argentina.

This monocenter, descriptive, prospective, non-interventional study evaluated the long-term immune responses following routine vaccination with one or 2 doses of a licensed inactivated hepatitis A (HA) vaccine (Avaxim® 80U Pediatric) at age 11-23 months in a cohort of children from Mendoza, Argentina. Antibodies to hepatitis A virus (anti-HAV) were quantified annually up to Y5, and at Y7. Children whose titer decreased to below the seroprotection threshold (defined as an anti-HAV antibody concentration of ≥ 10 mIU/mL in a microparticle enzyme immunoassay up to Y5, or ≥ 3 mIU/mL in an electrochemiluminescence immunoassay at Y7) received a routine booster dose of the same HA vaccine. This report summarizes the data at 7 year after the first vaccination. Of 546 participants initially included, 264 participants remained at Y7 and provided blood samples. Of these, 204 having received one HA primary dose as a toddler were still seroprotected at Y7; titers for a further 7 also having received one HA dose as a toddler fell to below the seroprotection threshold and they therefore received a booster; all 53 having received 2 HA doses as a toddler and still present at Y7 remained seroprotected at Y7. One or 2 primary doses of this HA vaccine in toddlers result in very good persistence of anti-HAV up to 7 year post-first vaccination.

Comparability of Antibody-Mediated Cell Killing Activity Between a Proposed Biosimilar RTXM83 and the Originator Rituximab.

BACKGROUND: Biosimilars are described as biological products that resemble the structure of original biologic therapeutic products, with no clinically meaningful differences in terms of safety and effectiveness from the original. A wide range of biosimilars are under development or are already licensed in many countries. Biosimilars are earning acceptance and becoming a reality for immunotherapy treatments mainly based on the alternatives for the commercial anti-CD20 monoclonal antibody rituximab. The most important mechanism of action reported for this antibody is the induction of antibody-dependent cell cytotoxicity (ADCC), which is associated with the polymorphisms present at the 158 position in the IgG receptor FcγRIIIa. OBJECTIVE: The aim of the study was to validate the functional comparability between the proposed rituximab biosimilar RTXM83 and the original product. To achieve this we assessed the binding capacity and ADCC induction of this biosimilar, taking into account the different FcγRIIIa-158 polymorphisms. METHODS: Binding capacity was evaluated by flow cytometry using CD20 positive cells and a wide range of antibody concentrations. The FcγRIIIa-158 polymorphisms were analyzed by polymerase chain reaction (PCR) followed by allele-specific restriction enzyme digestion. ADCC was measured by a colorimetric lactate dehydrogenase-release assay, using effector cells from donors with different FcγRIIIa-158 polymorphisms. RESULTS: Binding capacity assay showed no differences between both products. Regarding ADCC, a similar relative potency was obtained between both antibodies, showing a higher response for the FcγRIIIa-158 valine/valine (V/V) polymorphism compared to the phenylalanine/phenylalanine (F/F), for both rituximab and RTXM83. CONCLUSION: Our data strongly suggest the biocomparability between the proposed biosimilar and the originator rituximab, in antibody recognition and ADCC activity. Additionally, our results suggest that donors with the FcγRIIIa-158V/V polymorphism induce a higher ADCC response, as has been reported.

Persistence of immunity from 1 year of age after one or two doses of hepatitis A vaccine given to children in Argentina.

BACKGROUND: This study was done to determine the immunogenicity of a single dose of hepatitis A vaccine in children, providing needed clinical data on the flexibility of booster administration. METHODS: Participants had received one dose of inactivated hepatitis A vaccine (Avaxim™ 80 U Pediatric) at 12-23 months of age or two doses of the same vaccine at 12 and 18 months of age prior to enrolment. Anti-hepatitis A antibody concentrations were measured at the first, second, and third year after vaccination. Suspected cases of hepatitis A in participant families were assessed and family socioeconomic data were collected. RESULTS: A series of 546 participants were enrolled. Of 467 (85.5%) participants completing 3 years of follow-up, 365 had received a single vaccine dose and 94 had received two vaccine doses. Seropositivity (anti-HAV ≥ 10 mIU/mL) at 3 years was 99.7% after one dose and 100% after two doses. At one year, geometric mean concentrations were higher after two doses (1433.9 mIU/mL, 95% confidence interval [CI] 1108-1855) than one (209.7 mIU/mL, 95% CI 190.6-230.6). Geometric mean concentrations decreased in both groups during the study, but remained well above 10 mIU/mL through the third year. The geometric mean of 3-year to one-year anti-hepatitis A concentration ratios was 0.74 (95% CI 0.70-0.79) following one dose and 0.57 (95% CI 0.47-0.70) following two doses. The greatest decrease in geometric mean concentrations occurred during the third year, ie, 21.2% in the one-dose group and 40.8% in the two-dose group. Six participants became seronegative during follow-up and responded strongly to a booster dose. Anti-hepatitis A concentrations increased in 135 children (34.9%) in the second year and 50 (13.7%) in the third year; none lived in a family with a case of hepatitis A. Three confirmed cases of hepatitis A occurred in family members. Participants belonged to a middle-income, urban/suburban population with good sanitation facilities and water supplies. CONCLUSION: A single dose of hepatitis A vaccine at 12-23 months of age resulted in hepatitis A seropositivity in all but one vaccinee after 3 years. Increased anti-hepatitis A serum concentrations suggested exposure to wild-type hepatitis A virus in this middle-class socioeconomic environment. Continuing surveillance is required to confirm the effectiveness of a single-dose hepatitis A vaccination; however, the results of the first three years are encouraging.

Eficacia clínica de los macrólidos como parte del tratamiento empírico en neumonía de la comunidad que se interna: estudio piloto, aleatorizado, a doble-ciego / Clinical efficacy of macrolides as part of a combination therapy of community-acquired pneumonia: a pilot study

Introducción: A la fecha no se ha publicado un estudio aleatorizado que soporte las recomendaciones de tratamiento combinado en neumonía de la comunidad (NAC). El objetivo de este ensayo piloto fue evaluar los efectos clínicos de la adición de un macrólidoa la terapia empírica en pacientes con NAC. Materiales y métodos: Se aleatorizaron sesenta y dos pacientes hospitalizados por NAC a recibir ampicilina/sulbactam IV más azitromicina oral (n=32) versus ampicilina/sulbactam IV más placebo (n=30) a doble ciego. El punto final principal fue la cura precoz, evaluada al 5to día, definida como alta médica antes del 5to día ó estabilización clínica sin necesidad de cambios terapéuticos. Los puntos finales secundarios fueronestadía hospitalaria, fallo terapéutico y mortalidad. Resultados: La cura precoz fue mayor en el grupo macrólidos (81% vs 53%) (p=0.02), con una reducción de riesgo relativa de 60% (95% CI: 10-82%), una reducción absoluta de riesgo de 28% (95% CI: 5-50%) y un número necesario a tratar de 3 pacientes (95%CI: 2-18). La estadía hospitalaria fue menor en el grupo macrólidos (6,5 ± 2,3 vs 8,5 ±4,5 días, p=0.027). No hubo diferencias en cuanto a fallo terapéutico (3 versus 6 pacientes) ni mortalidad entre ambos grupos. Conclusiones: En este estudio piloto, el uso de azitromicina oral en combinación conbetalactámicos se asoció a una mayor tasa de cura precoz y menor estadía hospitalaria, sugiriendo una resolución clínica acelerada de la neumonía.

Background and objectives: To date, no randomized trials support the recommendation of combination therapy for community-acquired pneumonia (CAP). The aim of the pilot study was to determine the clinical efficacy of the addition of a macrolide as part of anempirical therapy of patients with CAP.Methods: Sixty-two patients admitted for non-severe CAP were randomized into two double-blind groups: intravenous ampicillin/sulbactam plus oral azithromycin (n=32) versusintravenous ampicillin/sulbactam plus placebo (n=30). The primary end point was early cure, evaluated at 5th day, and defined as 1) discharge before 5th day; or 2) clinical stability without changes in the antibiotic therapy. The secondary end points were lengthof stay, treatment failure and mortality. Results: The early cure rate was higher in the macrolide group than in the placebo group (81% vs 53%) (p = 0.02), with a relative risk reduction of 60% (95% CI: 10 - 82%), anabsolute risk reduction of 28% (95% CI: 5 - 50%) and a needed number of 3 patients to be treated (95% CI: 2 - 18). The length of stay was shorter in the macrolide group (6.5 ±2.3 vs 8.5 ± 4.5 days, p = 0.027), and there were no differences in treatment failure (3 vs6 patients) or mortality. Conclusion: The use of oral azithromycin in combination with ampicillin/sulbactam wasassociated with a higher early cure rate and a shorter length of stay, suggesting an accelerated clinical resolution of CAP.

Infección Respiratoria Aguda por metapneumovirus humano en Ushuaia, Argentina: descripción del primer caso / Acute lower respiratory infection by human metapneumovirus in Ushuaia, Argentina: description of the first patient

RESUMEN Hacia fines del año 2003, en la ciudad de Ushuaia resultó llamativa la cantidad de niños internados con cuadros respiratorios graves y estudios virológicos negativos. Sobre la base de publicaciones acerca de la circulación de un nuevo virus respiratorio, metapneumovirus humano, se decidió investigar su presencia en tres muestras respiratorias de niños internados con infección respiratoria aguda en el Hospital Regional de Ushuaia. Los aspirados nasofaríngeos, previamente negativos para los virus respiratorios comunes por la técnica de inmunofluorescencia, fueron estudiados mediante la técnica de transcripción inversa y amplificación genómica por reacción en cadena de la polimerasa para metapneumovirus humano. Una de las muestras resultó positiva para metapneumovirus humano. En ninguno de los pacientes se detectaron anticuerpos de clase IgM para Chlamydia spp y Mycoplasma pneumoniae, por la técnica de inmunofluorescencia. La descripción del presente caso enfatiza la necesidad de ampliar el espectro diagnóstico en niños internados que resulten negativos para los virus respiratorios más comunes. (AU)

Infección Respiratoria Aguda por metapneumovirus humano en Ushuaia, Argentina: descripción del primer caso / Acute lower respiratory infection by human metapneumovirus in Ushuaia, Argentina: description of the first patient

RESUMEN Hacia fines del año 2003, en la ciudad de Ushuaia resultó llamativa la cantidad de niños internados con cuadros respiratorios graves y estudios virológicos negativos. Sobre la base de publicaciones acerca de la circulación de un nuevo virus respiratorio, metapneumovirus humano, se decidió investigar su presencia en tres muestras respiratorias de niños internados con infección respiratoria aguda en el Hospital Regional de Ushuaia. Los aspirados nasofaríngeos, previamente negativos para los virus respiratorios comunes por la técnica de inmunofluorescencia, fueron estudiados mediante la técnica de transcripción inversa y amplificación genómica por reacción en cadena de la polimerasa para metapneumovirus humano. Una de las muestras resultó positiva para metapneumovirus humano. En ninguno de los pacientes se detectaron anticuerpos de clase IgM para Chlamydia spp y Mycoplasma pneumoniae, por la técnica de inmunofluorescencia. La descripción del presente caso enfatiza la necesidad de ampliar el espectro diagnóstico en niños internados que resulten negativos para los virus respiratorios más comunes.

Prevalence and characterization of astroviruses in Argentinean children with acute gastroenteritis.

Among viral agents causing gastroenteritis, human astroviruses (HAstVs) take second or third place, after rotaviruses and caliciviruses, as the most frequent cause of illness. The aims of this study were to determine the prevalence of HAstV infection and to characterize the circulating HAstV strains in children with diarrhea under 3 years of age treated between 1995 and 1998 at out- or in-patient facilities of the children's hospital in Mendoza, Argentina. Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme immunoassay (EIA) were used to detect HAstVs in stool specimens. Positive specimens were tested further by EIA and/or sequenced to type detected HAstV strains. HAstVs were detected in 40 (3.7%) of 1,070 samples that were rotavirus and calicivirus-negative: 14 (3.5%) of 402 from outpatients and 26 (3.9%) of 668 from inpatients. HAstV infection tended to be more severe in children during their first year of life: 18 (4.7%) of 383 HAstV-positive children 0-11 months old were hospitalized versus 8 (2.8%) of 285 children 1 year of age or older (P = 0.29). Type 1 (HAstV-1) was the most common type (41%), followed by HAstV-4 (25%), HAstV-2 (13%), HAstV-3 (13%), and HAstV-5 (8%). In this first epidemiological study of HAstV infection in this region, we confirmed HAstV to be a cause of severe gastroenteritis in children, more often among children younger than 12 months of age. HastV-4 caused 25% of HastV infections in Mendoza, although it has been detected commonly elsewhere. Distinct genetic lineages were apparent but their epidemiological significance remains to be demonstrated.

Sequence diversity of human caliciviruses recovered from children with diarrhea in Mendoza, Argentina, 1995-1998.

Human caliciviruses were detected by EIA and/or RT-PCR in stool specimens from children with diarrhea treated at out- or in-patient facilities between 1995 and 1998 in Mendoza, Argentina. Mexico virus-like strains detected by primers NV36/51 were transiently prevalent in 1995/1996. Significantly more human caliciviruses were detected when primers were designed from contemporaneously circulating strains. Nucleotide sequences of a highly conserved region in the RNA polymerase gene of 10 selected human caliciviruses were determined. Eight strains were Norwalk-like viruses and two strains were Sapporo-like viruses. Seven of the eight Norwalk-like viruses also were positive by the recombinant Mexico virus antigen EIA. The seven Mexico virus EIA-positive strains revealed two patterns in the RNA polymerase sequences: two strains were closest to Mexico virus and the other five strains were closest to Lordsdale virus. One of the five "Lordsdale" viruses was found to be a naturally occurring recombinant between the Mexico virus and Lordsdale human calicivirus genetic clusters [Jiang et al., (1999b) Archives of Virology 144:2377-2387]. The Mexico virus EIA-negative strain had 73-77% nucleotide identity with the closest related Norwalk-like viruses, indicating it might belong to a new genetic cluster of the Norwalk-like virus genus. The two Sapporo-like viruses were distinct genetically; one belonged to the Houston/90 or Parkville cluster and the other to a new cluster. Some strains appeared to have short periods of prevalence and locally adapted primer pairs significantly increased detection rates. The finding of high diversity of circulating strains, including recombinant strains and strains with previously unrecognized genetic identities, highlights a need for studies of human caliciviruses in these children and other populations.