Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.

Rotavirus produces diarrhea in children under 5 years old. Most of those conventional methods such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR) have been used for rotavirus detection. However, these techniques need a multi-step process to get the results. In comparison with conventional methods, the real-time RT-PCR is a highly sensitive method, which allows getting the results in only one day. In this study a real-time RT-PCR assay was tested using a panel of 440 samples from patients with acute gastroenteritis, and characterized by PAGE and RT-PCR. The results show that the real-time RT-PCR detected rotavirus from 73% of rotavirus-negative samples analyzed by PAGE and RT-PCR; thus, the percentage of rotavirus-positive samples increased to 81%. The results indicate that this real-time RT-PCR should be part of a routine analysis, and as a support of the diagnosis of rotavirus in Mexico.

Molecular characterization of human calicivirus associated with acute diarrheal disease in Mexican children.

BACKGROUND: Human caliciviruses (HuCV) are emerging enteric pathogens that are a common cause of diarrhea in humans worldwide. Due to the paucity of information on the molecular characterization of HuCV circulating in Mexico, the aim of this work was to investigate the diversity and molecular epidemiology of the HuCV infection associated with acute diarrheal disease in Mexican children aged up to 5 years. RESULTS: Of the 131/414 (32%) HuCV positive-specimens analyzed, 128 were identified as Norovirus (NoV) and three as Sapovirus (SaV). Of the NoV positive specimens, 118/128 (92%) were NoV GII and 10/128(8%) were untypeable by RT-PCR in both polymerase and capsid genes, whereas one SaV isolate was further confirmed by sequencing as GI.2. Phylogenetic analysis based on polymerase partial gene sequences from 89/131 (68%) HuCV isolates showed that 86/89 (97%) belong to NoV GII.4 with three main variant clusters of this genotype, 2/89 (2%) to NoV GII.2, and 1/89 (1%) to SaV GI.2. Furthermore, partial sequencing of the capsid gene VP1 of 63/131 (48%) strains indicated that 61/63 (97%) correlated with NoV GII.4, whereas only 2/63 (3%) clustered to NoV GII.2. HuCV infections were detected throughout the year, and the highest number of cases positive for NoV was found in children between 7 and 18 months of age (60%). CONCLUSIONS: This study highlights the usefulness of analyzing both polymerase and capsid genes for molecular characterization of HuCV and demonstrates the relatedness and predominance of NoV GII.4 with acute diarrheal disease in young Mexican children, thus contributing to better understanding of the molecular epidemiology of this disease.

Construction of an internal RT-PCR standard control for the detection of human caliciviruses in stool.

RT-PCR is the most sensitive assay for the detection of human caliciviruses (HuCV) in stool and environmental samples. However, false negative results are commonly obtained due to the presence of RT-PCR inhibitors. In order to exclude such false negative results, an internal control (IC) was developed for the assay by cloning a 319 nt sequence of the Norwalk virus (NV) polymerase containing a 156 nt cDNA insert. The RT-PCR assay was carried out using RNA derived from the constructed plasmid and a primer set previously described for calicivirus detection, resulting in a 475 nt product. Distinct bands of the internal control and the viral specific RT-PCR products (319 nt) were obtained when the internal control was added to the samples. Similar results were also obtained when both the control RNA and viral RNA were seeded into stool samples from asymptomatic volunteers, or when the internal control was included into positive samples. Since the primer set used in the assays can detect a wide range of strains in both norovirus and sapovirus genera, this internal control should have a broad application for the diagnosis of human caliciviruses diagnosis in both clinical and environmental samples.