Field Performance of Two Methods for Detection of Poliovirus in Wastewater Samples, Mexico 2016-2017.

To enhance our ability to monitor poliovirus circulation and certify eradication, we evaluated the performance of the bag-mediated filtration system (BMFS) against the two-phase separation (TPS) method for concentrating wastewater samples for poliovirus detection. Sequential samples were collected at two sites in Mexico; one L was collected by grab and ~ 5 L were collected and filtered in situ with the BMFS. In the laboratory, 500 mL collected by grab were concentrated using TPS and the sample contained in the filter of the BMFS was eluted without secondary concentration. Concentrates were tested for the presence of poliovirus and non-poliovirus enterovirus (NPEV) using Global Poliovirus Laboratory Network standard procedures. Between February 16, 2016, and April 18, 2017, 125 pairs of samples were obtained. Collectors spent an average (± standard deviation) of 4.3 ± 2.2 min collecting the TPS sample versus 73.5 ± 30.5 min collecting and filtering the BMFS sample. Laboratory processing required an estimated 5 h for concentration by TPS and 3.5 h for elution. Sabin 1 poliovirus was detected in 37 [30%] samples with the TPS versus 24 [19%] samples with the BMFS (McNemar's mid p value = 0.004). Sabin 3 poliovirus was detected in 59 [47%] versus 49 (39%) samples (p = 0.043), and NPEV was detected in 67 [54%] versus 40 [32%] samples (p < 0.001). The BMFS method without secondary concentration did not perform as well as the TPS method for detecting Sabin poliovirus and NPEV. Further studies are needed to guide the selection of cost-effective environmental surveillance methods for the polio endgame.

A one-step real-time RT-PCR helps to identify mixed rotavirus infections in Mexico.

Rotaviruses continue being the most important pathogens responsible of diarrhea in young children worldwide. Seminested reverse transcription polymerase chain reaction (RT-PCR) is used to determine rotavirus genotype; however, this technique employs multistep procedures. The real-time RT-PCR is a fast and reliable tool that can be used as rotavirus genotyping tool, especially in rotavirus outbreaks. In this study, we tested a real-time RT-PCR to identify rotavirus genotype using a panel of 252 samples from patients with diarrheal disease caused by G9P[4] and G12P[8] genotypes, which were identified as emerging rotaviruses in 2 outbreaks in Chiapas, Mexico. Our results show that the real-time RT-PCR assay detected these rotaviruses, and it allowed us to identify mixed genotype infections, G/P combinations, and the viral abundance in some samples in which the seminested assay could not identify them. Therefore, the real-time RT-PCR is a molecular tool that can be great support during rotavirus outbreaks.

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.

Diarrhea morbidity and mortality in Mexican children: impact of rotavirus disease.

AIM: To analyze changes in prevalence and seasonality of diarrhea morbidity and mortality and to evaluate the impact of rotavirus disease among Mexican children younger than 5 years old. METHODS: Diarrhea surveillance was performed from 1990 to 2002. Rotavirus testing was performed on stool specimens from 1996 to 2002. Data were obtained from different surveillance systems considering a nationwide representation in Mexico. Diarrhea morbidity and mortality rates were analyzed against time to determine trends or seasonal patterns. RESULTS: Improvement of surveillance for all diarrhea episodes denoted an initial morbidity increase from 1995 to 1999, followed by a decrease by 2002, without any seasonal pattern. However, from 1990 to 1995, morbidity for severe diarrhea decreased 63%. From 1996 to 2002, 62-68% of severe diarrhea episodes occurring during the fall-winter season (FWS) were rotavirus-positive compared with 6-12% in the spring-summer season (SSS). From 1990 to 2002, diarrhea mortality decreased 84%. Higher mortality rates for children younger than 1 year old coincided precisely during the FWS, annually. Both severe diarrhea episodes and diarrhea deaths denoted a changing seasonal pattern. In 1990-1991, 2 waves of increased diarrhea activity occurred. The increase in SSS was much more pronounced than that in FWS. From 1992 to 1995 for severe diarrhea and from 1993 to 2002 for diarrhea deaths, the SSS frequencies subsequently reduced, whereas the FWS peaks remained annually. CONCLUSIONS: A significant reduction in morbidity and mortality of severe diarrhea has occurred from 1990 and 2002 in Mexican children younger than 5 years old. This is a consequence of preventive programs initiated for cholera control since 1991, which had greater impact on SSS diarrhea and limited response for FWS diarrhea, when rotavirus is mainly present. Currently rotavirus diarrhea requires new prevention strategies and specific control measures, such as a specific national vaccine program.

Infectious diseases in Mexico. A survey from 1995-2000.

Data obtained at a central laboratory for emerging, re-emerging, and other infectious diseases in Mexico from 1995-2000 are presented. An outstanding increase of DEN-3 circulation was identified. Aedes aegypti, the dengue vector, is widely distributed. Leptospirosis has become the most important differential diagnosis for dengue. Identification of rabies virus variants allowed cataloging of new transmitters of rabies. Rotavirus showed a clear seasonal distribution, while different proportions of pathogenic classes of Escherichia coli under endemic and outbreak conditions were seen. Serotypes of several bacteria are reported as well as the sources of isolation and frequency of Shigella, Salmonella, and Vibrio cholerae. Rise and disappearance of cholera could be followed along the past decade. Influenza strains were identified, as were several pathogens causing sexually transmitted infections. Laboratory support was important for surveillance after Hurricane Mitch. Multidrug-resistant strains of Mycobacterium tuberculosis are emerging and primary resistance is very high. It is now mandatory to search for antibodies to Trypanosoma cruzi in blood banks. Triatoma barberi, a peridomestic bug, is the main vector of Chagas disease. Localized cutaneous leishmaniosis increased in regions having a guerrilla element in Chiapas. Modern immunodiagnostic techniques are used for control studies of cysticercosis and similar techniques were recently standardized for Trichinella spiralis detection. Low iodine values in children's urine were found in several Mexican states; therefore, use of iodized salt should be encouraged.