RESUMO
The prevalence of cystic echinococcosis is high in many livestock areas of Peru, where intermediate hosts such as sheep, cattle, and South American camelids can be infected. Several species of E. granulosus have been described in relation to its genetic diversity and distribution. The aim of this study was to determine the species of E. granulosus sensu lato (s.l.) metacestodes collected from sheep, cattle, swine and camelids at different localities in the department of Puno, in the southern highlands of Peru. One hundred and fifty-two echinococcal cysts were collected from 10 different locations. E. granulosus s.l. species were determined by amplification of the Internal transcribed spacer 1 of the ribosomal DNA using a Nested PCR-RFLP technique. The cytochrome C oxidase 1 gene (450 bp) was also amplified and sequenced in samples with different RFLP patterns. Cysts samples were collected from sheep (39.5%), cattle (32.9%), pigs (15.8%) and alpacas/llamas (11.8%). E. granulosus sensu stricto (G1 genotype) was mainly identified in all animal hosts, while, the E. canadensis (G7) was only identified in cysts from pigs and alpacas. This is the first report of E. granulosus sensu stricto and E. canadensis in llamas and alpacas, respectively. Knowledge of species and molecular epidemiology of E. granulosus s.l. in endemic areas in Peru may help to evaluate preventive programs, understand disease transmission, as well as improve vaccine and chemotherapy effectiveness.
Assuntos
Equinococose , Echinococcus granulosus , Echinococcus , Animais , Bovinos , Equinococose/epidemiologia , Equinococose/veterinária , Echinococcus granulosus/genética , Genótipo , Gado , Peru/epidemiologia , Ovinos , SuínosRESUMO
OBJECTIVES: To inform next steps in pediatric diarrhea burden reduction by understanding the shifting enteropathogen landscape after rotavirus vaccine implementation. METHODS: We conducted a case-control study of 1788 medically attended children younger than 5 years, with and without gastroenteritis, after universal rotavirus vaccine implementation in Peru. We tested case and control stools for 5 viruses, 19 bacteria, and parasites; calculated coinfection-adjusted attributable fractions (AFs) to determine pathogen-specific burdens; and evaluated pathogen-specific gastroenteritis severity using Clark and Vesikari scales. RESULTS: Six pathogens were independently positively associated with gastroenteritis: norovirus genogroup II (GII) (AF 29.1, 95% confidence interval [CI]: 28.0-32.3), rotavirus (AF 8.9, 95% CI: 6.8-9.7), sapovirus (AF 6.3, 95% CI: 4.3-7.4), astrovirus (AF 2.8, 95% CI: 0.0-4.0); enterotoxigenic Escherichia coli heat stable and/or heat labile and heat stable (AF 2.4, 95% CI: 0.6-3.1), and Shigella spp. (AF 2.0, 95% CI: 0.4-2.2). Among typeable rotavirus cases, we most frequently identified partially heterotypic strain G12P[8] (54 of 81, 67%). Mean severity was significantly higher for norovirus GII-positive cases relative to norovirus GII-negative cases (Vesikari [12.7 vs 11.8; P < .001] and Clark [11.7 vs 11.4; P = .016]), and cases in the 6- to 12-month age range relative to cases in other age groups (Vesikari [12.7 vs 12.0; P = .0002] and Clark [12.0 vs 11.4; P = .0016]). CONCLUSIONS: Norovirus is well recognized as the leading cause of pediatric gastroenteritis in settings with universal rotavirus vaccination. However, sapovirus is often overlooked. Both norovirus and sapovirus contribute significantly to the severe pediatric disease burden in this setting. Decision-makers should consider multivalent vaccine acquisition strategies to target multiple caliciviruses in similar countries after successful rotavirus vaccine implementation.
Assuntos
Gastroenterite/microbiologia , Gastroenterite/prevenção & controle , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus , Estudos de Casos e Controles , Pré-Escolar , Diarreia/microbiologia , Diarreia/prevenção & controle , Diarreia/virologia , Fezes/microbiologia , Fezes/virologia , Gastroenterite/parasitologia , Gastroenterite/virologia , Genótipo , Humanos , Norovirus/genética , Peru , Estudos Prospectivos , Rotavirus/genética , Sapovirus/genética , Índice de Gravidade de DoençaRESUMO
BACKGROUND: This study identified Trypanosoma cruzi discrete typing units (DTUs) in maternal and infant specimens collected from two hospitals in Bolivia, using conventional genotyping and DTU-specific serotyping. METHODS: Specimens from 142 mothers were used, including 24 seronegative and 118 seropositive individuals; 29 women transmitted T. cruzi to their infants. Maternal and infant parasite loads were determined by quantitative real-time PCR. Maternal sera were tested with an in-house parasite lysate ELISA and serotyped by a lineage-specific peptide ELISA, targeting the trypomastigote small surface antigen (TSSA). Trypanosoma cruzi genotypes in infected infants were determined by a triple PCR-RFLP assay. RESULTS: All infant specimens were genotyped as TcV. Maternal parasite loads and absorbance values by the lysate ELISA were significantly higher for transmitters compared with non-transmitters. Among seropositive mothers, 65.3% had positive results by the TSSA II/V/VI peptide ELISA. No significant difference in reactivity to TSSA II/V/VI was observed for transmitters compared with non-transmitters (79.3% vs 60.7%, respectively). CONCLUSIONS: Our findings reinforce the difficulty in obtaining sufficient sample numbers and parasite DNA to investigate the interaction between parasite genetics and the risk of congenital transmission and argue for the inclusion of DTU-specific serotyping in prospective studies.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Antígenos de Superfície , Bolívia/epidemiologia , Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Feminino , Humanos , Masculino , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/genéticaRESUMO
BACKGROUND: The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: A total of 265 match pair samples of whole blood-guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR. CONCLUSIONS: The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.
Assuntos
Doença de Chagas/diagnóstico , DNA de Protozoário/sangue , Trypanosoma cruzi/genética , Doença de Chagas/parasitologia , DNA de Protozoário/genética , Testes Diagnósticos de Rotina/métodos , Humanos , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Quantitative polymerase chain reaction (qPCR) for Toxoplasma gondii multicopy genes has emerged as a promising strategy for sensitive detection of parasite DNA. qPCR can be performed from blood samples, which are minimally invasive to collect. However, there is no consensus about what type of blood specimen yields the best sensitivity. The development of a novel protocol for qPCR detection of T. gondii using blood clot, involving an appropriate DNA extraction method and the use of an internal amplification control to monitor the reaction is presented in the current study. Assays directed to the B1 and REP529 genes were performed in spiked specimens of whole blood, guanidine-ethylenediaminetetraacetic acid blood, and clot. The clot-based qPCR was shown to be more sensitive when compared with other types of specimens, detecting five and 0.05 T. gondii genomes, using B1 and REP529 targets, respectively. Finally, a comparative analysis with samples from HIV patients with clinical suspicion of toxoplasmosis was performed, demonstrating the detection of four positive suspected cases with clots compared with only one using guanidine-ethylenediaminetetraacetic acid blood. The high analytical sensitivity and the cost-effective advantages offered by clot supports this methodology as a good laboratory tool to monitor parasite burden.
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Carga Parasitária/métodos , Reação em Cadeia da Polimerase/métodos , Trombose/parasitologia , Toxoplasma/isolamento & purificação , Toxoplasmose/diagnóstico , Adulto , DNA de Protozoário/genética , Genoma de Protozoário , Infecções por HIV/sangue , Infecções por HIV/parasitologia , Humanos , Técnicas de Diagnóstico Molecular/métodos , Sensibilidade e Especificidade , Toxoplasma/genética , Toxoplasmose/sangue , Adulto JovemRESUMO
BACKGROUND: Norovirus is a leading cause of acute gastroenteritis worldwide. Routine norovirus diagnosis requires stool collection. The goal of this study was to develop and validate a noninvasive method to diagnose norovirus to complement stool diagnostics and to facilitate studies on transmission. METHODS: A multiplex immunoassay to measure salivary immunoglobulin G (IgG) responses to 5 common norovirus genotypes (GI.1, GII.2, GII.4, GII.6, and GII.17) was developed. The assay was validated using acute and convalescent saliva samples collected from Peruvian children <5 years of age with polymerase chain reaction (PCR)-diagnosed norovirus infections (n = 175) and controls (n = 32). The assay sensitivity and specificity were calculated to determine infection status based on fold rise of salivary norovirus genotype-specific IgG using norovirus genotype from stool as reference. RESULTS: The salivary assay detected recent norovirus infections and correctly assigned the infecting genotype. Sensitivity was 71% and specificity was 96% across the evaluated genotypes compared to PCR-diagnosed norovirus infection. CONCLUSIONS: This saliva-based assay will be a useful tool to monitor norovirus transmission in high-risk settings such as daycare centers or hospitals. Cross-reactivity is limited between the tested genotypes, which represent the most commonly circulating genotypes.
Assuntos
Infecções por Caliciviridae/diagnóstico , Saliva/virologia , Anticorpos Antivirais/imunologia , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Estudos de Casos e Controles , Pré-Escolar , Fezes/virologia , Humanos , Imunoglobulina G/imunologia , Norovirus/genética , Norovirus/imunologia , Peru/epidemiologia , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saliva/imunologia , Sensibilidade e EspecificidadeRESUMO
Background: Congenital Trypanosoma cruzi infection accounts for an estimated 22% of new cases of Chagas disease in Latin America. However, neonatal diagnosis is challenging, as 9-month follow-up for immunoglobulin G testing is poor, quantitative polymerase chain reaction (qPCR) analysis is not routinely performed, and the micromethod misses ≥40% of congenital infections. Methods: Biorepository samples from new mothers and their infants from Piura, Peru, (an area of nonendemicity), and Santa Cruz, Bolivia (an area of endemicity) were accessed. Infant specimens were assessed using the micromethod, qPCR analysis, and a trypomastigote excretory secretory antigen (TESA) blot for detection of immunoglobulin M (IgM)-specific shed acute phase antigen (SAPA) bands, using qPCR as the gold standard. Results: When compared to qPCR, IgM TESA blot was both sensitive and specific for congenital Chagas disease diagnosis. Cumulative sensitivity (whether only 4 bands or all 6 bands were present) was 80% (95% confidence interval [CI], 59%-92%). Specificity was 94% (95% CI, 92%-96%) in the area of endemicity and 100% in the area of nonendemicity. SAPA bands occurred sequentially and in pairs, and parasite loads correlated highly with the number of SAPA bands present. The micromethod detected infection in fewer than half of infected infants. Conclusions: The IgM TESA blot for detection of SAPA bands is rapid, relatively inexpensive, and more sensitive than the micromethod and may be a useful point-of-care test for detection of congenital T. cruzi infection.
Assuntos
Doença de Chagas/congênito , Doença de Chagas/diagnóstico , Testes Diagnósticos de Rotina/métodos , Glicoproteínas/sangue , Immunoblotting/métodos , Imunoglobulina M/imunologia , Neuraminidase/sangue , Trypanosoma cruzi/imunologia , Anticorpos Antiprotozoários/imunologia , Bolívia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Peru , Gravidez , Sensibilidade e EspecificidadeRESUMO
Norovirus, a leading cause of acute gastroenteritis in humans, is a highly diverse virus. Here, we report the complete genome sequence of a nontypeable genogroup II (GII) norovirus that was detected in a symptomatic Peruvian child in 2008. This virus showed low nucleotide sequence identities (≤82%) against all known genotypes.
RESUMO
Background: Sapovirus is one of the primary viral causes of acute gastroenteritis (AGE), especially where rotavirus vaccination has been implemented. The characteristics and impact of natural infection at the community level, however, have not been well documented. Methods: Stool samples were analyzed from 100 children randomly selected from a community-based birth cohort study in Peru. All diarrheal and 1 nondiarrheal stools collected trimonthly from children up to age 2 years (n = 1669) were tested for sapovirus detection. Viral shedding duration was determined by testing additional weekly samples (n = 440) collected before and after a sapovirus-positive sample. Results: The incidence of sapovirus infection in the first and second years of life was 4.3 and 11.1 per 100 child-months, respectively. By age 2 years, 82% of children had at least 1 sapovirus infection, and 64% had at least 1 sapovirus-associated diarrhea episode. The median shedding period was 18.5 days. In 112 of 175 infections, 14 genotypes from 4 genogroups (GI, GII, GIV, and GV) were determined. Among genogroups, GI were more frequently found in symptomatic infections than in asymptomatic infections (odds ratio, 3.1; 95% confidence interval, 1.3-7.4). Fifty-nine children had serial sapovirus infections, but only 3 had repeated infection of the same genotype. Conclusions: Sapovirus was frequently detected in children with AGE at the community level during the first 2 years of life. Serial sapovirus infections by multiple genotypes in a child suggest genotype-specific immunity from each infection, which needs to be taken into account for vaccine development.
Assuntos
Infecções por Caliciviridae/epidemiologia , Diarreia/virologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Sapovirus/isolamento & purificação , Estudos de Coortes , Diarreia/epidemiologia , Fezes/virologia , Feminino , Genótipo , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Peru/epidemiologia , Filogenia , Saúde Pública , Eliminação de Partículas ViraisRESUMO
Human sapovirus has been shown to be one of the most important etiologies in pediatric patients with acute diarrhea. However, very limited data are available about the causative roles and epidemiology of sapovirus in community settings. A nested matched case-control study within a birth cohort study of acute diarrhea in a peri-urban community in Peru from 2007 to 2010 was conducted to investigate the attributable fraction (AF) and genetic diversity of sapovirus. By quantitative reverse transcription-real-time PCR (qPCR) sapovirus was detected in 12.4% (37/299) of diarrheal and 5.7% (17/300) of nondiarrheal stools (P = 0.004). The sapovirus AF (7.1%) was higher in the second year (13.2%) than in the first year (1.4%) of life of children. Ten known genotypes and one novel cluster (n = 5) within four genogroups (GI, GII, GIV, and GV) were identified by phylogenetic analysis of a partial VP1 gene. Further sequence analysis of the full VP1 gene revealed a possible novel genotype, tentatively named GII.8. Notably, symptomatic reinfections with different genotypes within the same (n = 3) or different (n = 5) genogroups were observed in eight children. Sapovirus exhibited a high attributable burden for acute gastroenteritis, especially in the second year of life, of children in a Peruvian community. Further large-scale studies are needed to understand better the global burden, genetic diversity, and repeated infections of sapovirus.