Multilocus Sequence Typing helps understand the genetic diversity of Cryptosporidium hominis and Cryptosporidium parvum isolated from Colombian patients.

Multilocus Sequence Typing has become a useful tool for the study of the genetic diversity and population structure of different organisms. In this study, a MLST approach with seven loci (CP47, MS5, MS9, MSC6-7, TP14, and gp60) was used to analyze the genetic diversity of Cryptosporidium hominis and Cryptosporidium parvum isolated from 28 Colombian patients. Five Cryptosporidium species were identified: C. hominis, C. parvum, Cryptosporidium felis, Cryptosporidium meleagridis, and Cryptosporidium suis. Unilocus gp60 analysis identified four allelic families for C. hominis (Ia, Ib, Id, and Ie) and two for C. parvum (IIa and IIc). There was polymorphic behavior of all markers evaluated for both C. hominis and C. parvum, particularly with the CP47, MS5, and gp60 markers. Phylogenetic analysis with consensus sequences (CS) of the markers showed a taxonomic agreement with the results obtained with the 18S rRNA and gp60 gene. Additionally, two monophyletic clades that clustered the species C. hominis and C. parvum were detected, with a higher number of subclades within the monophyletic groups compared to those with the gp60 gene. Thirteen MLG were identified for C. hominis and eight for C. parvum. Haplotypic and nucleotide diversity were detected, but only the latter was affected by the gp60 exclusion from the CS analysis. The gene fixation index showed an evolutionary closeness between the C. hominis samples and a less evolutionary closeness and greater sequence divergence in the C. parvum samples. Data obtained in this work support the implementation of MLST analysis in the study of the genetic diversity of Cryptosporidium, considering the more detailed information that it provides, which may explain some genetic events that with an unilocus approach could not be established. This is the first multilocus analysis of the intra-specific variability of Cryptosporidium from humans in South America.

Enteroinvasive Escherichia coli O96:H19 is an Emergent Biofilm-Forming Pathogen.

Enteroinvasive Escherichia coli (EIEC) is a diarrheagenic E. coli pathotype carrying a virulence plasmid that encodes a type III secretion system (TTSS) directly implicated in bacterial cell invasion. Since 2012, EIEC serotype O96:H19 has been recognized in Europe, Colombia, and most recently Uruguay. In addition to the invasion phenotype, the strains isolated from Colombian children with moderate-to-severe gastroenteritis had a strong biofilm formation phenotype, and as a result, they are referred to as biofilm-forming enteroinvasive E. coli (BF-EIEC). The objective of this study was to characterize the biofilm formation phenotype of the BF-EIEC O96:H19 strain 52.1 isolated from a child with moderate-to-severe gastroenteritis in Colombia. Random mutagenesis using Tn5 transposons identified 100 mutants unable to form biofilm; 20 of those had mutations within the pgaABCD operon. Site-directed mutagenesis of pgaB and pgaC confirmed the importance of these genes in N-acetylglucosamine-mediated biofilm formation. Both biofilm formation and TTSS-mediated host cell invasion were associated with host cell damage on the basis of cytotoxic assays comparing the wild type, invasion gene mutants, and biofilm formation mutants. Multilocus sequence typing-based phylogenetic analysis showed that BF-EIEC strain 52.1 does not cluster with classic EIEC serotype strains. Instead, BF-EIEC strain 52.1 clusters with EIEC serotype O96:H19 strains described in Europe and Uruguay. In conclusion, BF-EIEC O96:H19, an emerging pathogen associated with moderate-to-severe acute gastroenteritis in children under 5 years of age in Colombia, invades cells and has a strong biofilm formation capability. Both phenotypes are independently associated with in vitro cell cytotoxicity, and they may explain, at least in part, the higher disease severity reported in Europe and Latin America. IMPORTANCE Enteroinvasive Escherichia coli (EIEC), a close relative of Shigella, is implicated in dysenteric diarrhea. EIEC pathogenicity involves cell invasion mediated by effector proteins delivered by a type III secretion system (TTSS) that disrupt the cell cytoskeleton. These proteins and the VirF global regulator are encoded by a large (>200 kb) invasion plasmid (pINV). This study reports an emergent EIEC possessing a cell invasion phenotype and a strong polysaccharide matrix-mediated biofilm formation phenotype. Both phenotypes contribute to host cell cytotoxicity in vitro and may contribute to the severe disease reported among children and adults in Europe and Latin America.

Etiology of acute gastroenteritis among children less than 5 years of age in Bucaramanga, Colombia: A case-control study.

BACKGROUND: Acute gastroenteritis (AGE) is a major cause of morbidity and mortality in children aged less than 5 years in low- and middle-income countries where limited access to potable water, poor sanitation, deficient hygiene, and food product contamination are prevalent. Research on the changing etiology of AGE and associated risk factors in Latin America, including Colombia, is essential to understand the epidemiology of these infections. The primary objectives of this study were to describe etiology of moderate to severe AGE in children less than 5 years of age from Bucaramanga, Colombia, a middle-income country in Latin American, and to identify the presence of emerging E. coli pathotypes. METHODOLOGY/PRINCIPAL FINDINGS: This was a prospective, matched for age, case-control study to assess the etiology of moderate to severe AGE in children less than 5 years of age in Bucaramanga, Colombia, South America. We tested for 24 pathogens using locally available diagnostic testing, including stool culture, polymerase chain reaction, microscopy and enzyme-linked immunoassay. Adjusted attributable fractions were calculated to assess the association between AGE and each pathogen in this study population. The study included 861 participants, 431 cases and 430 controls. Enteric pathogens were detected in 71% of cases and in 54% of controls (p = <0.001). Co-infection was identified in 28% of cases and in 14% of controls (p = <0.001). The adjusted attributable fraction showed that Norovirus GII explained 14% (95% CI: 10-18%) of AGE, followed by rotavirus 9.3% (6.4-12%), adenovirus 3% (1-4%), astrovirus 2.9% (0.6-5%), enterotoxigenic Escherichia coli (ETEC) 2.4% (0.4-4%), Cryptosporidium sp. 2% (0.5-4%), Campylobacter sp. 2% (0.2-4%), and Salmonella sp.1.9% (0.3 to 3.5%). Except for Cryptosporidium, all parasite infections were not associated with AGE. Three emergent diarrheagenic E. coli pathotypes were identified in cases (0.7%), including an enteroaggregative/enterotoxigenic E.coli (EAEC/ETEC), an enteroaggregative/enteropathogenic E.coli (EAEC/EPEC), and an emergent enteroinvasive E. coli with a rare O96:H19. No deaths were reported among cases or controls. CONCLUSIONS/SIGNIFICANCE: Norovirus and rotavirus explained the major proportion of moderate to severe AGE in this study. Higher proportion of infection in cases, in the form of single infections or co-infections, showed association with AGE. Three novel E. coli pathotypes were identified among cases in this geographic region.

Case-Control Pilot Study on Acute Diarrheal Disease in a Geographically Defined Pediatric Population in a Middle Income Country.

INTRODUCTION: Acute diarrheal disease (ADD) is a common cause of morbidity and mortality in children under 5 years of age. Understanding of the etiology of ADD is lacking in most low and middle income countries because reference laboratories detect limited number of pathogens. The objective of this study was to determine the feasibility to conduct a comprehensive case-control study to survey diarrheal pathogens among children with and without moderate-to-severe ADD. MATERIALS AND METHODS: Microbiology and molecular-based techniques were used to detect viral, bacterial, and parasitic enteropathogens. The study was conducted in Bucaramanga, Colombia, after Institutional Review Board approval was obtained. RESULTS: Ninety children less than 5 years of age were recruited after a written informed consent was obtained from parents or guardians. Forty-five subjects served as cases with ADD and 45 as controls. Thirty-six subjects out of 90 (40.0%) were positive for at least one enteropathogen, that is, 20 (44.4%) cases and 16 (35.5%) controls. CONCLUSIONS: The three most common enteric pathogens were enteroaggregative E. coli (10.0%), Norovirus (6.7%), and Salmonella spp. (5.6%). The E. coli pathogens were 18.8% of all infections making them the most frequent pathogens. Half of ADD cases were negative for any pathogens.

Concordancia de dos pruebas serológicas para el diagnóstico de la enfermedad de Chagas / Concordance of two serological tests for the diagnosis of Chagas disease

Objetivo Establecer la concordancia entre un Ensayo Inmunoenzimático Ligado a una Enima Casera (ELISA) y la Inmunofluorescencia Indirecta (IFI) para el diagnóstico de infección por T. cruzi empleando eluidos sanguíneos. Metodología Se realizó un estudio de evaluación de tecnología diagnóstica y muestreo de corte transversal a 650 habitantes de una zona endémica de Colombia. Se determinó el área bajo la curva de operador-receptor (del inglés ROC) y se usó la IFI estandarizada en eluidos sanguíneos como gold standard. Se estableció el punto de corte para el ELISA, así como la concordancia entre las lecturas. Resultados El ELISA presentó una concordancia de 0,99 (IC95 %: 0,989-0,992) entre las lecturas realizadas y una curva ROC de 0,9795. El punto de corte establecido fue 0,5 de absorbancia en la prueba de ELISA. 16,6 % fueron positivas para anticuerpos anti-T. cruzi por ELISA y 10,9 % por IFI. Conclusiones El ELISA mostró buena concordancia frente a IFI, por lo tanto es una buena elección diagnóstica para la población que vive en áreas remotas.

Objetive Establish the concordance between in-house ELISA and IIF for the diagnosis of infection with T. cruzi using blood eluates. Methodology A study of diagnostic technology evaluation and cross-sectional sample of 650 residents of an endemic area of Colombia was conducted. It was determined the Receiver Operating Characteristic curve (ROC) and IIF was used as a gold standard. It was established the cutoff for the ELISA and the correlation between readings. Results The in-house ELISA it was an agreement of 0.99 (95 % CI: 0.989 to 0.992) between the two readings taken and the area for the ROC curve was 0.9795. The cutoff was set at 0.5 absorbance in the ELISA test. 16.6 % were positive by ELISA and 10.9 % by IIF. Conclusions The in-house ELISA showed good concordance compared to the IIF, so it is a good choice diagnostic for the population living in remote areas.

[Concordance of two serological tests for the diagnosis of Chagas disease]. / Concordancia de dos pruebas serológicas para el diagnóstico de la enfermedad de Chagas.

OBJECTIVE: Establish the concordance between in-house ELISA and IIF for the diagnosis of infection with T. cruzi using blood eluates. METHODOLOGY: A study of diagnostic technology evaluation and cross-sectional sample of 650 residents of an endemic area of Colombia was conducted. It was determined the Receiver Operating Characteristic curve (ROC) and IIF was used as a gold standard. It was established the cutoff for the ELISA and the correlation between readings. RESULTS: The in-house ELISA it was an agreement of 0.99 (95 % CI: 0.989 to 0.992) between the two readings taken and the area for the ROC curve was 0.9795. The cutoff was set at 0.5 absorbance in the ELISA test. 16.6 % were positive by ELISA and 10.9 % by IIF. CONCLUSIONS: The in-house ELISA showed good concordance compared to the IIF, so it is a good choice diagnostic for the population living in remote areas.

[Triatoma dimidiata populations' (Hemiptera: Reduviidae: Triatominae) feeding behaviour in an endemic zone and related epidemiological implications]. / Conducta alimentaria de poblaciones de Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) en una zona endémica y sus implicaciones epidemiológicas.

OBJECTIVE: Determining Triatoma dimidiata's feeding behaviour in domiciliary and extra-domiciliary habitats in an endemic area of the Santander department in Colombia. MATERIALS AND METHODS: The ELISA technique was used for processing the intestinal contents of 367 insects captured in rural areas around the municipalities of Capitanejo and Macaravita. 12 anti-animal species specific polyclonal anti-sera were used in ELISA. T. dimidiata hosts were determined by reactivity to each anti-serum; host percentages were established. RESULTS: 42.2% of the intestinal content processed by ELISA was reactive for blood proteins from one or more than 10 hosts. Domestic animal proteins were identified in all reactive intra-domiciliary and peridomestic insects, most often chicken blood, followed by that from goats, canines and humans. Blood from domestic animals like goats, chicken and horses was also detected in wild insects. Blood from animals such as armadillo and fara were identified in intra-domiciliary insects. Human host protein was found in 11% of intra-domiciliary and peri-domiciliary insects. CONCLUSIONS: The vector's eclectic nature in domiciliary and extra-domiciliary habitats was determined by identifying human blood in wild and domestic animals. The wild populations' mobility towards domiciliary and peri-domiciliary areas was demonstrated by finding domestic animals' blood in them and wild animals' blood in domestic and peri-domestic insects. These results contribute towards understanding Trypanosoma cruzi transmission-cycles.

Conducta alimentaria de poblaciones de Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) en una zona endémica y sus implicaciones epidemiológicas / Triatoma dimidiata populations' (Hemiptera: Reduviidae: Triatominae) feeding behaviour in an endemic zone and related epidemiological implications

Objetivo Determinar la conducta alimentaria de Triatoma dimidiata en hábitats domiciliarios y extradomiciliarios en una zona endémica de Santander, Colombia. Materiales y métodos Mediante la técnica de ELISA, se procesaron 367 contenidos intestinales de insectos capturados en zonas rurales de los municipios de Capitanejo y Macaravita. Estos fueron procesados por medio de la técnica de ELISA, con 12 antisueros policlonales anti-especie animal específicos. Los hospederos de T. dimidiata fueron determinados por la reactividad ante cada antisuero; se establecieron las proporciones de hospederos. Resultados El 42,2 por ciento de los contenidos intestinales procesados fueron reactivos en el ELISA para proteínas sanguíneas de uno o más de 10 hospederos. En la totalidad de los insectos reactivos de intradomicilio y peridomicilio se identificaron proteínas de animales domésticos, con mayor frecuencia la sangre de gallina, seguida de caprino, canino y humano; en los insectos silvestres también se detectó sangre de animales domésticos como cabra, gallina y equino. En los insectos intradomésticos, sangre de animales como fara y armadillo. El hospedero humano fue identificado en domicilio y peridomicilio en el 11 por ciento de los insectos. Conclusiones Se determinó el carácter ecléctico del vector en hábitats domiciliarios y extradomiciliarios, por la identificación de sangre humana, de animales silvestres y domésticos. Se evidenció movilidad de las poblaciones silvestres hacia el domicilio y peridomicilio por el hallazgo de sangre de animales domésticos en éstos y de animales silvestres en los insectos domésticos y peridomésticos. Estos resultados contribuyen a la comprensión de los ciclos de transmisión de T. cruzi.

Objective Determining Triatoma dimidiata's feeding behaviour in domiciliary and extra-domiciliary habitats in an endemic area of the Santander department in Colombia. Materials and methods The ELISA technique was used for processing the intestinal contents of 367 insects captured in rural areas around the municipalities of Capitanejo and Macaravita. 12 anti-animal species specific polyclonal anti-sera were used in ELISA. T. dimidiata hosts were determined by reactivity to each anti-serum; host percentages were established. Results 42.2 percent of the intestinal content processed by ELISA was reactive for blood proteins from one or more than 10 hosts. Domestic animal proteins were identified in all reactive intra-domiciliary and peridomestic insects, most often chicken blood, followed by that from goats, canines and humans. Blood from domestic animals like goats, chicken and horses was also detected in wild insects. Blood from animals such as armadillo and fara were identified in intra-domiciliary insects. Human host protein was found in 11 percent of intra-domiciliary and peri-domiciliary insects. Conclusions The vector's eclectic nature in domiciliary and extra-domiciliary habitats was determined by identifying human blood in wild and domestic animals. The wild populations' mobility towards domiciliary and peri-domiciliary areas was demonstrated by finding domestic animals' blood in them and wild animals' blood in domestic and peri-domestic insects. These results contribute towards understanding Trypanosoma cruzi transmission-cycles.