Determinación de la diversidad genética del citomegalovirus humano en diferentes poblaciones de pacientes en Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.La seroprevalencia de citomegalovirus es mayor del 95% en la población adulta de Costa Rica; la infección primaria ocurre muy temprano en la vida y es la infección congénita más frecuente en recién nacidos. El objetivo de este trabajo fue determinar la variabilidad genética y los genotipos del gene gB del citomegalovirus humano. Se recolectaron muestras de sangre de mujeres embarazadas, alcohólicos, pacientes con SIDA, niños con trastornos hemato-oncológicos, donadores de sangre y se incluyeron aislamientos de citomegalovirus de neonatos con enfermedad congénita. Se utilizó un sistema de PCR semi-anidado para obtener una banda de 293-296 pares de bases, la cual fue analizada por la técnica de Polimorfismo conformacional de banda simple (PCBS) y secuenciada para determinar los patrones genéticos polimórficos y los genotipos, respectivamente. La mayor diversidad polimórfica se encontró en los pacientes con SIDA con 14 patrones diferentes mientras que en los niños con trastornos hemato-oncológicos se demostró el mismo patrón en el 56% de los casos, sugiriendo una posible infección nosocomial en este grupo. En los neonatos se encontraron tres genotipos (gB1, gB2, gB3) mientras que en los pacientes con SIDA y en los donadores de sangre solo se demostró el gB2. En las muestras analizadas se determinaron los genotipos gB1, gB2 y gB3 y el gB2 se determinó en el 73% de los casos, no se detectaron infecciones mixtas. Los resultados de este trabajo indican que la técnica del PCBS puede ser una herramienta importante para detectar el citomegalovirus humano en infecciones intrahospitalarias y se sugiere la importancia de incluir poblaciones de estudio adicionales para determinar mejor la diversidad genética y su prevalencia

HBx M130K and V131I (T-A) mutations in HBV genotype F during a follow-up study in chronic carriers

BACKGROUND: Around 400 million people worldwide are chronically infected with Hepatitis B virus (HBV). An estimated 10% of these chronic patients develop progressive liver damage including cirrhosis and Hepatocellular Carcinoma (HCC). The HBx gene encodes a protein of 154 amino acids which is a transactivator and has been associated with HBV pathogenesis. A change in the amino acid sequences at positions 130 and 131 in the HBV-X protein (M130K and V131I) produced by T-A point mutations at the nucleic acids level has been associated with severe liver damage and HCC in patients from China and Africa. Further, such changes have been proposed as a prognostic marker for progressive liver damage and HCC. The purpose of this study was to determine if T-A mutations are present in HBV chronic carriers with genotype F (the major genotype in Costa Rica) and further, if these mutations are associated with HBV disease progression in Costa Rica HBV patients from 1972 to 1985. RESULTS: Serum samples from 50 HBV positive individuals were amplified and directly sequenced, 48 belonged to genotype F, 1 from genotype D and another was classified as D or E. T-;A mutations were absent in 17 acute patients who recovered, but was present in 12 of 29 chronic carrier samples (42.8%), in one sample the T-A mutations were detected as early as 29 days after clinical onset of disease. In 17 carriers with available liver biopsies, T-;A mutations were found in 8 sera of 13 (61.5%) classified as moderate or severe, and none in 4 biopsies with mild liver damage. However, it was not possible to demonstrate a statistical association between the presence of T-A mutations and moderate/severe liver damage, using a Fischer exact test, 1 tail, p = 0.05. In 4 patients HCC was diagnosed, and 2 of them presented the T-A mutations in their sera. CONCLUSION: T-A mutations were found in HBV genotype F in chronic carriers but not in patients who recovered from acute infection. These mutations could be developing early during infection although the possibility of infection with the mutant virus could not be excluded. More studies are necessary to establish if the T-A mutation can be used as a prognostic marker for severity of liver disease in patients infected with HBV

Aggression, Conflict and Peace ResearchMental health problems, family functioning and social support among children survivors of Colombia’s armed conflict

Purpose - Colombia presents with one of the largest armed conflicts in the world. Children exposed directly or indirectly to armed conflicts live the emotional footprints left by war. This study identified mental health problems among children survivors of Colombia’s armed conflict and associated factors. Design/methodology/approach – A cross-sectional study with (n=80) children aged 7 to 11 years (M=9.8 years; SD= 1.4) was conducted using the Child Behavior Checklist, Family APGAR, and MOS social support survey adaptation to children. Linear regression analyses were also performed with emotional and behavioral problems as the outcomes and related factors as the predictors. Findings - Clinical levels of emotional and behavioral problems were found in 56.3% of children. Internalizing problems (63.7%) were more common than externalizing problems (51.2%). Older children had greater emotional problems at the trend level, and those with higher functioning families had lower emotional problems. Children with higher perceived social support had lower behavior problems at the trend level. Research limitations/implications - This study includes a sample facing multiple risks and uses a holistic approach to consider family and social resources that may support children who are survivors of the armed conflict in Colombia. These results provide a foundation for future promotion and prevention programs related to children’s mental health problems to support peacebuilding within the framework of the Colombian post-conflict process. Originality/value – To the best of authors’ knowledge, this is the first study to collect empirical data on the mental health of children survivors of Colombia’s armed conflict focused in the Atlantic Department

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

<p>Abstract</p> <p>Background</p> <p>Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines.</p> <p>Results</p> <p>HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b). Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein.</p> <p>Conclusion</p> <p>This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.</p

Estudio de mutantes resistentes a los antiretrovirales en pacientes con VIH con falla terapeutica y efecto de los factores de riesgo en el tratamiento

INTRODUCTION: Information about HIV phenotypes of resistant to available ART and the influence of different risk factors on virological failures (VF) in Costa Rican HIV positive patients prior or during HAART is unknown. MATERIALS AND METHODS: Eighty nine samples, 72 VF and 17 basal (before treatment) were analyzed by examining resistant mutants in reverse transcriptase (RT) and protease (PT) regions using Trugene or LIPA genotyping kits. Sixty eight control patients were selected and relevant information was collected in a questionnaire. RESULTS: Poor adherence, presence of resistant mutations and number of treatment's changes were the only significant factors found (p = 0.006, 0.04 and 0.01 respectively). From 66 sequenced samples, 78%, 50% and 50% showed resistance to NRTI (nucleoside reverse transcriptase inhibitors), NNRT (non-nucleoside reverse transcriptase inhibitors) and PI (protease inhibitors), respectively. The most frequent mutations were M41L, M184V, and T215FY in RT and L62PI, L10FIRV and M36I in PT. DISCUSSION: The most important factor related to treatment response in this study was adherence to treatment. Mutations in RT were related to the treatment failure while the ones found in PT were secondary mutations which have been previously described to influence the selection of primary resistance mutations in these regions. The study reveals the urgency to detect resistant mutations in VF to be considered by physicians for selection of treatment schedule, to analyze basal HIV patients for monitoring of the spread of resistant mutations and the importance to reinforce the adherence in the patients for overall treatment outcome.En Costa Rica no se tiene información a cerca de genotipos de resistencia para los tratamientos anti-retrovirales disponibles y la influencia de diferentes factores de riego en la falla virológica (FV) de pacientes VIH positivos previo o durante su tratamiento. Ochenta y nueve muestras, 72 FV y 17 basales, fueron analizadas con Trugene o LIPA para la detección de mutantes de resistencia en la transcriptasa reversa (TR) y en la proteasa (PT) del VIH. Se seleccionaron sesenta y ocho controles y se recolectó información relevante en un cuestionario. La mala adherencia, la presencia de mutaciones y el número de cambios de tratamiento fueron los únicos factores con significancia encontrados. (p = 0.03, 0.04 and 0.04 respectively). De 66 muestras secuenciadas, 78%, 50% y 50% mostraron resistencia a los inhibidores análogos y no análogos de nucleótidos para la TR y la PT respectivamente. La mutaciones más frecuentes fueron M41L, M184V, y T215FY en la TR y L62PI, L10FIRV y M36I en la PT. La adherencia fue el factor más importante relacionado con la respuesta al tratamiento. Las mutaciones encontradas en la TR estaban relacionadas al tratamiento mientras que las de la PT fueron mutaciones secundarias que propician la aparición de las mutaciones asociadas a resistencia en esa región. Este estudio revela la necesidad de detectar mutantes de resistencia en pacientes con FV y de estudiar las muestras basales. Además la importancia de reforzar la adherencia en los pacientes para una mejor respuesta al tratamiento

\u3cem\u3eRickettsia felis\u3c/em\u3e in \u3cem\u3eCtenocephalides felis\u3c/em\u3e from Guatemala and Costa Rica

Rickettsia felis is an emerging human pathogen associated primarily with the cat flea Ctenocephalides felis. In this study, we investigated the presence of Rickettsia felis in C. felis from Guatemala and Costa Rica. Ctenocephalides felis were collected directly from dogs and cats, and analyzed by polymerase chain reaction for Rickettsia-specific fragments of 17-kDa protein, OmpA, and citrate synthase genes. Rickettsia DNA was detected in 64% (55 of 86) and 58% (47 of 81) of flea pools in Guatemala and Costa Rica, respectively. Sequencing of gltA fragments identified R. felis genotype URRWXCal2 in samples from both countries, and genotype Rf2125 in Costa Rica. This is the first report of R. felis in Guatemala and of genotype Rf2125 in Costa Rica. The extensive presence of this pathogen in countries of Central America stresses the need for increased awareness and diagnosis

A DNA pooling based system to detect Escherichia coli virulence factors in fecal and wastewater samples

The availability of a useful tool for simple and timely detection of the most important virulent varieties of Escherichia coli is indispensable. To this end, bacterial DNA pools which had previously been categorized were obtained from isolated colonies as well as selected in terms of utilized phenotype; the pools were assessed by two PCR Multiplex for the detection of virulent E. coli eaeA, bfpA, stx1, stx2, ipaH, ST, LT, and aatA genes, with the 16S gene used as DNA control. The system was validated with 66 fecal samples and 44 wastewater samples. At least one positive isolate was detected by a virulent gene among the 20 that were screened. The analysis of fecal samples from children younger than 6 years of age detected frequencies of 25% LT positive strains, 8.3% eae, 8.3% bfpA, 16.7% ipaH, as well as 12.5 % aatA and ST. On the other hand, wastewater samples revealed frequencies of 25.7% eaeA positive, 30.3% stx1, 15.1% LT and 19.7% aatA. This study is an initial step toward carrying out epidemiological field research that will reveal the presence of these bacterial varieties.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Distinct Salmonella Enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.

An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.This work was supported by the Wellcome Trust. We would like to thank the members of the Pathogen Informatics Team and the core sequencing teams at the Wellcome Trust Sanger Institute (Cambridge, UK). We are grateful to D. Harris for work in managing the sequence data

A somatic coliphage threshold approach to improve the management of activated sludge wastewater treatment plant effluents in resource-limited regions

Versión aceptada para publicaciónEffective wastewater management is crucial to ensure the safety of water reuse projects and 29 effluent discharge into surface waters. Multiple studies have demonstrated that municipal 30 wastewater treatment with conventional activated sludge processes is inefficient for the removal 31 of the wide spectrum of viruses in sewage. In this study, a well-accepted statistical approach was 32 used to investigate the relationship between viral indicators and human enteric viruses during 33 wastewater treatment in a resource-limited region. Influent and effluent samples from five urban 34 wastewater treatment plants (WWTP) in Costa Rica were analyzed for somatic coliphage and 35 human enterovirus, hepatitis A virus, norovirus genotype I and II, and rotavirus. All WWTP 36 provide primary treatment followed by conventional activated sludge treatment prior to 37 discharge into surface waters that are indirectly used for agricultural irrigation. The results 38 revealed a statistically significant relationship between the detection of at least one of the five 39 human enteric viruses and somatic coliphage. Multiple logistic regression and Receiver Operating Characteristic curve analysis identified a threshold of 3.0 ×103 40 (3.5-log10) somatic 41 coliphage plaque forming unit per 100 mL, which corresponded to an increased likelihood of encountering enteric viruses above the limit of detection (>1.83×102 42 virus target/100 mL). 43 Additionally, quantitative microbial risk assessment was executed for famers indirectly reusing 44 WWTP effluent that met the proposed threshold. The resulting estimated median cumulative 45 annual disease burden complied with World Health Organization recommendations. Future 46 studies are needed to validate the proposed threshold for use in Costa Rica and other regions.Universidad de Costa Rica/[]/UCR/Costa RicaNational Science Foundation/[OCE-1566562]/NSF/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data