C1q and HBHA-specific IL-13 levels as surrogate plasma biomarkers for monitoring tuberculosis treatment efficacy: a cross-sectional cohort study in Paraguay.

Introduction: New diagnostic tools are needed to rapidly assess the efficacy of pulmonary tuberculosis (PTB) treatment. The aim of this study was to evaluate several immune biomarkers in an observational and cross-sectional cohort study conducted in Paraguay. Methods: Thirty-two patients with clinically and microbiologically confirmed PTB were evaluated before starting treatment (T0), after 2 months of treatment (T1) and at the end of treatment (T2). At each timepoint plasma levels of IFN-y, 17 pro- and anti-inflammatory cytokines/chemokines and complement factors C1q, C3 and C4 were assessed in unstimulated and Mtb-specific stimulated whole blood samples using QuantiFERON-TB gold plus and recombinant Mycobacterium smegmatis heparin binding hemagglutinin (rmsHBHA) as stimulation antigen. Complete blood counts and liver enzyme assays were also evaluated and correlated with biomarker levels in plasma. Results: In unstimulated plasma, C1q (P<0.001), C4 (P<0.001), hemoglobin (P<0.001), lymphocyte proportion (P<0.001) and absolute white blood cell count (P=0.01) were significantly higher in PTB patients at baseline than in cured patients. C1q and C4 levels were found to be related to Mycobacterium tuberculosis load in sputum. Finally, a combinatorial analysis identified a plasma host signature comprising the detection of C1q and IL-13 levels in response to rmsHBHA as a tool differentiating PTB patients from cured TB profiles, with an AUC of 0.92 (sensitivity 94% and specificity 79%). Conclusion: This observational study provides new insights on host immune responses throughout anti-TB treatment and emphasizes the role of host C1q and HBHA-specific IL-13 response as surrogate plasma biomarkers for monitoring TB treatment efficacy.

Enfermedad de Chagas: Riesgo de transmisión por especies secundarias de triatominos capturados en etapa de vigilancia entomológica en las dos regiones del Paraguay

El principal vector intradomiciliar del Trypanosoma cruzi en el Cono Sur de América Latina ha sido el Triatoma infestans. En la última década se ha declarado la interrupción de transmisión de la enfermedad por esta especie en varias áreas endémicas. El T. cruzi interactúa con triatominos silvestres y reservorios mamíferos, por ello hay un riesgo permanente de la invasión de viviendas por especies secundarias como T. sordida y nativas de focos selváticos como: T. guasayana, T. guasu, Panstrongylus geniculatus, P. megistus que deben ser vigiladas para evitar el proceso de colonización de las viviendas. El objetivo fue evaluar el potencial riesgo de transmisión de la enfermedad de Chagas por especies secundarias de triatominos capturados en etapa de vigilancia entomológica en áreas endémicas de las regiones Oriental y Occidental del país. Se aplicaron técnicas moleculares asociadas a indicadores entomológicos a un total de 759 ejemplares de 4 especies de triatominos capturados en las dos regiones. Se detectó colonización del 19% por la especie T. sordida en viviendas del Departamento de Concepción. De las especies T. guasayana, T. guasu, P. geniculatus consideradas especies del ambiente selvático, se capturó al menos 1 ejemplar en cada departamento en el intradomicilio. De 759 ejemplares analizados, se detectaron 17 con infección natural con T. cruzi (2,2%), de los cuales 2 eran de P. geniculatus y 1 de T. guasayana, ambos del intradomicilio. Estos hallazgos ponen en evidencia que existe un potencial riesgo de transmisión de T. cruzi por estas especies de triatominos.

The main intradomiciliary vector of Trypanosoma cruzi in the Southern Cone of Latin America has been Triatoma infestans. In the last decade, the decrease in transmission of the disease by this species has been declared in endemic areas. T. cruzi interacts with wild triatomines and mammalian reservoirs, therefore there is a permanent risk of invasion of dwellings by secondary species such as T. sordida and native to jungle foci such as: T. guasayana, T. guasu, Panstrongilus geniculatus, P. megistus that should be monitored to avoid the process of colonization of dwellings. The objective of the study was to evaluate the potential risk of transmission of Chagas disease by secondary triatomine species captured in the entomological surveillance stage in endemic areas of the Eastern and Western regions of the country. Molecular techniques associated with entomological indicators were applied to a total of 759 specimens of 4 species of triatomines captured in the two regions. Colonization of 19% by the species T. sordida was detected in dwellings of the department of Concepción. At least one specimen of the species T. guasayana, T. guasu, P. geniculatus considered species of the jungle environment was captured in each department in the intradomiciliary environment. Of the total number of specimens analyzed (759), 17 were detected with natural infection with T. cruzi (2.2%), of which 2 were of P. geniculatus and 1 of T. guasayana both from intradomiciliary environment. These findings show that there is a potential risk of transmission of T. cruzi by these triatomine species.

Hisopado endocervical en mujeres con síntomas de COVID-19 provenientes de departamentos del Paraguay / Endocervical swabbing in women with COVID-19 symptoms from departments of Paraguay

INTRODUCCIÓN: El COVID-19 es eminentemente una infección de transmisión e inicio respiratorio, se discute la existencia de otras fuentes de contagio. El receptor viral ACE2 también ha sido detectado en el útero y en la vagina; de allí se ha planteado el compromiso del virus SARS-CoV-2 sobre el sistema genitourinario y sus posibles repercusiones en el embarazo. OBJETIVO: Determinar la presencia de SARS-CoV-2 en muestras endocervicales de mujeres con COVID-19 en departamentos del Paraguay. PACIENTES Y MÉTODOS: Diseño observacional prospectivo, de corte transverso. Se reclutaron 200 mujeres desde agosto 2020 hasta febrero 2021, con no más de 48/72 h de un resultado previo positivo de hisopado nasofaríngeo para SARS-CoV-2 por retrotranscriptasa reversa-reacción en cadena de la polimerasa (en inglés rt-RT-PCR) y que aceptaron ingresar al estudio. Se llenó un cuestionario clínico epidemiológico. Las tomas de muestras se realizaron en servicios de salud del Ministerio de Salud Pública y Bienestar Social (MSP y BS), domicilios y albergues de los distintos departamentos de Paraguay. Cada paciente fue sometida a un hisopado con hisopos de dacron o citobrush endocervical para la detección de SARS-CoV-2 por rt RT-PCR. Resultados: Las mujeres estudiadas tenían una edad media de 46,5 años (IC 95% 31,5-62,5). Refirieron contagio comunitario con SARS-CoV-2 en 75,5%, 13,5% en el hogar, 8,5% en el lugar de trabajo y 1,5% en el extranjero. Las manifestaciones clínicas fueron: 30%, síndrome gripal, fiebre 22,5%, tos 20%, anosmia 15,5%, trastornos digestivos 15,5%, y otros se presentaron con menor frecuencia. Las muestras de hisopados o citobrush endocervical sometidas a rt-RT-PCR para la deteccción de SARS Cov-2, resultaron negativas en las 200 mujeres de estudio. Discusión: Cabe destacar que las muestras vaginales fueron tomadas dentro de las 24-72 h de haber obtenido un resultado positivo para SARS-CoV-2 en el hisopado nasofaríngeo y que 62,5% de las mujeres se encontraban internadas en módulos respiratorios. Se discute la razón de la negatividad de los exámenes y su trascendencia. CONCLUSIÓN: No se detectó infección con SARS-CoV-2 en la región endocervical de 200 mujeres con manifestaciones clínicas de COVID 19 y evaluadas dentro de las 48/72 h de un resultado positivo nasofaríngeo para SARS Cov-2. Los resultados en la población de estudio concuerdan con otros estudios reportados en la literatura científica.

BACKGROUND: COVID-19 is an eminently respiratory transmissible infection of respiratory initiation, the existence of other sources of contagion is discussed. The ACE2 viral receptor has also been detected in the uterus and vagina; Hence, the involvement of the SARS-CoV-2 virus on the genitourinary system and its possible repercussions on pregnancy has been raised. AIM: To determine the presence of SARS-CoV-2 in endocervical samples of women with COVID-19 in the departments of Paraguay. METHODS: Designed as a prospective observational of transverse cohort. Two hundred women were recruited from August 2020 to February 2021, with no more than 48/72 hours of a previous positive nasopharyngeal swab result for SARS-CoV-2 by reverse transcriptase-polymerase chain reaction (rt-RT-PCR) and who agreed to participate in the study. A clinical epidemiological questionnaire was completed. The samples were taken in health services of the MSPYBS (Public Ministry of Health and Social Welfare), homes and shelters in the different departments of Paraguay. Each patient underwent a swab (dacron swabs) or endocervical cytobrush for the detection of SARS-CoV-2 by rt-RT-PCR. RESULTS: Women recruited had a mean age of 46.5 years (95% CI 31,562.5). They reported contagion with SARS-CoV-2: 75.5% in the community, 13.5% at home, 8.5% in the place of work and 1.5% abroad. The clinical manifestations were: 30% flu syndrome, 22.5% fever, 20% cough, 15.5% anosmia, 15.5% digestive disorders, among other symptoms. The swabs or endocervical cytobrush samples subjected to rt-RT-PCR for the detection of SARS-CoV-2 were negative in the 200 study women. Discussion: It should be noted that the vaginal samples were taken within 24-72 hours after obtaining a positive result for SARS-CoV-2 in the nasopharyngeal swab and that 62.5% of the women were hospitalized in respiratory modules. The reason for the negativity of the exams and their significance are discussed. CONCLUSION: No SARS Cov-2 infection was detected in the endocervical region of 200 women with clinical manifestations of COVID 19 and evaluated within 48/72 hours of a positive nasopharyngeal result for SARS Cov-2. The results in the study population agree with the findings of other studies reported in the literature.

[Endocervical swabbing in women with COVID-19 symptoms from departments of Paraguay]. / Hisopado endocervical en mujeres con síntomas de COVID-19 provenientes de departamentos del Paraguay.

BACKGROUND: COVID-19 is an eminently respiratory transmissible infection of respiratory initiation, the existence of other sources of contagion is discussed. The ACE2 viral receptor has also been detected in the uterus and vagina; Hence, the involvement of the SARS-CoV-2 virus on the genitourinary system and its possible repercussions on pregnancy has been raised. AIM: To determine the presence of SARS-CoV-2 in endocervical samples of women with COVID-19 in the departments of Paraguay. METHODS: Designed as a prospective observational of transverse cohort. Two hundred women were recruited from August 2020 to February 2021, with no more than 48/72 hours of a previous positive nasopharyngeal swab result for SARS-CoV-2 by reverse transcriptase-polymerase chain reaction (rt-RT-PCR) and who agreed to participate in the study. A clinical epidemiological questionnaire was completed. The samples were taken in health services of the MSPYBS (Public Ministry of Health and Social Welfare), homes and shelters in the different departments of Paraguay. Each patient underwent a swab (dacron swabs) or endocervical cytobrush for the detection of SARS-CoV-2 by rt-RT-PCR. RESULTS: Women recruited had a mean age of 46.5 years (95% CI 31,562.5). They reported contagion with SARS-CoV-2: 75.5% in the community, 13.5% at home, 8.5% in the place of work and 1.5% abroad. The clinical manifestations were: 30% flu syndrome, 22.5% fever, 20% cough, 15.5% anosmia, 15.5% digestive disorders, among other symptoms. The swabs or endocervical cytobrush samples subjected to rt-RT-PCR for the detection of SARS-CoV-2 were negative in the 200 study women. DISCUSSION: It should be noted that the vaginal samples were taken within 24-72 hours after obtaining a positive result for SARS-CoV-2 in the nasopharyngeal swab and that 62.5% of the women were hospitalized in respiratory modules. The reason for the negativity of the exams and their significance are discussed. Conclusión: No SARS Cov-2 infection was detected in the endocervical region of 200 women with clinical manifestations of COVID 19 and evaluated within 48/72 hours of a positive nasopharyngeal result for SARS Cov-2. The results in the study population agree with the findings of other studies reported in the literature.

Association of baseline white blood cell counts with tuberculosis treatment outcome: a prospective multicentered cohort study.

OBJECTIVES: Tuberculosis (TB) is the leading infectious cause of death in the world. Cheaper and more accessible TB treatment monitoring methods are needed. Here, we evaluated white blood cell (WBC) absolute counts, lymphocyte, and monocyte proportions during TB treatment, and characterized their association with treatment failure. METHODS: This multicentered prospective cohort study was based in Bangladesh, Georgia, Lebanon, Madagascar, and Paraguay. Adult, non-immunocompromised patients with culture-confirmed pulmonary TB were included and followed up after two months of treatment and at the end of therapy. Blood counts were compared to treatment outcome using descriptive statistics, logistic regression, and Receiver Operating Characteristic (ROC) analyses. RESULTS: Between December 2017 and August 2020, 198 participants were enrolled, and 152 completed treatment, including 28 (18.5%) drug-resistant patients. The rate of cure at the end of treatment was 90.8% (138/152). WBC absolute counts decreased, and lymphocyte proportions increased throughout treatment. In multivariate analyses, baseline high WBC counts and low lymphocyte proportions were associated with positive sputum culture results at the end of treatment (WBC > 11,450 cells/mm3: p = 0.048; lymphocytes <16.0%: p = 0.039; WBC > 11,450 cells/mm3 and lymphocytes <16.0%: p = 0.024). CONCLUSION: High WBC counts and low lymphocyte proportions at baseline are significantly associated with the risk of TB treatment failure.

Técnicas moleculares integradas a la vigilancia entomológica de vectores de la enfermedad de Chagas: Estudio del vector secundario Triatoma sordida en la Región Oriental del Paraguay / Molecular techniques integrated to the entomological surveillance of Chagas disease vectors: Study of the secondary vector Triatoma sordida in the Eastern Region of Paraguay

Las técnicas moleculares para la detección de infección natural y fuente de alimentación en vectores secundarios de la enfermedad de Chagas cuando son aplicadas a ejemplares capturados en áreas endémicas, históricamente ocupadas por Triatoma infestans, proporcionan a las investigaciones epidemiológicas respuestas más exactas con relación a la transmisibilidad de la enfermedad. El presente estudio tiene como objetivo emplear biomarcadores moleculares para evaluar el impacto de la infestación intra y peridomicilar de Triatoma sordida en viviendas bajo vigilancia entomológica de departamentos de la Región Oriental del Paraguay en el período 2007 al 2015. Un total de 559 ejemplares de T. sordida capturados en 253, 91 y 52 viviendas de los departamentos Paraguarí, San Pedro y Cordillera, respectivamente fueron analizados. La infestación detectada fue del 24% al 48% así como una elevada colonización intradomiciliar del 5% al 36% en los tres departamentos. La detección molecular de infección natural osciló entre el 14% y 44%; y en 111 ejemplares se determinó la fuente de alimentación. El marcador molecular citocromo b permitió demostrar por vez primera un elevado porcentaje de triatominos con sangre humana como fuente de alimentación, principalmente en Cordillera con un 82% (28/34 T. sordida capturados). Estos hallazgos dejan en evidencia el avance del T. sordida en la ocupación del nicho ecológico de T. cruzi y la capacidad de esta especie secundaria como vector en la transmisión de T. cruzi en comunidades de la Región Oriental

When molecular techniques for the detection of natural infection and blood meal source in secondary vectors of Chagas disease are applied to specimens captured in endemic areas, historically occupied by Triatoma infestans, provide more accurate answers to questions about transmissibility of the illness and further contribute to the epidemiological studies. The aim of this study was to evaluate the impact of intra and peridomiciliary infestation of Triatoma sordida in households from the departments of the Eastern Region of Paraguay, under entomological surveillance during the period 2007 to 2015, by using the molecular biomarkers technology. A total of 559 specimens of T. sordida captured in 253, 91 and 52 households from Paraguarí, San Pedro and Cordillera departments, respectively, were analyzed. The infestation detected was from 24% to 48% as well as a high intradomicialiary colonization from 5% to 36% in the three departments. The molecular detection of natural infections ranged from 14% to 44% and in 111 specimens the meal source was identified. The molecular marker cytochrome b allowed to demonstrate, for the first time, high frequency of triatomines with human blood as a food source, mainly in Cordillera as it was determined in 82% (28/34) of the T. sordida captured. These findings demonstrate a progress of T. sordida into the ecological niche of T. cruzi and the abillity of this secondary species as a vector of the transmission of T. cruzi in communities from the Eastern Region of Paraguay

IL-17A, a possible biomarker for the evaluation of treatment response in Trypanosoma cruzi infected children: A 12-months follow-up study in Bolivia.

BACKGROUND: The National Program for Chagas disease was implemented in Bolivia in 2006, and it greatly decreased the number of infections through vector control. Subsequently, a treatment regimen of benznidazole (BNZ) was started in seropositive school-age children living in certified vector control areas. METHODS AND FINDINGS: We conducted a 12-month follow-up study and seven blood samples were taken during and after the treatment. Serology, conventional diagnostic PCR (cPCR) and quantitative Real-time PCR (qPCR) were performed. Plasma Th1/Th2/Th17 cytokines levels were also determined. Approximately 73 of 103 seropositive children complied with BNZ, with three interruptions due to side effects. To evaluate each individual's treatment efficacy, the cPCR and qPCR values during the final 6 months of the follow-up period were observed. Among 57 children who completed follow-up, 6 individuals (11%) showed both cPCR(+) and qPCR(+) (non reactive), 24 (42%) cPCR(-) but qPCR(+) (ambiguous) and 27 (47%) cPCR(-) and qPCR(-) (reactive). Within 14 Th1/Th2/Th17 cytokines, IL-17A showed significantly higher levels in seropositive children before the treatment compared to age-matched seronegative children and significantly decreased to the normal level one-year after. Moreover, throughout the follow-up study, IL-17A levels were positively co-related to parasite counts detected by qPCR. At the 12 months' time point, IL-17A levels of non-reactive subjects were significantly higher than either those of reactive or ambiguous subjects suggesting that IL-17A might be useful to determine the reactivity to BNZ treatment. CONCLUSIONS: Plasma levels of IL-17A might be a bio-marker for detecting persistent infection of T. cruzi and its chronic inflammation.

Exploring the "Latin American Mediterranean" family and the RDRio lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela.

BACKGROUND: The Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RDRio) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RDRio genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C103 SNP and the regions of difference RDRio and RD174. IS6110-RFLP results were also used when available. RESULTS: Genotyping of 413 M. tuberculosis isolates from three Latin-American countries detected LAM (46%) and the ill-defined T clade (16%) as the most frequent families. The highest clustering rate was detected in the sample population from the city of Caracas in Venezuela. We observed considerable differences in the presence of the RDRio lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C103, MIRU02-MIRU40 signature) of the RDRio lineage were essentially confirmed. For the LAM family, the most polymorphic loci were MIRU40, MIRU31, MIRU10, MIRU26, MIRU16 and the least polymorphic MIRU24, MIRU20, MIRU04, MIRU23. CONCLUSIONS: Our results suggest a differential adaptation of LAM-sublineages in neighboring populations and that RDRio strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RDRio) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.

Burden of Influenza in Less Than 5-Year-Old Children Admitted to Hospital with Pneumonia in Developing and Emerging Countries: A Descriptive, Multicenter Study.

This descriptive 4-year study reports the proportion of detection of influenza viruses in less than 5-year-old children hospitalized for pneumonia in eight developing and emerging countries and describes clinical and microbiological characteristics of influenza-related pneumonia cases. Hospitalized children presenting radiologically confirmed pneumonia aged 2-60 months were prospectively enrolled in this observational standardized study. Mean proportion of isolated influenza virus was 9.7% (95% confidence interval: 7.9-11.8%) among 888 pneumonia children analyzed, with moderate heterogeneity between countries-ranging from 6.2% in Cambodia to 18.8% in Haiti. The clinical characteristics of children with influenza-related pneumonia were not substantially different from those of other pneumonia cases. Influenza A H1N1-related pneumonia cases appeared as more severe than pneumonia cases related to other strains of influenza. Streptococcus pneumoniae was detected more often in blood samples from influenza-related cases than in those without detected influenza viruses (19.7% versus 9.5%, P = 0.018). Influenza-related pneumonia is frequent among children less than 5 years old with pneumonia, living in developing and emerging countries. Influenza might be a frequent etiologic agent responsible for pneumonia or a predisposing status factor for pneumococcal-related pneumonia in this population.

Fuente de alimentación de ejemplares de Triatoma sordida en un área con alto riesgo de domiciliación en el Chaco Paraguayo / Blood meal source of specimens of Triatoma sordida in an area with high risk of domiciliation in the Paraguayan Chaco

En el presente estudio evaluamos indicadores entomológicos (índices de infección, colonización e infección natural) de ejemplares de Triatoma sordida capturados en el intra y peridomicilio de viviendas del Chaco Paraguayo en el período 2014 al 2016. El objetivo fue comparar con los hallazgos previamente reportados en el periodo 2010 al 2013 en la misma región. Además se ha incorporado la detección de fuente de alimentación como complemento para definir el riesgo de domiciliación de este vector secundario. Se aplicaron técnicas moleculares asociados a indicadores entomológicos y epidemiológicos a 220 ejemplares de T. sordida capturados en 67 viviendas de 24 localidades del Chaco. Se detectó infestación y colonización por T. sordida en el intradomicilio 13/67 (19%) y 5/13 (38%) y en el peridomicilio 54/67 (81%) y 43/54 (80%), respectivamente. Se detectó infección con T. cruzi en un 17,3% de los ejemplares analizados. La fuente de alimentación pudo detectarse en tan solo 13 de 220 ejemplares (6%), todos resultaron positivos para sangre de gallina y correspondían a captura en el peridomicilio. El índice de infestación intradomiciliar fue del 19%, superior al de años anteriores y similar a las zonas históricamente endémicas de la Región Occidental (18%-20%). El riesgo de transmisión intradomiciliar sigue siendo elevado porque en 3 de 5 viviendas con ninfas de T. sordida, los ejemplares estaban infectados con T. cruzi. Los indicadores entomológicos obtenidos en el presente estudio concuerdan con resultados previos de nuestro equipo, y confirman la capacidad vectorial de este triatomino secundario en la transmisión de la enfermedad de Chagas(AU)

Computational analysis of a species D human adenovirus provides evidence of a novel virus.

A human adenovirus (HAdV) species D, was isolated from a hospitalised child with severe lower respiratory infection. It was initially detected in the nasopharyngeal aspirate of the child followed by conventional PCR amplification of the hexon, penton base, and fibre genes. Sanger DNA sequencing and phylogenetic analyses showed characteristics of a recombinant genome not described before. Next Generation Sequencing analysis was performed to reconstruct its complete DNA genome after viral isolation in adenocarcinoma human cell line (A549). A complete genomic sequence of 35.2 kb in length, with a G+C content of 57 % was obtained, related to HAdV-D29 (96 % identity). Imputed serology analysis demonstrated its novel type with a nucleotide sequence identity of 95.3 % (hexon loop 1) and 96 % (hexon loop 2) to HAdV-D9. The penton base gene showed a novel sequence, distantly related to HAdV-D44. The E3 and E4 regions evolved significantly from their ancestors. The fibre gene was almost identical to the knob region of HAdV-D15 but showed an unrelated shaft sequence. In conclusion the genomics of this novel HAdV, designated the HAdV-D83 [P83H9F15] prototype and bearing a new penton base gene, supports the importance of viral evolution to understand modified tissue tropism, enhanced transmission, or altered virulence.

Severity of Pneumonia in Under 5-Year-Old Children from Developing Countries: A Multicenter, Prospective, Observational Study.

Pneumonia is the leading cause of death in children. The objectives were to evaluate the microbiological agents linked with hypoxemia in hospitalized children with pneumonia from developing countries, to identify predictors of hypoxemia, and to characterize factors associated with in-hospital mortality. A multicenter, observational study was conducted in five hospitals, from India (Lucknow, Vadu), Madagascar (Antananarivo), Mali (Bamako), and Paraguay (San Lorenzo). Children aged 2-60 months with radiologically confirmed pneumonia were enrolled prospectively. Respiratory and whole blood specimens were collected, identifying viruses and bacteria by real-time multiplex polymerase chain reaction (PCR). Microbiological agents linked with hypoxemia at admission (oxygen saturation < 90%) were analyzed by multivariate logistic regression, and factors associated with 14-day in-hospital mortality were assessed by bivariate Cox regression. Overall, 405 pneumonia cases (3,338 hospitalization days) were analyzed; 13 patients died within 14 days of hospitalization. Hypoxemia prevalence was 17.3%. Detection of human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) in respiratory samples was independently associated with increased risk of hypoxemia (adjusted odds ratio [aOR] = 2.4, 95% confidence interval [95% CI] = 1.0-5.8 and aOR = 2.5, 95% CI = 1.1-5.3, respectively). Lower chest indrawing and cyanosis were predictive of hypoxemia (positive likelihood ratios = 2.3 and 2.4, respectively). Predictors of death were Streptococcus pneumoniae detection by blood PCR (crude hazard ratio [cHR] = 4.6, 95% CI = 1.5-14.0), procalcitonin ≥ 50 ng/mL (cHR = 22.4, 95% CI = 7.3-68.5) and hypoxemia (cHR = 4.8, 95% CI = 1.6-14.4). These findings were consistent on bivariate analysis. hMPV and RSV in respiratory samples were linked with hypoxemia, and S. pneumoniae in blood was associated with increased risk of death among hospitalized children with pneumonia in developing countries.

Microorganisms Associated With Pneumonia in Children <5 Years of Age in Developing and Emerging Countries: The GABRIEL Pneumonia Multicenter, Prospective, Case-Control Study.

Background: Pneumonia, the leading infectious cause of child mortality globally, mainly afflicts developing countries. This prospective observational study aimed to assess the microorganisms associated with pneumonia in children aged <5 years in developing and emerging countries. Methods: A multicenter, case-control study by the GABRIEL (Global Approach to Biological Research, Infectious diseases and Epidemics in Low-income countries) network was conducted between 2010 and 2014 in Cambodia, China, Haiti, India (2 sites), Madagascar, Mali, Mongolia, and Paraguay. Cases were hospitalized children with radiologically confirmed pneumonia; controls were children from the same setting without any features suggestive of pneumonia. Nasopharyngeal swabs were collected from all subjects; 19 viruses and 5 bacteria were identified by reverse-transcription polymerase chain reaction. Associations between microorganisms and pneumonia were quantified by calculating the adjusted population attributable fraction (aPAF) after multivariate logistic regression analysis adjusted for sex, age, time period, other pathogens, and site. Results: Overall, 888 cases and 870 controls were analyzed; ≥1 microorganism was detected in respiratory samples in 93.0% of cases and 74.4% of controls (P < .001). Streptococcus pneumoniae, Mycoplasma pneumoniae, human metapneumovirus, rhinovirus, respiratory syncytial virus (RSV), parainfluenza virus 1, 3, and 4, and influenza virus A and B were independently associated with pneumonia; aPAF was 42.2% (95% confidence interval [CI], 35.5%-48.2%) for S. pneumoniae, 18.2% (95% CI, 17.4%-19.0%) for RSV, and 11.2% (95% CI, 7.5%-14.7%) for rhinovirus. Conclusions: Streptococcus pneumoniae, RSV, and rhinovirus may be the major microorganisms associated with pneumonia infections in children <5 years of age from developing and emerging countries. Increasing S. pneumoniae vaccination coverage may substantially reduce the burden of pneumonia among children in developing countries.

Chagas disease: national survey of seroprevalence in children under five years of age conducted in 2008

BACKGROUND Since the early 1990s, programs to control Chagas disease in South America have focused on eradicating domiciliary Triatoma infestans, the main vector. Seroprevalence studies of the chagasic infection are included as part of the vector control programs; they are essential to assess the impact of vector control measures and to monitor the prevention of vector transmission. OBJECTIVE To assess the interruption of domiciliary vector transmission of Chagas disease by T. infestans in Paraguay by evaluating the current state of transmission in rural areas. METHODS A survey of seroprevalence of Chagas disease was carried out in a representative sample group of Paraguayans aged one to five years living in rural areas of Paraguay in 2008. Blood samples collected on filter paper from 12,776 children were tested using an enzyme-linked immunosorbent assay. Children whose serology was positive or undetermined (n = 41) were recalled to donate a whole blood sample for retesting. Their homes were inspected for current triatomine infestation. Blood samples from their respective mothers were also collected and tested to check possible transmission of the disease by a congenital route. FINDINGS A seroprevalence rate of 0.24% for Trypanosoma cruzi infection was detected in children under five years of age among the country’s rural population. Our findings indicate that T. cruzi was transmitted to these children vertically. The total number of infected children, aged one to five years living in these departments, was estimated at 1,691 cases with an annual incidence of congenital transmission of 338 cases per year. MAIN CONCLUSION We determined the impact of vector control in the transmission of T. cruzi, following uninterrupted vector control measures employed since 1999 in contiguous T. infestans-endemic areas of Paraguay, and this allowed us to estimate the degree of risk of congenital transmission in the country.

Chagas disease: national survey of seroprevalence in children under five years of age conducted in 2008.

BACKGROUND: Since the early 1990s, programs to control Chagas disease in South America have focused on eradicating domiciliary Triatoma infestans, the main vector. Seroprevalence studies of the chagasic infection are included as part of the vector control programs; they are essential to assess the impact of vector control measures and to monitor the prevention of vector transmission. OBJECTIVE: To assess the interruption of domiciliary vector transmission of Chagas disease by T. infestans in Paraguay by evaluating the current state of transmission in rural areas. METHODS: A survey of seroprevalence of Chagas disease was carried out in a representative sample group of Paraguayans aged one to five years living in rural areas of Paraguay in 2008. Blood samples collected on filter paper from 12,776 children were tested using an enzyme-linked immunosorbent assay. Children whose serology was positive or undetermined (n = 41) were recalled to donate a whole blood sample for retesting. Their homes were inspected for current triatomine infestation. Blood samples from their respective mothers were also collected and tested to check possible transmission of the disease by a congenital route. FINDINGS: A seroprevalence rate of 0.24% for Trypanosoma cruzi infection was detected in children under five years of age among the country's rural population. Our findings indicate that T. cruzi was transmitted to these children vertically. The total number of infected children, aged one to five years living in these departments, was estimated at 1,691 cases with an annual incidence of congenital transmission of 338 cases per year. MAIN CONCLUSION: We determined the impact of vector control in the transmission of T. cruzi, following uninterrupted vector control measures employed since 1999 in contiguous T. infestans-endemic areas of Paraguay, and this allowed us to estimate the degree of risk of congenital transmission in the country.

Coinfección leishmania infantum, VIH e histoplasma capsulatum: manifestaciones dermatológicas / Leishmania infantum, HIV and hystoplasma capsulatum coinfection: dermatologic manifestations

La coinfección Leishmania/VIH es frecuente y por lo general son casos de leishmaniosis visceral (LV), sin embargo, Leishmania infantum, el agente etiológico, también puede causar formas cutáneas en pacientes VIH-positivos. Los parásitos llegan a la piel por difusión, en una nueva infección o reactivación de una infección latente. Existen técnicas moleculares que confirman el diagnóstico y caracterizan a la especie. En estos pacientes también se presenta como infección oportunista la histoplasmosis, reconocida como marcador de SIDA y causada por Histoplasma capsulatum. En este trabajo describimos un caso de coinfección SIDA, histoplasmosis y LV asociado a lesiones cutáneas. Paciente de sexo masculino, adulto, residente en J.A. Saldívar, VIH positivo (2010), sin tratamiento antirretroviral. En el 2012 es internado en el Instituto de Medicina Tropical (IMT) con fiebre, anemia, hepato-esplenomegalia, frotis de médula ósea y rk39 negativos para Leishmania, recibe tratamiento con Anfotericina B. En el 2013 presenta síndrome febril prolongado y en médula ósea se encuentran amastigotes de Leishmania sp. En el 2014 presenta úlceras en el rostro en las que se observan es porosmicóticos de H. capsulatum y amastigotes de Leishmania sp. Se confirma L. infantum por técnicas de biología molecular. En el caso estudiado concluimos que la presentación dérmica es causada por L. infantum, caracterizada por técnicas moleculares, eH. capsulatum que se presenta en concomitancia debido al estado inmunológico. Cabe resaltar la importancia del diagnóstico diferencial para aplicar el tratamiento correcto y además la manifestación clínica que normalmente no es asociada a esta especie de Leishmania.

Leishmania/HIV coinfection is frequent and generally they are visceral leishmaniasis cases(VL). However, Leishmania infantum, the etiological agent, could also cause cutaneousforms in HIV-positive patients. The parasites reach the skin through diffusion, a newinfection or a reactivation of a latent infection. There are molecular techniques that confirmthe diagnosis and characterize Leishmania species. These patients also present otheropportunistic infections like histoplasmosis, well known as an AIDS marker and caused byHistoplasma capsulatum. In this work, we describe one HIV/histoplasmosis/VL coinfectioncase associated to cutaneous lesions. The patient was a male, adult, coming from the city ofJ.A. Saldívar, HIV positive (since 2010), without antiretroviral treatment. He was hospitalized in 2012 at the Instituto de Medicina Tropical (IMT) with fever, anemia,hepatosplenomegaly, bone marrow smear and rK39 both negative for Leishmania. He wastreated with Amphotericin B. In 2013, he presented a prolonged febrile syndrome andLeishmania amastigotes were observed in the bone marrow. In 2014, the patient presentedulcerations on the face where H. capsulatum fungal spores and Leishmania amastigoteswere observed. L. infantum was confirmed by molecular methods. In the case under studywe can conclude that the dermal manifestation was caused by L. infantuma ccording to thecharacterization by molecular techniques, and simultaneously H. capsulatum was presentdue to his immunological status. It should be emphasized the importance of the differentialdiagnosis in order to apply the right treatment and also the uncommon clinicalmanifestation that is not associated to this Leishmania species.

Triatoma sordida: indicadores de adaptación y transmisión de trypanosoma cruzi en intradomicilio del Chaco Paraguayo / Triatoma sordida: indicators of adaptive capacity and trypanosoma cruzi intradomicile transmission in the Paraguayan Chaco

El gran Chaco es una eco-región que incluye Argentina, Bolivia y Paraguay; donde la trasmisión vectorial de la enfermedad de Chagas por Triatoma infestans (vector principal), constituye hasta la fecha un problema de salud pública. El Chaco paraguayo ocupa el 25% de esta región, caracterizada por una baja densidad poblacional y localidades dispersas. El objetivo de este estudio fue determinar el rol potencial de la especie Triatoma sordida (vector secundario) en el ciclo doméstico de transmisión de Trypanosoma cruzi. Se aplicaron técnicas moleculares asociadas a indicadores entomológicos y epidemiológicosa 436 ejemplares de T. sordida capturados en 147 viviendas del Chaco Paraguayo. Se detectó infestación y colonización en el intradomicilio y peridomicilio por T. sórdida en 12 (8.2%) y 79 (53.7%) viviendas de las 147 evaluadas, respectivamente. Al menos un ejemplar infectado con T. cruzi fue detectado por PCR en las 12 viviendas con colonización intradomiciliar y en dos de ellas por caracterización molecular se detectó en ninfas el genotipo TC2. Adultos y ninfas en el peridomicilio de 4 viviendas dieron positivo para el genotipo TC1. Se estima un elevado riesgo de transmisión de T. cruzi intradomiciliar del 87%. Estos resultados evidencian capacidad adaptativa de esta especie en el domicilio, y un incremento de su potencial vectorial para transmitir la enfermedad de Chagas en el Chaco Paraguayo.

The Gran Chaco is an eco-region that includes Argentina, Bolivia and Paraguay; where the vectorial transmission of Chagas disease by Triatoma infestans (main vector) is a public health problem up to date. The Paraguayan Chaco occupies 25% of this region, characterized by low population density and dispersed locations. The aim of this study wasto determine the potential role of the specie Triatoma sordida (secondary vector) in the domestic cycle of transmission of Trypanosoma cruzi. Molecular techniques associated with entomological and epidemiological indicators were applied in 436 specimens of T. sordida captured in 147 dwellings of the Paraguayan Chaco. Infestation rate and colonization by T. Sordida was detected in the intradomicile and peridomiciliary in 12 (8.2%) and 79 (53.7%)of the 147 dwellings, respectively. In 12 houses were colonization was observed, at leastone specimen was PCR-T. cruzi positive, and in two of them genotype TC2 of T. cruzi were detected innymphs by molecular characterization. Adults and nymphs in the peridomiciliary of 4 houses were positive for genotype TC1.A high risk of indoor transmission of T. cruzi isestimated in 87%. These results demonstrate adaptive capacity of this specie in the intradomicile, and increased vector potential to transmit Chagas disease in the Paraguayan Chaco.

Absence of asymptomatic cases of malaria in a historically endemic indigenous locality of the Department of Caaguazú, Paraguay: moving toward elimination.

INTRODUCTION:: Paraguay was among the 16 countries that reported zero indigenous malaria cases in 2014. METHODS:: A cross-sectional observational descriptive study was performed in 100 adults from Santa Teresa, Paraguay. Parasite detection was carried out using seminested multiplex polymerase chain reaction (PCR) and microscopy. RESULTS: Among the participants, 44% were female and 56% were male, and 89% had a malaria history. No parasites were detected with either of the methods. CONCLUSIONS:: There were no asymptomatic cases in Santa Teresa, and this finding is very promising. A longitudinal study should be performed to confirm that there are no asymptomatic cases in this locality.

Análisis filogenético y de presión evolutiva de secuencias nucleotídicas del gen VP4 de especies de enterovirus humanos / Phylogenetic and evolutive pressure analyses of nucleotide sequences of VP4 gene of human enterovirus species

El género Enterovirus es un grupo viral que afecta a un amplio rango de hospederos, entre ellos los humanos (especies A, B, C, y D), causan enfermedades respiratorias, gastrointestinales, neurológicas, y otras, y son altamente contagiosos. Los síntomas pueden ser leves o graves. El objetivo del trabajo fue analizar la variación nucleotídica, filogenética y de presión evolutiva de secuencias nucleotídicas del gen VP4 de las cuatro especies que afectan a los humanos. Se emplearon 92 secuencias nucleotídicas disponibles en la base de datos GenBank; éstas se editaron con el software BioEdit y se alinearon con Clustal W; las relaciones filogenéticas se determinaron con MEGA6, y las presiones evolutivas con los algoritmos SNAP y SLAC. Se encontró que la identidad nucleotídica mínima intra-especie fue de 43,2% (especie B) a 72,6% (especie D). Los genotipos más variables por especie fueron EV-71 (A), Echovirus 2 (B), EV-118 (C), y EV-94 (D). El análisis de presión evolutiva mostró que el gen VP4 en las cuatro especies evoluciona bajo presión selectiva negativa. Esto indicaría que la alta tasa mutacional y eventos de recombinación no tienen un rol significativo en la evolución de este gen, debido probablemente a la localización interna de la proteína VP4.

The Enterovirus genus is a viral group that affects a wide host range, including humans (species A, B, C and D), cause respiratory, gastrointestinal, and neurologic disease, amongothers, and are highly contagious. The symptoms range from mild to severe. The objectiveof this study was to perform a nucleotidic variation, phylogenetic and selective pressureanalyses of the VP4 gene from the four enterovirus species that affect humans. Ninety-twonucleotide sequences (available in the GenBank database) were employed; they were edited with Bio Edit software and aligned with Clustal W; the phylogenetic relationships weredetermined with MEGA6, and the evolutive pressures with SNAP and SLAC algorithms. Itwas found an intra-species nucleotide identity of at least 43,2% (species B) to 72,6% (species D). The more variable genotypes by species were EV-71 (A), Echovirus 2 (B), EV-118 (C), and EV-94 (D). The selective pressure analysis showed that VP4 gene of the fourspecies evolves by negative pressure. This would indicate that the high mutation rate andrecombination events do not have a significant role in the evolution of this gene, probablydue to the internal localization of the VP4 protein.

Detección molecular de belactamasas de espectro extendido (BLEE) en enterobacterias aisladas en Asunción / Molecular detection of extended spectrum beta-lactamase (esbl) in enterobacteria isolated in Asunción

Las betalactamasas de espectro extendido (BLEE), son enzimas responsables de la hidrólisis del anillo betalactámico de penicilinas y cefalosporinas, excepto carbapemenes, inhibiendo así su actividad terapéutica. Si bien es posible la detección fenotípica de este mecanismo de resistencia por métodos convencionales, sólo los métodos moleculares permiten la identificación del gen responsable de dicha resistencia. El objetivo de este estudio descriptivo retrospectivo fue identificar los genes blaCTX-M2, blaPER-2, blaSHV y blaTEM, en aislamientos de enterobacterias productoras de BLEE,; de muestras clínicas colectadas entre julio 2007 y abril 2008, provenientes de dos hospitales de referencia de Asunción, Paraguay. La detección molecular de los genes se realizó por reacción en cadena de la polimerasa empleando oligonucleótidos específicos. De los 232 aislados BLEE analizados, el 83% (n=192) portó al menos un gen bla, en el 17% (n=40) restante no fue detectado ninguno de los genes incluido en el estudio. Se observaron las siguientes frecuencias: 49% (94/192) blaCTX-M2, 45% (86/192) blaSHV, 40% (77/192) blaTEM y 7% (13/192) blaPER-2. En el 47% (90/192) se detectó más de un gen, siendo la combinación blaCTX-M2+blaTEM+blaSHV, la más frecuente observada en 32 aislados. El blaCTX-M2 como el gen más frecuente en este estudio; concuerda con lo reportado en nuestro país y en Argentina. Este es el primer reporte de la presencia de blaTEM y blaSHV en Paraguay. Es de gran importancia el estudio de otros genes codificantes de resistencia, considerando la emergencia de otras BLEE en la región como blaCTX-M15 con actividad predominantemente ceftazidimasa.

Extended spectrum beta-lactamases (ESBLs), are enzymes responsible for thehydrolysis of the beta-lactam ring and resistance to both cephalosporins and penicillins,except carbapenems, therefore inhibiting its therapeutics activity. Even though, detectionof the phenotypic resistance mechanism by conventional methods is possible, onlymolecular methods allow identification of the gene responsible for the resistance. Theobjective of this retrospective study was to identify the blaCTX-M2, blaPER-2, blaSHV, blaTEMgenes in ESBL-producing enterobacteriaceae isolates, recovered from clinical samples collected between July 2007 and April 2008, from two reference hospitals in Asunción,Paraguay. Molecular gene detection was performed by polymerase chain reaction usingspecifics oligonucleotides. Out of the tested 232 ESBL-producing isolates, 83% (n=192)carried at least one of the bla genes as follows; 49% (94/192) blaCTX-M2, 45% (86/192)blaSHV, 40% (77/192) blaTEM and 7% (13/192) blaPER-2. In the rest 17% (n=40) none of thegenes included in this study was detected; in 47% (90/192) more than one gene wasdetected, resulting blaCTX-M2 + blaTEM + blaSHV as the most frequent combination in 32isolates. The presence of blaCTX-M2, as the most frequent codifying genes of BLEE is inagreement with previous reports in Paraguay and Argentina. This is the first report of thepresence of blaTEM and blaSHV circulating in Paraguay. It is of much importance the study ofothers codifying resistance genes, taking into account the emergence of other BLEE in theregion, such as blaCTX-M15, predominantly with ceftazidimase activity.