Wilma Basualdo

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

Summary In a multicenter, prospective case-control study involving 1758 children aged <5 years in developing and emerging countries, the main microorganisms associated with pneumonia were Streptococcus pneumoniae, human metapneumovirus, rhinovirus, and respiratory syncytial virus.

Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study

Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.

Phylogeny-based classification of human rhinoviruses detected in hospitalized children with acute lower respiratory infection in Paraguay, 2010–2011

Human rhinovirus (HRV), a single‐stranded, positive‐sense RNA virus, is associated with mild upper respiratory tract infections in children. The aim of this study was to carry out a molecular characterization and phylogeny‐based classification of the circulating genotypes of HRV in hospitalized children with clinical manifestations of acute lower respiratory infection in Paraguay. Nasopharyngeal aspirates were collected from 101 children under 5 years of age, hospitalized with symptoms of acute lower respiratory infection, between May 2010 and December 2011, at the largest public pediatric hospital in the Central Department of Paraguay. Detection was performed by a real‐time polymerase chain reaction, followed by conventional amplification of the VP4/VP2 genomic region, sequencing, and phylogenetic analysis. Rhinovirus was detected in 33.7% of the samples. Amplification of 18 samples showed the presence of all three species (HRV‐A, ‐B, and ‐C). Different genotypes were found for each species: 11 for HRV‐A (‐9, ‐12, ‐22, ‐30, ‐36, ‐43, ‐59, ‐61, ‐68, ‐88, and ‐89), one for HRV‐B (‐4), and four for HRV‐C (‐C2, ‐C3, ‐C6, and ‐C9). In South America, information about HRV diversity is scarce. This is the first report on HRV genotype diversity in South America. J. Med. Virol. 85:1645–1651, 2013.

Severity of pneumonia in under 5-year-old children from developing countries: A multicenter, prospective, observational study

Abstract. 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.

[Community-acquired Staphylococcus aureus isolated from Paraguayan children: clinical, phenotypic and genotypic characterization].

INTRODUCTION The prevalence of Staphylococcus aureus in the community has increased, being the pediatric population the most affected. This fact highlights the need for epidemiological surveillance. AIM To characterize clinical, phenotypic and genotypic isolates of S. aureus childrens samples with community-acquired infections, collected in hospitals of Asuncion and the Central Department, between November 2009 and December 2010. MATERIALS AND METHODS Descriptive and transverse analysis with analytical component. Clinical data collected by medical records, antibiotic susceptibility according to CLSI criteria and detection of mecA (encoding methicillin resistance) and luk-PV genes (encoding Panton Valentine leucocidin) by PCR using specific oligonucleotides. RESULTS 123 isolates of S. aureus, 76% came from skin and soft tissue infections and 20% from sepsis. 18.7% (n = 23) were resistant to methicillin (MRSA). The presence of the mecA gene, a variant there and the PVL was detected in 12.2 and 48 isolates respectively. 43% of MRSA (n = 10) was carrying luk-PV. The clinical and demographic differences between patients infected with MRSA or MSSA were not statistically significant. DISCUSSION This study constitutes the first phenotypic and genotypic characterization of S. aureus associated with pediatric patients in Paraguay.

Estandarización del análisis multi-locus de número variable de repeticiones en tándem para el estudio de Staphylococcus aureus resistentes a meticilina aislados de la comunidad en Paraguay

Staphylococcus aureus is a pathogen that can produce several infections with a wide range of severity and it has the hability to adapt to different tissues. The epidemiology is complex, by circulation of many differents clones worlwide, so the analysis for its identification requires reproducible and high discriminatory powder molecular methods. The aim of this study was to standardize the molecular technique multiple-locus variable number of tandem repeat analysis (MLVA) for the genetic variability analysis of S. aureus isolates, previously characterized by pulsed field gel electrophoresis (PFGE). The MLVA was made by PCR amplification of seven VNTR locus (clfA, clfB, sdrC, sdrD, sdrE, sspA y spA). A high level of reproducibility has reached in the study. The use of isolates previously typified by multi-locus secuencing typing (MLST), PFGE, locus spa and cassette SCCmec, allowed to validate the MLVA clusters comparatively. The isolates that were clustered by MLVA as the same isolate, showed the same results by other molecular techniques, and the MLVA can distinguish isolates with identical PFGE patterns. This technique have all criteria of a usefull molecular typification technique.

Presence of ON1 and BA HRSV-genotypes in Paraguay, 2010-2013.

Human respiratory syncytial virus (HRSV), a member of the aramyxoviridae family, is a nonsegmented, single-stranded RNA irus [1]. HRSV is the main viral cause of acute and severe lower resiratory tract infections in children worldwide. It has a single type, ith two subgroups (A and B) that are further divided into several enotypes based on the variability of the G-protein gene. For HRSV , 11 genotypes have been described, which are GA1–GA7, NA1, A2, SAA1, and ON1 [2]. HRSV B has 17 genotypes, designated as B1–GB4, SAB1–SAB3, and BA1–BA10 [3]. In Paraguay, we have previously found that children with severe cute respiratory infection required longer time of hospitalization 5–15 days) when HRSV was present alone or in co-infection with denovirus, rhinovirus, or coronavirus [4]. The lack of genetic inforation about these Paraguayan HRSV isolates led us to carry out molecular characterization of the circulating genotypes in the entral Department. A total of 36HRSV were detected out of 280 nasopharyngeal spirates, collected from children under 5 years of age with sympoms of severe acute respiratory infection, between May 2010 nd December 2013, admitted to the Hospital General Pediátrico iños de Acosta Ñu. HRSV detection was performed by real-time CR (Fast-Track Respiratory Pathogens Plus Kit, FTD, Luxembourg). enetic amplification of the attachment G-protein gene was caried out by Polymerase Chain Reaction. Phylogenetic relationships ere reconstructed by the neighbor-joining method with Kimura’s wo-parameters as the model of nucleotide substitution, as incororated in MEGA v5 [5]. Partial nucleotide sequences obtained in his study were deposited in the GenBank database (accession numers: KM508816–KM508827). Sequencing/phylogenetic analyses of 12 samples showed the resence of HRSV subgroups A (11 samples) and B (1 sample) Figs. 1 and 2). Concerning HRSV subgroup A, genotype NA1 cirulated between 2010 and 2012 (three seasons). In 2012 both enotypes NA1 and GA5 co-circulated. In 2013 the novel genotype N1 circulated (Fig. 1); this genotype has a 72-nt duplication of