Genetic diversity of Pneumocystis jirovecii in colonized Cuban infants and toddlers.

Pneumocystis jirovecii is a leading cause of opportunistic infections among immunocompromised patients. The aim of this study was to determine the genetic diversity of P. jirovecii from colonized Cuban infants and toddlers by analysis of four genetic loci: mitochondrial large subunit (mtLSU) rRNA, cytochrome b (CYB), superoxide dismutase (SOD) and ß-tubulin (ß-tub). We determined the multilocus profiles based on concatenated genotype data (multilocus genotype; MLG) and nucleotide sequences (multilocus sequence analysis; MLSA) respectively, calculated the discriminatory power of each analysis, and investigated possible associations with demographic and clinical data. Sixteen of 51 PCR-positive nasopharyngeal swab specimens (years 2010-2013) with high P. jirovecii load were selected for downstream analysis. In mixed allelic profiles all genotypes/nucleotide sequence patterns were considered separately. All samples could be genotyped based on mtLSU, CYB and ß-tub locus. However, the SOD locus could be successfully amplified in only 7/16 (44%) specimens. Eight different P. jirovecii MLGs were identified among the 16 cases and eight samples presented identical MLG (MLG 1). Seventeen MLSA profiles were distinguished. No statistical association between genotypes or MLGs and demographic or clinical data could be identified. For MLSA the higher discriminatory power (S=0.976) was observed. The combination of mtLSU, CYB and ß-tub loci proved to be useful for molecular epidemiology studies of P. jirovecii. A total of 17 different MLSA profiles observed in 16 specimens indicated high genetic variability of P. jirovecii circulating in colonized Cuban infants and toddlers.

Prevalence and genotype distribution of Pneumocystis jirovecii in Cuban infants and toddlers with whooping cough.

This study describes the prevalence and genotype distribution of Pneumocystis jirovecii obtained from nasopharyngeal (NP) swabs from immunocompetent Cuban infants and toddlers with whooping cough (WC). A total of 163 NP swabs from 163 young Cuban children with WC who were admitted to the respiratory care units at two pediatric centers were studied. The prevalence of the organism was determined by a quantitative PCR (qPCR) assay targeting the P. jirovecii mitochondrial large subunit (mtLSU) rRNA gene. Genotypes were identified by direct sequencing of mtLSU ribosomal DNA (rDNA) and restriction fragment length polymorphism (RFLP) analysis of the dihydropteroate synthase (DHPS) gene amplicons. qPCR detected P. jirovecii DNA in 48/163 (29.4%) samples. mtLSU rDNA sequence analysis revealed the presence of three different genotypes in the population. Genotype 2 was most common (48%), followed in prevalence by genotypes 1 (23%) and 3 (19%); mixed-genotype infections were seen in 10% of the cases. RFLP analysis of DHPS PCR products revealed four genotypes, 18% of which were associated with resistance to sulfa drugs. Only contact with coughers (prevalence ratio [PR], 3.51 [95% confidence interval {CI}, 1.79 to 6.87]; P = 0.000) and exposure to tobacco smoke (PR, 1.82 [95% CI, 1.14 to 2.92]; P = 0.009) were statistically associated with being colonized by P. jirovecii. The prevalence of P. jirovecii in infants and toddlers with WC and the genotyping results provide evidence that this population represents a potential reservoir and transmission source of P. jirovecii.

Haemophilus influenzae: Caracterización de aislamientos recuperados de enfermedades invasivas en Cuba durante el período 2008-2011 / Haemophilus influenzae: Characterization of the isolates recovered from invasive diseases in Cuba from 2008-2011

Con el objetivo de caracterizar los aislamientos de Haemophilus influenzae, responsables de enfermedades invasivas en Cuba, en la etapa posterior a la vacunación se estudió el total de los recuperados durante el período 2008-2011, remitidos al Instituto Pedro Kourí desde diferentes provincias del país. La confirmación de especie y determinación de serotipos se realizó atendiendo al requerimiento de los factores V y X y empleando el método de aglutinación en lámina, respectivamente. Se definieron los biotipos a través de las pruebas de indol, urea y ornitina; se determinó la concentración mínima inhibitoria (CMI) mediante la prueba del elipsómetro para la ampicilina, la ceftriaxona, el cloranfenicol y la rifampicina. Para 23 aislamientos se corroboró la identificación como H influenzae : 21 fueron serotipables y 2 no tipables (HNT). El 90,4por ciento de los serotipables fueron serotipo b y se detectaron dos serotipos a. Nueve aislamientos de H influenzae b pertenecieron al biotipo I y los aislamientos, serotipo a y HNT, al biotipo II. En una cepa se demostró producción de la enzima ß-lactamasa y resistencia para la ampicilina y el cloranfenicol, con valores de CMI= 8 y 12 µg/mL, respectivamente. Se puso en evidencia que a pesar de la disminución de la incidencia de la enfermedad invasiva por Hib, este serotipo aún constituye el más frecuente y se alerta sobre la necesidad de una vigilancia sostenida que permita detectar fallos vacunales. La susceptibilidad antimicrobiana demostrada para este período pudiera considerarse como un beneficio adicional de la introducción de la vacunación en Cuba(AU)

In order to characterize Haemophilus influenzae isolates responsible for invasive disease (ID) in Cuba during the post-vaccination period, we studied the total isolates recovered from 2008 to 2011 and submitted to the Institute Pedro Kouri from different provinces. Specie confirmation and serotype determination were performed by considering the factors V and X requirements and using the agglutination plate method, respectively. Biotypes were defined by testing indol, urea and ornithine. The minimum inhibitory concentration (MIC) was assessed by ellipsometer test for ampicillin, ceftriaxone, chloramphenicol and rifampicin. Twenty three isolates were identified as H. influenzae; of them, 21 were serotyped and two non-typable (HNT). Near ninety percent of the serotyped isolates were classified as serotype b and only 2 as serotype a. Nine isolates of H. influenzae b belonged to biotype I while serotype a and HNT isolates corresponded to biotype II. In one strain it was demonstrated production of ß -lactamase enzyme as well as resistance to ampicillin (MIC=8 µg/mL) and chloramphenicol (MIC=12 µg/mL). It was evident that despite the decline in the incidence of Hib ID, this serotype is still the most common in Cuba, and highlights the need for ongoing monitoring to detect vaccine failures. The antimicrobial susceptibility observed during this period might be considered as an additional benefit of vaccination(AU)

Phase I clinical evaluation of a synthetic oligosaccharide-protein conjugate vaccine against Haemophilus influenzae type b in human adult volunteers.

Since 1989, we have been involved in the development of a vaccine against Haemophilus influenzae type b. The new vaccine is based on the conjugation of synthetic oligosaccharides to tetanus toxoid. Our main goals have been (i) to verify the feasibility of using the synthetic antigen and (ii) to search for new production alternatives for this important infant vaccine. Overall, eight trials have already been conducted with adults, children (4 to 5 years old), and infants. We have described herein the details from the first two phase I clinical trials conducted with human adult volunteers under double blind, randomized conditions. The participants each received a single intramuscular injection to evaluate safety and initial immunogenicity. We have found an excellent safety profile and an antibody response similar to the one observed for the control vaccine.

Phase I Clinical Evaluation of a Synthetic Oligosaccharide-Protein Conjugate Vaccine against Haemophilus influenzae Type b in Human Adult Volunteers

Since 1989, we have been involved in the development of a vaccine against Haemophilus influenzae type b. The new vaccine is based on the conjugation of synthetic oligosaccharides to tetanus toxoid. Our main goals have been (i) to verify the feasibility of using the synthetic antigen and (ii) to search for new production alternatives for this important infant vaccine. Overall, eight trials have already been conducted with adults, children (4 to 5 years old), and infants. We have described herein the details from the first two phase I clinical trials conducted with human adult volunteers under double blind, randomized conditions. The participants each received a single intramuscular injection to evaluate safety and initial immunogenicity. We have found an excellent safety profile and an antibody response similar to the one observed for the control vaccine(AU)

Desde 1989, hemos participado en el desarrollo de una vacuna contra el Haemophilus influenzae tipo b. La nueva vacuna se basa en la conjugación de oligosacáridos sintéticos a toxoide tetánico. Nuestros principales objetivos han sido (i) para verificar la viabilidad de la utilización de la síntesis de antígenos y (ii) búsqueda de nuevas alternativas de producción para este importante vacuna infantil. En total, ocho ensayos ya se han realizado con adultos, niños (de 4 a 5 años), y los lactantes. Hemos descrito en este documento los detalles de los dos primeros ensayos clínicos de fase I realizado con voluntarios adultos humanos en virtud de doble ciego, randomizado condiciones. Los participantes recibieron una única inyección intramuscular para evaluar la seguridad y la inmunogenicidad inicial. Hemos encontrado un excelente perfil de seguridad y una respuesta de anticuerpos similar a la observada para el control de vacunas

Nasopharyngeal colonization by Moraxella catarrhalis and study of antimicrobial susceptibility in healthy children from Cuban day-care centers.

The prevalence of nasopharyngeal carriage of Moraxella catarrhalis was determined for the first time in Cuba. One-hundred fifty healthy children attending three day-care centers in the municipality of Marianao, Havana City were studied. The percentage of recovering bacteria in nasal and pharyngeal swabs was compared. Antimicrobial susceptibilities to ampicillin, trimethoprim-sulfamethoxazole, tetracycline, cefotaxime, ceftriaxone, chloramphenicol, erythromycin, azithromycin, amoxicillin/clavulanate, and norfloxacin were determined by the disk diffusion method according to recommendations of the National Committee for Clinical Laboratory Standards. Sixty-five percent of the children studied carried Moraxella catarrhalis. The nasal cavity was the main isolation site for this organism (81% of positive cultures). Most strains were highly susceptible to the antimicrobial agents tested, except to ampicillin (53.6% resistance). This study provides evidence of the need for continued surveillance of antimicrobial susceptibility of Moraxella catarrhali, in order to determine optimal empiric therapy for community-acquired respiratory tract infections produced by this pathogen.

A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b.

Glycoconjugate vaccines provide effective prophylaxis against bacterial infections. To date, however, no commercial vaccine has been available in which the key carbohydrate antigens are produced synthetically. We describe the large-scale synthesis, pharmaceutical development, and clinical evaluation of a conjugate vaccine composed of a synthetic capsular polysaccharide antigen of Haemophilus influenzae type b (Hib). The vaccine was evaluated in clinical trials in Cuba and showed long-term protective antibody titers that compared favorably to licensed products prepared with the Hib polysaccharide extracted from bacteria. This demonstrates that access to synthetic complex carbohydrate-based vaccines is feasible and provides a basis for further development of similar approaches for other human pathogens.

Portadores nasales de Staphylococcus aureus resistente a la meticilina entre niños cubanos que asisten a círculos infantiles / Nasal carriers of methicillin-resistant Staphylococcus aureus among cuban children attending day-care centers

FUNDAMENTO. Recientes pero escasos informes de cepas de Staphylococcuss aureus resistentes a la meticilina (SARM) entre niños no expuestos a los factores de riesgos asociados con su adquisición, nos indujeron a investigar su circulación en la comunidad. PACIENTES Y MÉTODOS. Durante los meses de septiembre y octubre de 1997 se tomaron exudados nasales y faríngeos a 358 niños menores de 5 años, atendidos en tres círculos infantiles del municipio Marianao de la Ciudad de La Habana, Cuba. Las cepas de S. aureus aisladas fueron caracterizadas por susceptibilidad a antimicrobianos utilizando el método de Kirby-Bauer. Se confirmó la resistencia a la meticilina a través del método " Oxacillin Salt-Agar Screening-Plate" recomendado por el Comité Nacional de Procedimientos Estándar del Laboratorio Clínico. RESULTADOS. El 18,7 por ciento de los niños portaban cepas de S. aureus en el tracto respiratorio superior. En el 2,2 por ciento eran cepas de SARM. Los índices más altos de resistencia se revelaron para la eritromicina (50,7 por ciento) y tetraciclina (29,9 por ciento). Todas las cepas fueron susceptibles a la ciprofloxacina. CONCLUSIONES. Nuestros resultados proporcionan evidencias sobre el aislamiento de cepas de SARM entre niños sanos atendidos en círculos infantiles, e indican la posibilidad de que éstas se establezcan y diseminen de forma acelerada en la comunidad (AU)