RESUMO
BACKGROUND: Measles vaccine (MV) may have non-specific beneficial effects for child health and particularly seems to prevent respiratory infections. Streptococcus pneumoniae is the leading cause of bacterial pneumonia among children worldwide, and nasopharyngeal colonization precedes infection. OBJECTIVE: We investigated whether providing early MV at 18 weeks of age reduced pneumococcal colonization and/or density up to 9 months of age. METHOD: The study was conducted in 2013-2014 in Guinea-Bissau. Pneumococcal vaccine was not part of the vaccination program. Infants aged 18 weeks were block-randomized 2:1 to early or no early MV; at age 9 months, all children were offered MV as per current policy. Nasopharyngeal swabs were taken at baseline, age 6.5 months, and age 9 months. Pneumococcal density was determined by q-PCR. Prevalence ratios of pneumococcal colonization and recent antibiotic treatment (yes/no) by age 6.5 months (PR6.5) and age 9 months (PR9) were estimated using Poisson regression with robust variance estimates while the pneumococcal geometric mean ratio (GMR6.5 and GMR9) was obtained using OLS regression. RESULTS: Analyses included 512 children; 346 early MV-children and 166 controls. At enrolment, the pneumococcal colonization prevalence was 80% (411/512). Comparing early MV-children with controls, the PR6.5 was 1.02 (95%CI = 0.94-1.10), and the PR9 was 1.04 (0.96-1.12). The GMR6.5 was 1.02 (0.55-1.89), and the GMR9 was 0.69 (0.39-1.21). Early MV-children tended to be less frequently treated with antibiotics prior to follow up (PR6.5 0.60 (0.34-1.05) and PR9 0.87 (0.50-1.53)). Antibiotic treatment was associated with considerably lower colonization rates, PR6.5 0.85 (0.71-1.01) and PR9 0.66 (0.52-0.84), as well as lower pneumococcal density, GMR6.5 0.32 (0.12-0.86) and GMR9 0.52 (0.18-1.52). CONCLUSION: Early MV at age 18 weeks had no measurable effect on pneumococcal colonization prevalence or density. Higher consumption of antibiotics among controls may have blurred an effect of early MV. TRIAL REGISTRATION: clinicaltrials.gov NCT01486355.
Assuntos
Vacina contra Sarampo/imunologia , Infecções Pneumocócicas/prevenção & controle , Antibacterianos/uso terapêutico , DNA Bacteriano/metabolismo , Feminino , Guiné-Bissau/epidemiologia , Humanos , Lactente , Masculino , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Distribuição de Poisson , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
El diagnóstico de las infecciones por Mycoplasma genitalium mediante métodos bacteriológicostradicionales resulta laborioso y poco práctico. Es por ello que los métodos moleculares basados en laamplificación del ADN se utilizan con fines diagnósticos de infecciones causadas por este microorganismo.En Cuba no existen informes de la implementación y utilización de los métodos de reacción en cadena dela polimerasa cuantitativa (qPCR) para la detección y cuantificación de este patógeno. En este trabajo seimplementó una qPCR para la detección y cuantificación de M genitalium mediante la amplificación de unfragmento del gen mgpB que codifica para la proteína adhesina celular, mediante la tecnología LightCycler® (Roche), utilizando los métodos de qPCR SYBR Green I y TaqMan. Se evaluó la especificidad y sensibilidad de dicha PCR utilizando ADN de M genitalium y de otros micoplasmas de origen humanos. Se logró la implementación de ambos métodos de qPCR con un límite de detección de 3,6 geq/μL, siendo la plataforma TaqMan la que mostró mejor eficiencia. Ambos métodos mostraron una alta especificidad para la detección de M genitalium y no se detectaron reacciones cruzadas con otros micoplasmas de origen humano. Se implementó por primera vez en Cuba una qPCR para la detección de M genitalium. El método TaqMan mostró mejor desempeño para la futura aplicación de esta metodología en muestras clínicas. El presente trabajo permitirá realizar estudios de caracterización genética y antigénica de las cepas circulantes en Cuba, útiles para conformar un inmunógeno vacunal(AU)
The diagnosis of M genitalium infections by bacteriological culture is not feasible due to slow growth and that is time-consuming. Consequently, molecular methods using DNA amplification are widely used in theinfection diagnosis procedure. There are no reports in Cuba of the implementation and use of quantitativePolymerase Chain Reaction methods for the detection and quantification of this pathogen. The aim of thisstudy was to implement a qPCR method for the detection of M genitalium by the amplification of the mgpBadhesin gene using two LightCycler® (Roche) protocols, SYBR Green I and TaqMan. The specifity andsensitivity were evaluated by using DNA of M. genitalium as well as other mycoplasms of human origin. Inboth qPCR-protocols, a limit of detection of 3.6 genome equivalents per μL template (geq/μL) was reached.The TaqMan protocol showed better efficiency than the SYBR Green assay. Both protocols showed a highspecificity for the detection of M. genitalium, without cross-reactions with other mycoplasmas of humanorigin. For the first time in Cuba, a qPCR for detection of M. genitalium was implemented. The TaqManmethod showed a better performance than the SYBR method and should be used for future applications inclinical samples. The present work will allow performing future studies of genetic and antigenic characterization of the circulating strains in Cuba, useful as vaccine immunogen(AU)
Assuntos
Mycoplasma genitalium/genética , Mycoplasma genitalium/imunologia , Infecções por Mycoplasma/diagnóstico , Reação em Cadeia da Polimerase/métodosRESUMO
El diagnóstico de las infecciones por Mycoplasma genitalium mediante métodos bacteriológicos tradicionales resulta laborioso y poco práctico. Es por ello que los métodos moleculares basados en la amplificación del ADN se utilizan con fines diagnósticos de las infecciones causadas por este microorganismo. En Cuba se han realizado pocos estudios sobre la presencia de M genitalium en el tracto urogenital. El objetivo de la presente investigaciónfue detectar M genitalium en individuos cubanos sexualmente activos mediante la implementación de métodosde PCR simple. Se implementaron dos PCR simples para la detección de fragmentos de 427 pb del gen ARNribosomal 16S y 281 pb del gen de la adhesina celular MgPa de M genitalium, que se evaluaron en muestras de exudado endocervical provenientes de 300 mujeres con sintomatología urogenital y muestras de orina de 49 hombres asintomáticos sexualmente activos. Se logró un límite de detección de la PCR del ARNr 16S de aproximadamente 5 copias de genoma por reacción, mientras que para la PCR MgPa se logró la amplificación de solo 50 copias de genoma por reacción. El 3por ciento (10/300) de los exudados endocervicales y el 24,5 por ciento (12/49) de lasmuestras de orina de hombres asintomáticos resultaron positivas mediante ambas PCR. El mayor porcentaje demuestras positivas correspondió a las muestras de orina provenientes de hombres asintomáticos, que resultó superior a lo esperado. El presente trabajo permitirá realizar estudios futuros de caracterización genética y antigénica de las cepas de Mycoplasma genitalium circulantes en Cuba, útiles para conformar un inmunógenovacunal(AU)
The diagnosis of Mycoplasma genitalium infections by bacteriological culture is not feasible due to slow growthand that is time-consuming. Consequently, molecular tools using DNA amplification are widely used in the infectiondiagnosis. There are few reports in Cuba on infections caused by this pathogen. The aim of this study was todetect M genitalium in clinical samples from sexually active individuals, by two PCR assays. PCR-methods wereimplemented and evaluated on clinical samples for the detection of M genitalium using fragments of the 16SrRNA (427 pb) and mgpB adhesion genes (281 pb) as targets. The PCR assays were used on 300 endocervicalswabs from female patients with urogenital symptoms and on 49 first-void-urine samples from asymptomaticmales. The limit of detection of 16S PCR and MgPa PCR-assays were of 5 and 50 geq/μL, respectively. For theanalyzed clinical samples, 3 percent (10/300) of female swabs and 24,5 percent (12/49) of urine samples from asymptomaticmen were positive for M. genitalium by the two PCR assays. In contrast to the anticipated results, male urinesamples had the highest positive rate. Two PCR assays for the detection of M. genitalium were implemented andsuccessfully used on clinical samples, with the unexpected finding of a high positive rate in specimens fromasymptomatic men. The current work will allow performing future studies of genetic and antigenic characterizationof the circulating Mycoplasma genitalium-strains in Cuba, useful as vaccine immunogen(AU)
