Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples

Abstract Background Several in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans. Methods A total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae. Results All used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains. Conclusions The in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.

Meningite por Haemophilus influenzae tipo f / Meningitis due to Haemophilus influenzae type f

Com o declínio da taxa de infecções causadas pelo Haemophilus influenzae tipo b após a ampla introdução da vacina, sorotipos não-b devem ser considerados agentes patogênicos potenciais em crianças menores de 5 anos com doença invasiva. Relatamos um caso de meningite por Haemophilus influenzae tipo f em um lactente imunocompetente de 1 ano. O agente foi identificado em líquido cefalorraquidiano e hemocultura. A sorotipagem foi realizada por testes com soros policlonais e confirmada por reação em cadeia de polimerase. Todas as cepas de Haemophilus influenzae associadas à doença invasiva deveriam ser sorotipadas e notificadas, a fim de possibilitar uma análise adequada das mudanças e tendências na distribuição de sorotipos desta doença.

With the decline in the rate of infections caused by Haemophilus influenzae serotype b since the widespread vaccination, non-b serotypes should be considered as potential pathogenic agents in children with invasive disease younger than 5 years old. We report the case of an immunocompetent 1-year-old boy with Haemophilus influenzae type f meningitis. The agent was identified in cerebrospinal fluid and blood cultures. Serotyping was performed by tests using polyclonal sera and confirmed by polymerase chain reaction. All Haemophilus influenzae isolates associated with invasive disease should be serotyped and notified as a way to evaluate the changes and trends in serotype distribution of this disease.

Impact of Haemophilus influenzae type b conjugate vaccine on bacterial meningitis in the Dominican Republic / Impacto de la vacuna conjugada contra Haemophilus influenzae tipo b sobre la meningitis bacteriana en la República Dominicana

OBJECTIVES: Widespread use of Haemophilus influenzae type b (Hib) vaccines has dramatically reduced the burden of Hib disease throughout the Americas. Few studies have evaluated the impact of Hib vaccination on non-culture-confirmed disease. This study analyzed trends in probable bacterial meningitis before and after the introduction of Hib vaccine in the Dominican Republic and estimated vaccine effectiveness against Hib meningitis. METHODS: Meningitis cases among children < 5 years of age were identified from admission records of the main pediatric hospital in Santo Domingo during 1998-2004. Laboratory criteria were used to classify meningitis cases with probable bacterial etiology; confirmed cases had positive bacterial culture or antigen detection in cerebrospinal fluid. Cumulative incidence rates of confirmed and probable bacterial meningitis were calculated for children living in the National District. Confirmed cases of Hib meningitis were enrolled in a case-control study with age- and neighborhood-matched control children to calculate vaccine effectiveness. RESULTS: Before vaccine introduction, annual rates of meningitis with probable bacterial etiology were 49 cases per 100 000 children < 5 years old; Hib accounted for 60 percent of confirmed bacterial cases. During 2002-2004, after vaccine introduction, annual rates of probable bacterial meningitis were 65 percent lower at 16 cases per 100 000, and Hib accounted for 26 percent of confirmed cases. Rates of Hib meningitis and probable bacterial meningitis with no determined etiology declined by 13 and 17 cases per 100 000, respectively. CONCLUSIONS: Introduction of Hib vaccine substantially reduced the incidence of confirmed and probable bacterial meningitis in the Dominican Republic. The estimated impact of Hib vaccination was twice as great when non-culture-confirmed disease was included.

OBJETIVOS: El uso generalizado de la vacuna contra Haemophilus influenzae tipo b (Hib) ha permitido reducir radicalmente la carga de enfermedad por Hib en las Américas. Pocos estudios han evaluado el impacto de la vacunación contra Hib sobre los casos no confirmados mediante cultivo. En este estudio se analizaron las tendencias en el número de casos probables de meningitis bacteriana antes y después de la introducción de la vacuna contra Hib en la República Dominicana y se estimó la eficacia de la vacuna contra la meningitis. MÉTODOS: Se identificaron los casos de meningitis en niños menores de 5 años a partir de los registros de ingreso del principal hospital pediátrico de Santo Domingo entre 1998 y 2004. Los casos de meningitis con probable etiología bacteriana se clasificaron según criterios de laboratorio; los casos confirmados contaban con cultivo bacteriano positivo o detección de antígenos específicos en el líquido cefalorraquídeo. Se calcularon las tasas de incidencia acumulada de casos confirmados y probables de meningitis en los niños que vivían en el Distrito Nacional. Los casos confirmados de meningitis por Hib se incorporaron a un estudio de casos y controles -pareados según la edad y el barrio de residencia- para calcular la eficacia de la vacuna. RESULTADOS: Antes de la introducción de la vacuna, la tasa anual de meningitis de posible etiología bacteriana era de 49 casos por 100 000 niños menores de 5 años; de los casos confirmados de origen bacteriano, 60 por ciento fue por Hib. En el período 2002-2004, después de la introducción de la vacuna, la tasa anual de meningitis de posible etiología bacteriana fue de 16 casos por 100 000, es decir 65 por ciento más baja, y 26 por ciento de los casos confirmados correspondieron a Hib. Las tasas de meningitis por Hib y de posible origen bacteriano de etiología desconocida se redujeron en 13 y 17 casos por 100 000, respectivamente. CONCLUSIONES: La introducción de la vacuna contra Hib redujo sustancialmente la incidencia de casos de meningitis confirmados y probables en la República Dominicana. El impacto estimado de la vacunación contra Hib fue dos veces mayor cuando se consideraron los casos no confirmados mediante cultivo.

Diseño y evaluación de una reacción de polimerasa en cadena (RPC) múltiple, para la identificación de bacterias causantes de meningitis aguda en líquido cefalorraquídeo de niños chilenos / Multiplex PCR assay in spinal fluid to identify simultaneously bacterial pathogens associated to acute bacterial meningitis in Chilean children

Background: Acute bacterial meningitis (ABM) is a serious disease that needs rapid diagnosis for an accurate treatment. The most important etiological agents are: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae type b. Overall pathogen detection rate in patients with ABM in Chile is 83 percent. Aim: To evaluate a Polymerase Chain Reaction (PCR) protocol for simultaneous detection of several pathogens in patients with ABM. Material and methods: We designed and evaluated a multiplex PCR protocol for simultaneous specific genes identifications of S pneumoniae (¡ytA and ply genes), N meningitidis (ctrA, crgA) and H influenzae (bexA) in cerebrospinal fluid (CSF) samples from pediatric patients with suspected diagnosis of ABM. Sensitivity, specificity and minimum detection levels of DNA were determined. Amplifications ofrDNA 16S gene was done to confirm extraction of bacterial DNA. Results: Ninety nine CSF samples were studied, 90 from children with fever and negative CSF culture, and 9 from ABM and positive culture patients. The PCR protocol had a sensitivity of 89 percent, specificity of 100 percent, positive predictive value 100 percent and negative predictive value 99 percent. Conclusions: We observed a high concordance (89 percent) between bacteriological cultures and the PCR protocol results. This diagnostic tool could increase identification of agents in specific settings such as patients previously treated with antibiotics.

Vacinação contra Haemophilus influenzae tipo b: proteção a longo prazo / Haemophilus influenzae type b vaccination: long term protection

OBJETIVO: Identificar as evidências sobre o impacto da vacina conjugada para Haemophilus influenzae tipo b (Hib) na epidemiologia da doença invasiva por Hib. FONTE DOS DADOS: Pesquisa nas bases de dados do MEDLINE, LILACS, publicações técnicas de organizações internacionais, diretrizes nacionais e internacionais, nos últimos 15 anos (1991-2005), utilizando os seguintes unitermos: Haemophilus influenzae type b, immunization, impact, effectiveness. Foram incluídas as publicações que apresentaram informação para atender o objetivo deste artigo. Artigos publicados em período anterior ao da pesquisa e citados em referências dos artigos incluídos foram analisados quanto à apresentação de informação de interesse. SíNTESE DOS DADOS: A introdução da vacina conjugada para Hib produziu grande declínio na incidência de casos de doença invasiva por Hib nos diversos países em que seu uso foi incorporado à rotina de vacinação das crianças. No entanto, o ressurgimento de casos com doença invasiva por Hib tem mobilizado vários investigadores na busca das possíveis explicações para esses eventos, bem como a identificação das medidas a serem implementadas para evitar o reaparecimento da doença. CONCLUSÕES: O uso da vacina conjugada para Hib em escala populacional tem sido extremamente efetivo. No entanto, mudanças no esquema vacinal poderão ser necessárias para a manutenção do controle da doença invasiva por Hib, frente ao atual cenário epidemiológico das infecções pelo Hib.

Aspectos dos líquidos cefalorraquidianos nas meningites bacterianas / The cerebrospinal fluid in bacterial meningitis

Samples of 1815 cerebrospinal fluid (CSF) were studied in a meningitis outbreak during 1989 in S ao Paulo, Brazil. Neisseria meningitis 56 per cent with 44 per cent type B, Haemophilus influenzae 17 per cent , from which 72 per cent in children (days to 3-year-old) and Streptococcus pneumoniae 14 per cent from which 60 per cent in children (day to 1-year-old) of 443 (24 per cent ) of all strains. Cytochemistry study showed: purulent or turbidity aspects in 70 to 79 per cent positive bacterioscopy or culture of CSF; white cells count > 500/mm3; glucose < 45 mg/dl; protein > 90 mg/dl in 90 per cent of all patients. We concluded that: CSF prognostic factors: (aspect and cytochemistry) were correlated with bacterial meningitis. Bacterioscopy and positive cultures were correlated to NM, SP and HI isolation from these patients (Goodman Test)

Multiply resistant Haemophilus influenzae type b causing meningitis.

The isolation from patients of meningitis, of two multidrug resistant strains of H. influenzae is of relevance to the empirical treatment of meningitis patients. The isolates produced beta lactamase and had higher MICs as compared to the four H. influenzae strains sensitive to the drugs commonly used for the treatment of meningitis. The cephalosporins and gentamicin were found to be effective antibiotic agents. The occurrence of resistance to ampicillin, chloramphenicol, cloxacillin, cotrimoxazole, tetracycline, penicillin and erythromycin is of concern.