The use of oligonucleotide probes for meningococcal serotype characterization.

In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs). Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs) are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A) and 615U (VR3-B) used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

Meningococcal disease caused by Neisseria meningitidis serogroup B serotype 4 in Säo Paulo, Brazil, 1990 to 1996

Epidemia de doença meningococica por sorogrupo B tem ocorrido na Grande Säo Paulo desde 1988. Uma vacina cubana, baseada em proteina de membrana externa de uma cepa de Neisseria meningitidis sorogrupo B : sorotipo 4: sorosubtipo P1.15 (B:4:P1.15), foi administrada em cerca de 2,4 milhöes de crianças entre 3 meses a 6 anos de idade durante 1989 e 1990. A administraçäo da vacina teve pouco ou nenhum efeito na evoluçäo da epidemia. Para detectar mudança clonal que poderia explicar o continuo aumento da doença depois da vacinaçäo, foram sorotipadas 834 cepas isoladas entre 1990 e 1996 de pacientes com doença sistemica. Cepas B:4:P1.15 isoladas desde 1977, tem sido o fenotipo mais prevalente desde 1988. Essas cepas ainda säo prevalentes na regiäo e foram responsaveis por cerca de 68 por cento dos 834 casos por sorogrupo B nos ultimos 7 anos. Foram analisados 438 (52 por cento) dessas cepas por RFLP do rRNA gene (ribotipagem), e o perfil mais frequente foi o Rbl (68 por cento). Concluimos que o clone B:4:P1.15 - Rbl foi o mais prevalente e responsável pelo continuo aumento da doenca meningococica na Grande Säo Paulo durante os ultimos 7 anos apesar da campanha de vacinaçäo

A rapid and sensitive PCR strategy employed for amplification and sequencing of porA from a single colony-forming unit of Neisseria meningitidis.

The predicted amino acid sequence was determined for the class-1 outer membrane protein, PorA, from a B:15:P1.7,3 strain of Neisseria meningitidis that is currently causing an epidemic of meningitis in Northern Chile. The P1.7,3 PorA showed a unique sequence in the exposed loop 4 of the putative porin structure that is different from all the reported PorA sequences. Based on the nucleotide (nt) sequence of the P1.7,3 porA, we designed two sets of PCR (polymerase chain reaction) primers that specifically amplified porA from any N. meningitidis strain, and a third set of primers that amplified porA only from the P1.7,3 strain. Using these primers, we developed a sensitive double hot-start nested PCR (HNPCR) strategy that could amplify porA and generate nt sequence from as low as a single colony-forming unit. This strategy consisted of three phases of PCR. The first two phases were designed to generate amplified target DNA that could be directly visualized by ethidium bromide staining starting from one to two molecules of Neisseria genome. The third phase was designed to generate a sequence of several hundred nt directly from the amplified DNA. A number of culture-negative cerebrospinal fluid samples from individuals suspected of meningitis during a vaccine trial were analyzed by this strategy to obtain more accurate information on the actual number of cases that occurred in the study and the non-study populations. The basic HNPCR strategy described here could be applied to amplify and sequence target DNAs from any low-copy-number biological sample.