Comparative performances of seven quantitative Reverse - Transcription Polymerase Chain Reaction assays (RT-qPCR) for detecting SARS-CoV-2 infection in samples from individuals suspected of COVID-19 in São Paulo, Brazil

Introduction Brazil is the second largest country with COVID-19 positive cases worldwide. Due to the potent spread of the virus and the scarcity of kits and supplies, the Brazilian Ministry of Health has granted authorization for the use of kits available during this emergency, without an accurate evaluation of their performance. This study compared the performance and cost-effectiveness of seven molecular assays/kits available in São Paulo, Brazil, for SARS-CoV-2 diagnosis Materials and methods A total of 205 nasopharyngeal/oropharyngeal samples from suspected cases of COVID-19, were tested using the following assays: (i) GeneFinder COVID-19 plus RealAmp kit; (ii) 2019-nCoV RNA PCR-Fluorescence Probing, Da An Gene Co.; (iii) in-house RT-qPCR SARS-CoV-2 IAL; (iv) 2019-nCoV kit, IDT; (v) molecular SARS-CoV-2 (E) kit, Bio-Manguinhos; (vi) Allplex 2019-nCoV modified Assay, Seegene Inc, and (vii) Biomol one-step COVID-19 kit, IBMP. The criteria for determining a SARS-CoV-2 true positive result included the cycle threshold cut-off values, the characteristics of exponential/linear curves, the gene target diversity, and a positive result in at least two assays Results The overall sensitivity of the assays listed were GeneFinder 83.6%, Da An Gene 100.0%, IAL 90.4%, IDT 94.6%, Bio-Manguinhos 87.7%, Allplex 97.3%, and IBMP 87.7%. The minor sensitive gene target was RdRP. Although all assays had a Cohen's Kappa index ≥0.893, the best tests used multiplex assays identifying N-gene and/or E-gene targets Conclusion All assays tested accurate for diagnosis, but considering cost-effectiveness (cost, time consumption, number of samples tested, and performance), the in-house IAL assay was ideal for COVID-19 diagnosis in São Paulo, Brazil.

Evaluation of Haemophilus influenzae type b carrier status among children 10 years after the introduction of Hib vaccine in Brazil

The aim of the present study was to assess the prevalence of Haemophilus influenzaetype b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.

Ocorrência de resultados falso-negativos na reação de PCR em Tempo Real (PCR-TR) no diagnóstico da doença meningocócica / False-negative results in Real-Time PCR (RT-PCR) formeningococcal disease diagnosis

Factors responsible for false-negative results (F-N) in RT-PCR assay for detecting N. meningitidis in serumand CSF samples were investigated. As the meningococcal disease should be rapidly treated because ofits high mortality and epidemic potential, the F-N in diagnostic testing may cause treatment failuresand/or on disease restraint in community. Thus, it is crucial to learn the factors which cause F-N in RTPCRassays. The F-N were induced by inhibition, low quantity of target DNA in extracted samples, andinadequate temperature employed at PCR annealing procedure. As bacterial DNA concentration in samplesmight be highly variable, the ideal sample volume to be extracted could not be defined. As previouslyrecommended for N. meningitidis gene-grouping by RT-PCR assay, the annealing temperature at 60 °Cwas not suitable for B and W135 genogroups. Altogether, these factors induced F-N in 31 samples; therefore,30 % of N. meningitidis detected by RT-PCR were classified as non-genogrouped. The inhibitors and/orthe low amount of target DNA induced F-N on RT-PCR, independently of the specimen volume used forextracting DNA. However, adjustments on the PCR annealing temperature and amount of extracted DNAadded into the reaction might avoid the occurrence of the majority of F-N.

Caracterización de aislamientos invasivos de S. pneumoniae, H. influenzae y N. meningitidis en América Latina y el Caribe: SIREVA II, 2000-2005 / Characterization of invasive isolates of S. pneumoniae, H. influenzae, and N. meningitidis in Latin America and the Caribbean: SIREVA II, 2000-2005

OBJETIVOS: Analizar las características fenotípicas y la susceptibilidad a antibióticos de las cepas circulantes de Streptococcus pneumoniae, Haemophilus influenzae y Neisseria meningitidis en América Latina y el Caribe entre 2000 y 2005. Se evaluó la cobertura potencial de las vacunas conjugadas. MÉTODOS: Se estudió por métodos convencionales la distribución de los serotipos o serogrupos de 17 303 cepas de S. pneumoniae, 2 782 cepas de H. influenzae y 6 955 cepas de N. meningitidis aisladas de casos de neumonía, meningitis, sepsis, bacteriemias y otros procesos invasivos. Se evaluó la susceptibilidad a los antibióticos de las cepas estudiadas. Los aislamientos procedían de 453 centros centinelas de 19 países de América Latina y 4 del Caribe, como parte del proyecto SIREVA II. RESULTADOS: El serotipo 14 de S. pneumoniae fue el más frecuentemente aislado (21,1 por ciento), especialmente en niños menores de 6 años (29,1 por ciento). Las coberturas potenciales de las vacunas conjugadas antineumocócicas hepta, nona, deca y tridecavalentes fueron de 59,0 por ciento, 73,4 por ciento, 76,5 por ciento y 85,9 por ciento, respectivamente. De los aislamientos, 63,3 por ciento eran sensibles a la penicilina. El serotipo b de H. influenzae estuvo presente en 72,2 por ciento de los aislamientos en niños menores de 2 años, mientras 8,6 por ciento correspondieron a los serotipos a, c, d, e y f; 19,2 por ciento resultaron no serotipables. La proporción de cepas de H. influenzae productoras de betalactamasa en aislamientos en niños menores de 2 años fue de 16,3 por ciento. Los serogrupos de N. meningitidis más frecuentes fueron el B (69,0 por ciento) y el C (25,7 por ciento); 65,8 por ciento y 99,2 por ciento de las cepas fueron sensibles a la penicilina y a la rifampicina, respectivamente. CONCLUSIONES: Estos resultados resaltan la importancia de la vigilancia epidemiológica integral de S. pneumoniae, H. influenzae y N. meningitidis en América Latina...

OBJECTIVES: To analyze the phenotypical characteristics and the susceptibility to antibiotics of the circulating strains of Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis circulating in Latin America and the Caribbean from 2000-2005. Potential coverage by conjugate vaccines was evaluated. METHODS: Conventional methods were used to study the distribution of the serotypes or serogroups of 17 303 strains of S. pneumoniae, 2 782 strains of H. influenzae, and 6 955 strains of N. meningitidis isolated from cases of pneumonia, meningitis, sepsis, bacteriemias, and other invasive processes. The antimicrobial susceptibilities of the study strains were evaluated. The isolates came from 453 sentinel surveillance sites in 19 countries in Latin America and four in the Caribbean, as part of the SIREVA II (Network Surveillance System for the Bacterial Agents Responsible for Pneumonia and Meningitis) project. RESULTS: S. pneumoniae serotype 14 was the most frequently isolated (21.1 percent), especially in children under 6 years of age (29.1 percent). The potential coverages by hepta-, nona-, deca-, and trideca-valent antipneumonia conjugate vaccines were 59.0 percent, 73.4 percent, 76.5 percent, and 85.9 percent, respectively. Of the isolates, 63.3 percent were sensitive to penicillin. H. influenzae serotype b was present in 72.2 percent of the isolations from children under 2 years of age, whereas 8.6 percent produced serotypes a, c, d, e, and f, and 19.2 percent could not be serotyped. The rate of H. influenzae beta-lactamase-producing strains isolated from children under 2 years of age was 16.3 percent. The most frequent N. meningitidis serogroups were B (69.0 percent) and C (25.7 percent); 65.8 percent and 99.2 percent of the strains were susceptible to penicillin and rifampicin, respectively. CONCLUSIONS: These results highlight the importance of comprehensive epidemiological surveillance of S. pneumoniae, H. influenzae...

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

Use of oligonucleotide probes for meningococcal serotype characterization

No presente trabalho nos examinamos o uso potencial de sondas de oligonucleotideos para caracterizar sorotipos de Neisseria meningitidis sem o uso de anticorpos monoclonais (MAbs). A diversidade antigenica da proteina PorB forma a base do metodo de sorotipagem, todavia, o atual painel de MAbs utilizados, sub-estima em no minimo 50 por cento a diversidade desta proteina devido a falta de reagentes para as varias regiöes variáveis (VRs) da proteina PorB ou porque varias variantes das VRs nao sao reagentes com os MAbs disponíveis. Nos analisamos o uso de sondas de oligonucleotideos para caracterizar os sorotipos 10 e 19 de N. meningitidis. O gene porB da cepa prototipo do sorotipo 10 foi sequenciado e alinhado com outras 7 sequencias de diferentes sorotipos, e as individuais VRs foram entäo analisadas. Os resultados com as sondas 21U (VRI-A) e 615U (VR3-B) contra 72 cepas de N. meningitidis os MAbs 19 e 10 respectivamente. E possivel o uso de sondas para a caracterizaçäo dos sorotipos e podemos tipar 100 por cento da diversidade da VR do gene porB. Trata-se de um metodo simples, rapido, e especialmente util para a analise de um grande numero de amostras

Genetic structure of Neisseria meningitidis serogroup C epidemic strains in south Brazil

No presente estudo, nos reportamos os resultados de uma analise, baseada na sorotipagem, multilocus enzimatico (MEE) e ribotipagem de N. meningitidis sorogrupo C isoladas de paciente com doenca meningococica no Rio Grande do Sul (RS) e Santa Catarina (SC), onde o Centro de Controle Epidemiologico do Ministerio da Saude detectou um aumento de casos de doenca meningococica (DM) devido a este sorogrupo nos ultimos 2 anos (1992-1993)...

Genetic relationship of Corynebacterium diphteriae strains isolated from diphteria case and carriers by restriction fragment length polymorphism of rRNA genes

No presente estudo, nos reportamos os resultados de uma analise, baseada na ribotipagem de cepas de C. diphtheriae intermedius isoladas de uma crianca de 9 anos com difteria e seus 5 contatos. Analise quantitativa por RFLP de rRNA foi usada para determinar a relacao destas 7 cepas de C.diphtheriae fornecendo dados de interesse epidemiologico...

Monoclonal antibody to serotype 17 of Neisseria Meningitidis and-their prevalence in brazilian states

Neisseria meningitidis sao diplococos Gram negativos responsaveis por casos de doencas meningococica em todo o mundo. O potencial epidemico de N. meningitidis sorogrupos B e C e claramente mais uma funcao de seus antigenos de sorotipo que de seu polissacaride capsular. Ate recentemente soros hiperimune foram usados para detectar antigenos de sorotipo em bacterias. O advento de anticorpos monoclonais ofereceu a oportunidade de eliminar muitas das reacoes cruzadas e tem melhorado a acuracidade e reprodutibilidade da sorotipagem de meningococo. Nos produzimos um anticorpo monoclonal contra proteina de membrana externa do sorotipo 17 que ate entao tem sido detectado atraves do uso de soro policlonal. A prevalencia deste epitopo de sorotipo e baixa nas cepas brasileiras. Usando-se este anticorpo monoclonal em cepas brasileiras, nao pudemos diminuir a porcetagem de cepas sorogrupo C nao tipaveis, entretanto, houve uma diminuicao de 13 por cento em cepas sorogrupo B nao tipaveis e 25 por cento em cepas de outros sorogrupos.