ABSTRACT The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS-CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer’s instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925).

A meningite bacteriana (MB) é uma doença grave e ainda representa um sério problema de saúde pública, com altas taxas de morbidade e mortalidade. Os casos mais comuns de MB em todo o mundo, principalmente no Brasil, tem sido causados por Neisseria meningitidis, Streptococcus pneumoniae e Haemophilus influenzae tipo b. Cultura bacteriana é a técnica padrão-ouro para a confirmação de MB, mas cerca de 50% dos casos suspeitos não são confirmados por cultura, devido a problemas relacionados ao transporte inadequado e semeadura ou antibioticoterapia prévia. Métodos imunológicos apresentam baixa sensibilidade e têm possibilidade de reações cruzadas. PCR em tempo real (qPCR) é uma técnica molecular e tem sido utilizada com êxito para o diagnóstico de MB no Instituto Adolfo Lutz, em São Paulo, Brasil, desde 2007. A incorporação da qPCR na rotina de vigilância em Saúde Pública em nosso estado resultou na diminuição de 50% dos casos de MB indeterminadas. Nossos esforços estão focados na implementação da qPCR na rotina diagnóstica de MB em todo o Brasil.

Bacterial meningitis (BM) is a severe disease and still represents a serious public health problem with high rates of morbidity and mortality. The most common cases of BM around the world, mainly in Brazil, have been caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae type b. Bacterial culture is the gold-standard technique for BM confirmation, but approximately 50% of suspected cases are not culture-confirmed, due to problems related to improper transportation and seeding or previous antibiotic treatment. Immunological methods present low sensitivity and have possibility of cross-reactions. Real time PCR (qPCR) is a molecular technique and has been successful used for BM diagnosis at Instituto Adolfo Lutz in São Paulo State, Brazil, since 2007. The incorporation of qPCR in the Public Health surveillance routine in our state resulted in diminishing 50% of undetermined BM cases. Our efforts are focused on qPCR implementation in the BM diagnostic routine throughout Brazil.

We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR) assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.

No presente trabalho nós examinamos o uso potencial de sondas de oligonucleotídeos para caracterizar sorotipos de Neisseria meningitidis sem o uso de anticorpos mono-clonais (MAbs). A diversidade antigênica da proteína PorB forma a base do método de sorotipagem, todavia, o atual painel de MAbs utilizados, sub-estima em no mínimo 50% a diversidade desta proteína devido a falta de reagentes para as várias regiões variáveis (VRs) da proteína PorB ou porque várias variantes das VRs não são reagentes com os MAbs disponíveis. Nós analisamos o uso de sondas de oligonucleotídeos para caracterizar os sorotipos 10 e 19 de N. meningitidis. O gene porB da cepa protótipo do sorotipo 10 foi sequenciado e alinhado com outras 7 sequências de diferentes sorotipos, e as individuais VRs foram então analisadas. Os resultados com as sondas 21U (VR1-A) e 615U (VR3-B) contra 72 cepas de N. meningitidis confirmaram que VR1-A e VR3-B codificam epítopos para os MAbs 19 e 10 respectivamente. É possível o uso de sondas para a caracterização dos sorotipos e podemos tipar 100% da diversidade da VR do gene porB. Trata-se de um método simples, rápido, e especialmente útil para a análise de um grande número de amostras.

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 MAbs12. 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.

Epidemia de doença meningocócica por sorogrupo B tem ocorrido na Grande São Paulo desde 1988. Uma vacina cubana, baseada em proteína 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 milhões de crianças entre 3 meses a 6 anos de idade durante 1989 e 1990. A administração da vacina teve pouco ou nenhum efeito na evolução da epidemia. Para detectar mudança clonal que poderia explicar o contínuo aumento da doença depois da vacinação, foram sorotipadas 834 cepas isoladas entre 1990 e 1996 de pacientes com doença sistêmica. Cepas B:4:P1.15 isoladas desde 1977, têm sido o fenótipo mais prevalente desde 1988. Essas cepas ainda são prevalentes na região e foram responsáveis por cerca de 68% dos 834 casos por sorogrupo B nos últimos 7 anos. Foram analisados 438 (52%) dessas cepas por RFLP do rRNA gene (ribotipagem), e o perfil mais frequente foi o Rbl (68%). Concluímos que o clone B:4:P1.15 - Rbl foi o mais prevalente e responsável pelo contínuo aumento da doença meningocócica na Grande São Paulo durante os últimos 7 anos apesar da campanha de vacinação.

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988.21 A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

No presente estudo, nós reportamos os resultados de uma análise, baseada na ribotipagem de cepas de C. diphtheriae intermedius isoladas de uma criança de 9 anos com difteria e seus 5 contatos. Análise quantitativa por RFLP de rRNA foi usada para determinar a relação destas 7 cepas de C. diphtheriae fornecendo dados de interesse epidemiológico. Nós também testamos estas cepas para toxicidade in vitro usando método de difusão de Elek e in vivo usando método de cultura celular com células VERO. Os resultados de hibridização revelaram que as 5 cepas de C. diphtheriae isoladas dos contatos e uma isolada do caso (cepa isolada do nariz) tiveram padrões idênticos com as 4 enzimas de restrição usadas, ribotipo B. A distância genética deste ribotipo e o ribotipo A (cepa isolada da garganta do caso), que nós inicialmente assumimos ser responsável pela doença do paciente, foi de 0,450 mostrando pouca relação genética entre estes dois ribotipos. Nós não encontramos diferenças relativas à produção de toxina usando método de cultura celular entre as cepas. Em conclusão, o uso de RLFP de rRNA gene foi satisfatório em detectar pequenas diferenças em cepas de C. diphtheriae toxigênicas epidemiologicamente relacionadas e em fornecer informação sobre a relação genética entre elas.

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

No presente estudo, nós reportamos os resultados de uma análise, baseada na sorotipagem, multilocus enzimático (MEE) e ribotipagem de N. meningitidis sorogrupo C isoladas de paciente com doença meningocócica no Rio Grande do Sul (RS) e Santa Catarina (SC), onde o Centro de Controle Epidemiológico do Ministério da Saúde detectou um aumento do número de casos de doença meningocócica (DM) devido a este sorogrupo nos últimos 2 anos (1992-1993). Nós demonstramos que a DM devido a cepas de N. meningitidis sorogrupo C no RS e SC que ocorreram nos últimos 4 anos foi devido principalmente por um clone (ET 40), com isolados indistinguíveis por sorogrupo, sorotipo, subtipo e até por ribotipagem. Um pequeno número de casos que não foram devidos a cepas do ET 40 representaram um grupo geneticamente relacionado, que provavelmente é uma nova linhagem gerada do clone ET 40, referido como complexo ET 11 A. Nós também analisamos cepas de N. meningitidis sorogrupo C isoladas na grande São Paulo em 1976 como um grupo representativo do primeiro ano pós-epidêmico na região. A ribotipagem, bem como MEE, puderam fornecer informações sobre as características clonais das cepas isoladas no período pós-epidêmico e também no Sul do Brasil. As cepas de 1976 possuem mais similaridades com as cepas endêmicas atuais do que com as cepas epidêmicas (1992-1993) por ribotipagem, sensibilidade a sulfonamida e MEE. Em conclusão, sorotipagem com anticorpos monoclonais (C:2b:P1.3), MEE (complexo ET11 e ET11A) e ribotipagem usando a enzima de restrição ClaI, foram úteis em caracterizar estas cepas epidêmicas de N. meningitidis relacionadas com o aumento da incidência da DM em diferentes estados do sul do Brasil. É muito provável que estas cepas de N. meningitidis sorogrupo C possuam pouca ou nenhuma correlação genética com as cepas epidêmicas sorogrupo C de 1971-1975. A similaridade genética dos membros do complexo ET 11 e ET 11A foram confirmadas por ribotipagem usando-se 3 enzimas de restrição.

In the present study we report the results of an analysis, based on serotyping, multilocus enzyme electrophoresis (MEE), and ribotyping of N. meningitidis serogroup C strains isolated from patients with meningococcal disease (MD) in Rio Grande do Sul (RS) and Santa Catarina (SC) States, Brazil, as the Center of Epidemiology Control of Ministry of Health detected an increasing of MD cases due to this serogroup in the last two years (1992-1993). We have demonstrated that the MD due to N.meningitidis serogroup C strains in RS and SC States occurring in the last 4 years were caused mainly by one clone of strains (ET 40), with isolates indistinguishable by serogroup, serotype, subtype and even by ribotyping. One small number of cases that were not due to an ET 40 strains, represent closely related clones that probably are new lineages generated from the ET 40 clone referred as ET 11A complex. We have also analyzed N.meningitidis serogroup C strains isolated in the greater São Paulo in 1976 as representative of the first post epidemic year in that region. The ribotyping method, as well as MEE, could provide useful information about the clonal characteristics of those isolates and also of strains isolated in south Brazil. The strains from 1976 have more similarity with the actual endemic than epidemic strains, by the ribotyping, sulfonamide sensitivity, and MEE results. In conclusion, serotyping with monoclonal antibodies (C:2b:P1.3), MEE (ET 11 and ET 11A complex), and ribotyping by using ClaI restriction enzyme (Rb2), were useful to characterize these epidemic strains of N.meningitidis related to the increased incidence of MD in different States of south Brazil. It is mostly probable that these N.meningitidis serogroup C strains have poor or no genetic corelation with 1971-1975 epidemic serogroup C strains. The genetic similarity of members of the ET 11 and ET 11A complex were confirmed by the ribotyping method by using three restriction endonucleases.

Neisseria meningitidis são diplococos Gram negativos responsáveis por casos de doença meningocócica em todo o mundo. O potencial epidêmico de N. meningitidis sorogrupos B e C é claramente mais uma função de seus antígenos de sorotipo que de seu polissacáride capsular. Até recentemente soros hiperimune foram usados para detectar antígenos de sorotipo em bactérias. O advento de anticorpos monoclonais ofereceu a oportunidade de eliminar muitas das reações cruzadas e têm melhorado a acuracidade e reprodutibilidade da sorotipagem de meningococo. Nós produzimos um anticorpo monoclonal contra proteína de membrana externa do sorotipo 17 que até então tem sido detectado através do uso de soro policlonal. A prevalência deste epítopo de sorotipo é baixa nas cepas brasileiras. Usando-se este anticorpo monoclonal em cepas brasileiras, não pudemos diminuir a porcentagem de cepas sorogrupo C não tipáveis, entretanto, houve uma diminuição de 13% em cepas sorogrupo B não tipáveis e 25% em cepas de outros sorogrupos.

Neisseria meningitidis are gram-negative diplococci responsible for cases of meningococcal disease all over the world. The epidemic potential of N. meningitidis serogroup B and C is clearly a function of their serotype antigens more than of their capsular polysaccharides. Until recently, hiperimmune sera were used to detect typing antigens on the bacteria. The advent of monoclonal antibodies (MAbs) offered the opportunity to eliminate many of the cross-reactions and have improved the accuracy and reproducibility of meningococcal serotyping. We have produced a MAb to the outer membrane protein of the already existent serotype 17 that have been detected by the use of hiperimmune rabbit sera. The prevalence of this serotype epitope is low in the Brazilian strains. By using the MAb 17 we could not decrease the percentage of nontypeable serogroup C strains. However, there were a decreasing in nontypeable strains to 13% into serogroup B strains and to 25% into the other serogroups.