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.

COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols

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 SARS­CoV-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).

Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting

Abstract Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting.

Comparação de testes laboratoriais para o diagnóstico de infecção por vírus linfotrópicos de células T humanas do tipo 1 (HTLV-1)e tipo 2 (HTLV-2) em pacientes infectados por HIV-1 / Comparison of laboratorial tests for the diagnosis of human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) in HIV-1 infected patients

O presente estudo pesquisou o melhor algoritmo de testes laboratoriais para efetuar o diagnóstico de infecção por vírus linfotrópicos de células T humanas dos tipos 1 (HTLV-1) e 2 (HTLV-2) em pacientes HIV-1 positivos. Amostras de sangue de 1.608 pacientes do CRT DST/Aids-SP foram analisadas quanto à presença de anticorpos específicos usando-se dois ensaios de triagem (EIA Murex HTLV-I+II e Gold ELISA HTLV-I/II), dois confirmatórios [HTLV Blot 2.4 (Western Blot – WB) e INNO-LIA HTLV I/II (Line ImmunoAssay - LIA)] e um molecular (PCR em tempo real pol). Na triagem foram detectados 51(Murex) e 49 (Gold ELISA) soros reagentes. Pelo WB, 23 soros confirmaram infecção por HTLV-1, 12 HTLV-2, seis HTLV e nove apresentaram perfis indeterminados. O LIA detectou 24 soros HTLV-1 positivos, 20 HTLV-2 e seis HTLV. A PCR evidenciou segmento pol de HTLV-1 em 18 e HTLV-2 em 12 amostras de sangue. Pelos testes confirmatórios, em 50 pacientes foi confirmada a infecção por HTLV: 25 HTLV-1 (1,55 %), 21 HTLV-2 (1,31 %) e quatro HTLV (0,25 %). As sensibilidades do LIA, WB e PCR foram de 96 %, 76 % e 60 %, respectivamente. Considerando-se apenas o custo, o melhor algoritmo diagnóstico para população infectada pelo HIV-1 foi o uso da PCR seguida do LIA...

Evolution of bacterial meningitis diagnosis in Sao Paulo State-Brazil and future challenges / Evolucao do diagnostico das meningites bacterianas no Estado de Sao Paulo-Brasil e os futuros desafios

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.

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.

Avaliação de resultados discrepantes obtidos na execução de PCR em tempo real em amostras de pacientes com suspeita clínica de meningite bacteriana / Inconsistent results in real-time PCR in analyzing samples from patients with suspected bacterial meningitis

O princípio básico para obter resultado confiável é a compatibilidade entre as réplicas e suareprodutibilidade. Na rotina diagnóstica por PCR em tempo real (PCR-TR), em que centenas de amostrassão processadas, a obtenção de resultados com Cts tardios ou réplicas que diferem entre si por mais de trêsunidades, são inevitáveis. Das 3.000 amostras processadas em 2010, em duplicata, na rotina diagnósticadas meningites bacterianas por PCR-TR na pesquisa de N. meningitidis, S. pneumoniae e H. influenzae,157 (5,2 %), apresentaram inconsistência entre as réplicas (diferença entre Cts maior do que 3) e/ou altosvalores de Cts; e os ensaios foram retestados. O presente trabalho investigou estes resultados obtidos, osbenefícios destas repetições e as possíveis razões da ocorrência dos resultados discrepantes. Verificouseque, apenas 18 (11 %) das amostras submetidas à repetição, apresentaram resultados positivos. Erroshumanos inerentes à pipetagem, como o uso de pipetas não calibradas, a baixa concentração de DNAalvo nas amostras, a degradação da sonda ou mesmo a possível contaminação aleatória são fatores quecontribuem para induzir resultados discrepantes. A realização do ensaio de PCR-TR com amostras emduplicata e a repetição de ensaios com resultados discordantes é um artifício eficiente para avaliar e definirestes resultados.

Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

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.

Implantação e otimização da PCR em tempo real para o diagnóstico da influenza A (H1N1) pandêmica no Instituto Adolfo Lutz e perspectivas para 2010 / Implementation and optimization of real time PCR for diagnosis of pandemic influenza A (H1N1) at Instituto Adolfo Lutz and perspectives for 2010

Os autores apresentam sua experiência no diagnóstico laboratorial de InfluenzaA (H1N1) em 37.240 amostras clínicas obtidas de pacientes com suspeita de gripe, encaminhadas ao Instituto Adolfo Lutz para análise. Eles apresentam os algoritmos de testes moleculares empregados, comparam a eficiência dos mesmos quanto à sensibilidade, especificidade e custo e, finalmente sugerem um novo algoritmo para ser usado em caso de nova epidemia de Influenza A (H1N1) em 2010.

The authors present their experience with the molecular diagnosis of Influenza A (H1N1) with 37.240 clinicalsamples obtained from individuals suspected of flu, sent to Instituto Adolfo Lutz for analysis. They show the used algorithms, compare their efficiency in terms of sensitivity, specificity and cost, and suggest a new algorithm to be employed in case of an outbreak of Influenza A (H1N1) in 2010.