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2.
Microb Drug Resist ; 2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-32155381

RESUMO

Acinetobacter baumannii is the main species of the Acinetobacter genus; however, non-baumannii Acinetobacter (NBA) species causing infections have been described for the past years, as well as antimicrobial resistance. In this study, we describe the occurrence of two multidrug-resistant (MDR) IMP-1-producing Acinetobacter bereziniae isolates recovered from bloodstream infections in different patients but in the same intensive care unit among 134 carbapenem-resistant Acinetobacter screened. Antimicrobial susceptibility testing revealed resistance to carbapenems, extended spectrum, and antipseudomonad cephalosporins, amikacin, and trimethoprim-sulfamethoxazole. Both A. bereziniae isolates shared the same ApaI-pulsed-field gel electrophoresis (PFGE) pattern. Whole-genome sequencing of both isolates revealed that blaIMP-1 was embedded into an In86 Class I integron carrying also sul1, aac(6')-31, and aadA genes. A new sequence type (ST1309 Pasteur) was deposited. The virulence genes lpxC and ompA, seen in A. baumannii, were detected in the A. bereziniae strains. Recognition of A. bereziniae causing invasive MDR infection underscores the role of NBA species as human pathogens especially in at-risk patients.

3.
PLoS One ; 14(8): e0221525, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31437226

RESUMO

We aimed to investigate the nasopharyngeal colonization (NPC) by Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in the elderly population and to assess the demographic factors associated with NPC. This was an observational cohort study in which outpatients aged ≥60 years were enrolled from April to August 2017, with a follow-up visit from September through December 2017. Nasopharyngeal (NP) swabs were collected, bacteria were detected and isolated, and isolates were subjected to phenotypic and molecular characterization using standard microbiological techniques. At enrolment, the rates of S. aureus, methicillin-resistant S. aureus (MRSA), H. influenzae, and S. pneumoniae among 776 elderly outpatients were 15.9%, 2.3%, 2.5%, and 2.2%, respectively. Toxin production was detected in 21.1% of methicillin-susceptible S. aureus, and three SCCmec types were identified: II/IIb, IVa, and VI. At the follow-up visit, all carriage rates were similar (p > 0.05) to the rates at enrolment. Most of S. pneumoniae serotypes were not included in pneumococcal conjugate vaccines (PCVs), except for 7F, 3, and 19A. All strains of H. influenzae were non-typeable. Previous use of antibiotics and 23-valent pneumococcal polysaccharide vaccination (p < 0.05) were risk factors for S. aureus and MRSA carriage; S. aureus colonization was also associated with chronic kidney disease (p = 0.021). S. pneumoniae carriage was associated with male gender (p = 0.032) and an absence of diabetes (p = 0.034), while not receiving an influenza vaccine (p = 0.049) and chronic obstructive pulmonary disease (p = 0.031) were risk factors for H. influenzae colonization. The frailty of study participants was not associated with colonization status. We found a higher S. aureus carriage rate compared with the S. pneumoniae- and H. influenzae-carriage rates in a well-attended population in a geriatric outpatient clinic. This is one of the few studies conducted in Brazil that can support future colonization studies among elderly individuals.

4.
Int J Infect Dis ; 81: 191-195, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30849581

RESUMO

BACKGROUND: The primary method of molecular subtyping for the identification and investigation of outbreaks has been pulsed-field gel electrophoresis (PFGE). In some cases, this technique has not been able to show discrimination between the unrelated strains that can be achieved by whole genome sequencing (WGS). METHODS: The aim of this study was to determine the strengths and drawbacks of WGS using different analytic approaches compared to traditional typing method, PFGE, for retrospectively typing clusters cases of 28 S. Typhi. RESULTS: We evaluated three analytical approaches on the WGS data set (Nucleotide Difference (ND), (SNPs) and Whole genome multi locus sequence typing (wgMLST) that identically classified the clusters-related strains into two clusters, cluster A (with strains from 2017), and Cluster B (with strains from 2007). CONCLUSIONS: In this study WGS based typing, was able to compete with PFGE for differentiation of the clusters of S. Typhi strains.


Assuntos
Salmonella typhi/genética , Salmonella typhi/isolamento & purificação , Febre Tifoide/microbiologia , Antibacterianos/administração & dosagem , Brasil/epidemiologia , Pré-Escolar , Surtos de Doenças , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Masculino , Tipagem de Sequências Multilocus , Estudos Retrospectivos , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/fisiologia , Febre Tifoide/tratamento farmacológico , Febre Tifoide/epidemiologia , Sequenciamento Completo do Genoma
5.
PLoS One ; 12(2): e0172794, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28235065

RESUMO

BACKGROUND: The lack of information regarding the burden of acute bacterial meningitis in Latin America leads to a reduction in the estimated incidence rates of the disease, and impairs public health decisions on the use and follow-up of preventive interventions, particularly, the evaluation of existing vaccination policies. The use of the real-time PCR in diagnostic routine procedures has resulted in a substantial increase in confirmed bacterial meningitis cases. However, in resource-poor countries, these assays are only available in reference laboratories. Sample transportation to these laboratories is a critical constraint, as it requires specialized, high cost courier services. To overcome this barrier we evaluated the use of FTATM Elute filter paper cards for the conservation and processing of samples under normal environmental conditions, as they would be when transported from remote and under-equipped healthcare facilities to the reference centers. A total of 401 samples received in 2015 as part of Sao Paulo's national surveillance for routine diagnosis were selected for this study. METHODS: The sensitivity and specificity of real-time PCR were evaluated using fresh serum and cerebrospinal fluid (CSF) samples processed using our laboratory's standard DNA extraction, and processing the same samples after being dried and stored on FTATM card, and DNA extracted following the manufacturer's instructions. RESULTS: The sensitivities for detection of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from CSF dried and stored on FTATM cards were 98%, 92%, and 100%, respectively, and with serum samples were 73%, 88%, and 100%, respectively. When compared to our laboratory's standard methodology, results showed high concordance, with Kappa index ranges of 0.9877-1.00 for CSF, and 0.8004-1.00 for serum samples. CONCLUSION: The use of FTATM cards for CSF and serum conservation and transport represents a rapid, reliable, and cost-effective alternative that will allow obtaining valuable epidemiological information that would otherwise be lost.


Assuntos
Haemophilus influenzae/isolamento & purificação , Meningites Bacterianas/sangue , Meningites Bacterianas/líquido cefalorraquidiano , Neisseria meningitidis/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Brasil/epidemiologia , Feminino , Haemophilus influenzae/patogenicidade , Humanos , Masculino , Meningites Bacterianas/epidemiologia , Meningites Bacterianas/microbiologia , Neisseria meningitidis/patogenicidade , Streptococcus pneumoniae/patogenicidade
6.
AIDS Research and Human Retroviruses ; 31(5): 543-549, maio. 1, 2015.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IALPROD, Sec. Est. Saúde SP, SESSP-IALACERVO | ID: ses-30666

RESUMO

During the 1990s, high prevalences of HIV/human T lymphotropic virus type 1 (HTLV-1) and HIV/human T lymphotropic virus type 2 (HTLV-2) coinfections were detected in São Paulo, Brazil in association with intravenous drug use (IDU). The current prevalences and risk factors for HIV/HTLV-1/-2 were evaluated in 1,608 patients attending the AIDS/STD Reference and Training Center in São Paulo. Blood samples were analyzed for HTLV-1/2-specific antibodies using enzyme immunoassays (EIA Murex HTLV-I+II, Diasorin, and Gold ELISA HTLV-I+II, REM) and immunoblotting (HTLV Blot 2.4, MP Biomedicals and INNO-LIA HTLV-I/II, Innogenetics) and for the pol proviral DNA segments of HTLV-1 and HTLV-2 by “in-house” real-time PCR. These analyses revealed that 50 (3.11%) of the samples were HTLV positive, including 25 (1.55%) that were HTLV-1 positive, 21 (1.31%) that were HTLV-2 positive, and 4 (0.25%) that were HTLV positive (untypeable). The median age of the HIV/HTLV-coinfected individuals was 50 years versus 44 years in the overall population (p=0.000). The risk factors associated with HIV/HTLV-1/-2 coinfections were female gender (OR 3.26, 1.78–5.95), black/pardo color (OR 2.21, 1.21–4.03), infection with hepatitis B virus (HBV) (OR 4.27, 2.32–7.87) or hepatitis C virus (HCV) (OR 24.40, 12.51–48.11), and intravenous drug use (IDU) (OR 30.01, 15.21–59.29). The current low prevalence of HTLV-1/2 in HIV-infected patients in São Paulo could be explained in part by programs providing IDUs with sterile needles and syringes and changes in the drug usage patterns of individuals from injecting cocaine to smoking crack cocaine. (AU)


Assuntos
Humanos , Vírus Linfotrópico T Tipo 1 Humano , Vírus Linfotrópico T Tipo 2 Humano , Prevalência , Fatores de Risco , Síndrome de Imunodeficiência Adquirida/epidemiologia , HIV/patogenicidade , Pacientes , Brasil/epidemiologia
7.
AIDS Res Hum Retroviruses ; 31(5): 543-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25464979

RESUMO

During the 1990s, high prevalences of HIV/human T lymphotropic virus type 1 (HTLV-1) and HIV/human T lymphotropic virus type 2 (HTLV-2) coinfections were detected in São Paulo, Brazil in association with intravenous drug use (IDU). The current prevalences and risk factors for HIV/HTLV-1/-2 were evaluated in 1,608 patients attending the AIDS/STD Reference and Training Center in São Paulo. Blood samples were analyzed for HTLV-1/2-specific antibodies using enzyme immunoassays (EIA Murex HTLV-I+II, Diasorin, and Gold ELISA HTLV-I+II, REM) and immunoblotting (HTLV Blot 2.4, MP Biomedicals and INNO-LIA HTLV-I/II, Innogenetics) and for the pol proviral DNA segments of HTLV-1 and HTLV-2 by "in-house" real-time PCR. These analyses revealed that 50 (3.11%) of the samples were HTLV positive, including 25 (1.55%) that were HTLV-1 positive, 21 (1.31%) that were HTLV-2 positive, and 4 (0.25%) that were HTLV positive (untypeable). The median age of the HIV/HTLV-coinfected individuals was 50 years versus 44 years in the overall population (p=0.000). The risk factors associated with HIV/HTLV-1/-2 coinfections were female gender (OR 3.26, 1.78-5.95), black/pardo color (OR 2.21, 1.21-4.03), infection with hepatitis B virus (HBV) (OR 4.27, 2.32-7.87) or hepatitis C virus (HCV) (OR 24.40, 12.51-48.11), and intravenous drug use (IDU) (OR 30.01, 15.21-59.29). The current low prevalence of HTLV-1/2 in HIV-infected patients in São Paulo could be explained in part by programs providing IDUs with sterile needles and syringes and changes in the drug usage patterns of individuals from injecting cocaine to smoking crack cocaine.


Assuntos
Infecções por HIV/complicações , Infecções por HTLV-I/epidemiologia , Infecções por HTLV-II/epidemiologia , Adulto , Idoso , Animais , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Estudos Transversais , Feminino , Produtos do Gene pol/genética , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prevalência , Fatores de Risco
8.
Bepa - Boletim Epidemiológico Paulista ; 11(130): 1-10, outubro 2014. tab
Artigo em Português | Sec. Est. Saúde SP, SESSP-CTDPROD, Sec. Est. Saúde SP, SESSP-CVEPROD, Sec. Est. Saúde SP | ID: ses-36294

RESUMO

Os HTLV-1, HTLV-2 e HIV compartilham as mesmas vias de transmissão e as prevalências de coinfecção HIV/HTLV-1 e HIV-HTLV-2 variam de acordo com a região geográfica, a população de estudo e a época em que foi realizada a pesquisa. Altas taxas de coinfecção foram detectadas em pacientes com Aids em São Paulo na década de 1990 e foram associadas ao uso de drogas injetáveis (UDI). Neste estudo foi determinada a prevalência e os fatores de risco para a coinfecção HIV/HTLV em pacientes do CRT-DST/Aids de São Paulo. Amostras de sangue de 1.608 pacientes que aceitaram participar do estudo foram encaminhadas ao Instituto Adolfo Lutz para pesquisa de anticorpos anti-HTLV-1/2 por ensaio imunoenzimático e Western Blot (WB) e para pesquisa de DNA proviral pela PCR em tempo real pol. Na triagem sorológica, 51 soros resultaram reagentes para HTLV. Destes, pelo WB, 23 (1,43%) confirmaram infecção HTLV-1, 12 (0,75%) HTLV-2 e 6 (0,37%) HTLV não tipado. Pela PCR houve detecção de mais um caso de HTLV-1 (total 1,49%) e cinco casos de HTLV-2 (total 1,06%). Houve associação entre infecção HTLV-1/2 e gênero feminino (p=0.0027), cor negro/pardo (p=0.0332), infecção pelo HBV (p=0.0019), HCV e UDI (p<0.0000). A PCR em tempo real foi útil para confirmar casos com resultado HTLV não tipado e Indeterminado pelo WB e pode ser usada como primeiro teste confirmatório seguido do WB. A baixa prevalência de coinfecção HIV/HTLV no presente estudo parece estar relacionada a mudanças na população exposta ao HIV e na troca de cocaína injetável por crack no momento atual...(AU)


Assuntos
Humanos , Vírus Linfotrópico T Tipo 1 Humano , Vírus Linfotrópico T Tipo 2 Humano , HIV , Infecção , Pacientes
9.
Emerg Infect Dis ; 20(5): 806-11, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24751156

RESUMO

During 2010, outbreaks of serogroup C meningococcal (MenC) disease occurred in 2 oil refineries in São Paulo State, Brazil, leading to mass vaccination of employees at 1 refinery with a meningococcal polysaccharide A/C vaccine. A cross-sectional study was conducted to assess the prevalence of meningococci carriage among workers at both refineries and to investigate the effect of vaccination on and the risk factors for pharyngeal carriage of meningococci. Among the vaccinated and nonvaccinated workers, rates of overall meningococci carriage (21.4% and 21.6%, respectively) and of MenC carriage (6.3% and 4.9%, respectively) were similar. However, a MenC strain belonging to the sequence type103 complex predominated and was responsible for the increased incidence of meningococcal disease in Brazil. A low education level was associated with higher risk of meningococci carriage. Polysaccharide vaccination did not affect carriage or interrupt transmission of the epidemic strain. These findings will help inform future vaccination strategies.


Assuntos
Portador Sadio/epidemiologia , Meningite Meningocócica/classificação , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/imunologia , Adolescente , Adulto , Brasil/epidemiologia , Estudos Transversais , Surtos de Doenças , História do Século XXI , Humanos , Incidência , Meningite Meningocócica/genética , Meningite Meningocócica/imunologia , Infecções Meningocócicas/história , Tipagem de Sequências Multilocus , Fatores de Risco , Sorotipagem , Vacinação , Adulto Jovem
10.
Arq Neuropsiquiatr ; 71(9B): 672-6, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24141502

RESUMO

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.


Assuntos
Meningites Bacterianas/diagnóstico , Brasil , Contraimunoeletroforese , Previsões , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo
11.
Arq. neuropsiquiatr ; 71(9B): 672-676, set. 2013. graf
Artigo em Inglês | LILACS | ID: lil-688539

RESUMO

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.


Assuntos
Humanos , Meningites Bacterianas/diagnóstico , Brasil , Contraimunoeletroforese , Previsões , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo
12.
Mem Inst Oswaldo Cruz ; 108(2): 246-7, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23579808

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Líquidos Corporais/microbiologia , Infecções Meningocócicas/diagnóstico , Neisseria meningitidis/genética , Portador Sadio/microbiologia , Humanos , Neisseria meningitidis/isolamento & purificação , Faringe/microbiologia , Reação em Cadeia da Polimerase em Tempo Real
13.
Rev. Inst. Adolfo Lutz ; 72(2): 161-164, 2013. tab
Artigo em Português | LILACS | ID: lil-729376

RESUMO

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.


Assuntos
Diagnóstico , Laboratórios , Meningites Bacterianas , Reação em Cadeia da Polimerase em Tempo Real , Haemophilus influenzae , Neisseria meningitidis , Streptococcus pneumoniae
14.
Rev. Inst. Adolfo Lutz ; 72(1): 65-71, 2013. tab
Artigo em Inglês | LILACS | ID: lil-729390

RESUMO

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.


Assuntos
Líquido Cefalorraquidiano , Técnicas e Procedimentos Diagnósticos , Reações Falso-Negativas , Neisseria meningitidis , Reação em Cadeia da Polimerase em Tempo Real
15.
Rev. Inst. Adolfo Lutz ; 72(1): 65-71, 2013. tab
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-CTDPROD, Sec. Est. Saúde SP, SESSP-IALPROD, Sec. Est. Saúde SP | ID: ses-29754

RESUMO

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.(AU)


Assuntos
Reação em Cadeia da Polimerase em Tempo Real , Neisseria meningitidis , Reações Falso-Negativas , Técnicas e Procedimentos Diagnósticos , Líquido Cefalorraquidiano
16.
Rev. Inst. Adolfo Lutz ; 72(2): 161-164, 2013. tab
Artigo em Português | Sec. Est. Saúde SP, SESSP-CTDPROD, Sec. Est. Saúde SP, SESSP-IALPROD, Sec. Est. Saúde SP | ID: ses-29768

RESUMO

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. (AU)


Assuntos
Reação em Cadeia da Polimerase em Tempo Real , Meningites Bacterianas , Laboratórios , Diagnóstico , Neisseria meningitidis , Streptococcus pneumoniae , Haemophilus influenzae
17.
Mem Inst Oswaldo Cruz ; 107(7): 903-8, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23147147

RESUMO

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.


Assuntos
Antituberculosos/farmacologia , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Reação em Cadeia da Polimerase em Tempo Real
18.
Mem. Inst. Oswaldo Cruz ; 107(7): 903-908, Nov. 2012. tab
Artigo em Inglês | LILACS | ID: lil-656047

RESUMO

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.


Assuntos
Humanos , Antituberculosos/farmacologia , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Mutação , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Reação em Cadeia da Polimerase em Tempo Real
19.
BEPA - Boletim Epidemiológico Paulista ; 9(103): 16-20, jul. 2012.
Artigo em Português | Sec. Est. Saúde SP, SESSP-CTDPROD, Sec. Est. Saúde SP, SESSP-ACVSES, SESSP-CVEPROD, Sec. Est. Saúde SP, SESSP-IALPROD, Sec. Est. Saúde SP, SESSP-IALACERVO | ID: ses-28039

RESUMO

A incorporação do ensaio “triplex” de PCR em tempo-real (PCR-TR) àvigilância das meningites bacterianas a partir de 2007 aumentou a detecção de S. pneumoniae em 52%, de N. meningitidis em 85% e de H. influenzae em 20%. Entretanto, a detecção do H. influenzae se limitou às cepas capsuladas de 4 sorotipos (“a”, “b”, “c”, “d”). Em 2011, um novo ensaio para detectar todos os sorotipos de H. influenzae, inclusive os H. influenzae não tipáveis, foi proposto,com a substituição do gene bexA pelo gene hpd no ensaio triplex (“triplexmodificado”) responsável pela proteína D de H. influenzae. Foram analisadas1619 amostras clínicas de líquido cefalorraquidiano e/ou sangue de pacientes com suspeita de meningite bacteriana do município de São Paulo no período de junho a dezembro de 2011, nos dois formatos de PCR-TR “triplex”. O novo ensaio “triplex modificado” (com o gene hpd) detectou 13 casos adicionais de H. influenzae, não registrados pelo outro formato (com o gene bexA). Destes 13 casos adicionais, 12 foram Hi-nt e um do sorotipo “f”. Não houve modificação na sensibilidade em detectar amostras positivas para N. meningitidis ou S. pneumoniae. O emprego deste novo formato “triplex modificado” irá aprimorar o diagnóstico e a vigilância epidemiológica das meningites bacterianas, contribuindo com a redução dos casos de meningite sem etiologia definida(AU)


Assuntos
Meningites Bacterianas , Reação em Cadeia da Polimerase em Tempo Real , Haemophilus influenzae
20.
BEPA - Boletim Epidemiológico Paulista ; 9(102): 13-20, jun. 2012. tab
Artigo em Português | Sec. Est. Saúde SP, SESSP-CTDPROD, Sec. Est. Saúde SP, SESSP-ACVSES, SESSP-IALPROD, Sec. Est. Saúde SP, SESSP-IALACERVO | ID: ses-28031

RESUMO

A contraimunoeletroforese (CIE) é uma técnica amplamente empregada no Brasil para o diagnóstico laboratorial indireto das meningites causadas por Neisseria meningitidis (Men) dossorogrupos A, B, C e W135 ou Haemophilus influenzae sorotipo b (Hib). O objetivo deste trabalho foi validar a CIE empregando-se 301 amostras de líquido cefalorraquidiano (LCR) e 236amostras de soro de pacientes com suspeita de meningite bacteriana dos municípios de São Paulo e Campinas no período de 4 de junho de 2007 a 30 de maio de 2009. Em amostras de LCR, a CIE apresentou sensibilidade de 62,7% (42/67) (IC 95%, 50%-74,2%) e especificidade de 88,9% (208/234) (IC 95%, 84,1%-92,6%) para a detecção de Men. Em soro, a sensibilidade foi de35,3% (6/17) (IC 95%, 14,2%-61,7%) e a especificidade de 90,9% (199/219) (IC 95%, 86,3%-94,4%) Estes mesmos parâmetros não foram calculados para o componente Hib, devido à indisponibilidade de número significativo de amostras com cultura positiva para esta bactéria. Osresultados demonstraram que a técnica de CIE, embora tenha apresentado alta especificidade, apresentou baixa sensibilidade, especialmente em amostras de soro; assim, o uso da CIE não é recomendado como teste único para o diagnóstico das meningites bacterianas. Portanto, ressaltasea necessidade do emprego de outros testes indiretos com alta sensibilidade e especificidade, como a PCR em tempo real, para a melhoria do diagnóstico e do sistema de vigilância da doença(AU)


Assuntos
Estudos de Validação como Assunto , Meningites Bacterianas , Neisseria meningitidis
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