Identificação de Burkholderia pseudomallei baseada em PCR

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.As técnicas de amplificação de DNA estão sendo cada vez mais utilizadas em laboratórios clínicos para a confirmação da identificação de bactérias que têm importância médica. Um método de identificação de Burkholderia pseudomallei baseado em PCR tem sido usado em nosso centro há 10 anos e foi utilizado para confirmar a identificação de bactérias isoladas de casos de melioidose no Ceará desde 2003. Este método particular tem sido usado como padrão ouro para métodos menos discriminatórios. Nesse estudo, avaliamos três métodos de identificação de B. pseudomallei baseados em PCR e usamos seqüenciamento de DNA para solucionar discrepâncias entre os resultados baseados em PCR e os métodos de identificação fenotípica. O estabelecido protocolo de PCR semi-nested para a região espacial 16-23s da B. pseudomallei produziu um consistente resultado negativo para um de nossos 100 isolados testados (BCC#99), mas identificou corretamente todos os outros 71 isolados de B. pseudomallei. Uma variação anômala da seqüência foi detectada na região interna do sítio de ligação do primer reverso para este método. Métodos de PCR foram desenvolvidos para a detecção de outros dois genes bacterianos metabólicos de B. pseudomallei. O protocolo de PCR IpxO convencional teve sensibilidade de 0,89 e especificidade de 1,0, enquanto que o PCR em tempo real mostrou-se ainda melhor, com sensibilidade de 1,0 e especificidade de 1,0. Este método identificou todos os isolados de B. pseudomallei, incluindo o isolado discrepante que teve o PCR negativo. O protocolo de PCR phaC detectou o gene de todos os B. pseudomallei e em todos exceto três isolados de B. cepacia, tornando este método de identificação de B. pseudomallei baseado em PCR inadequado. Esta experiência com métodos de identificação de B. pseudomallei baseados em PCR indica que devemos ter precaução quando estes forem utilizados sozinhos para identificação dessa bactéria e que eles necessitam ser interpretados em conjunto com métodos fenotípicos e moleculares alternativos, tais como seqüenciamento genético

An Economical Tandem Multiplex Real-Time PCR Technique for the Detection of a Comprehensive Range of Respiratory Pathogens

This study used real-time PCR assays to screen small sample volumes for a comprehensive range of 35 respiratory pathogens. Initial thermocycling was limited to 20 cycles to avoid competition for reagents, followed by a secondary real-time multiplex PCR. Supplementary semi-nested human metapneumovirus and picornavirus PCR assays were required to complete the acute respiratory pathogen profile. Potential pathogens were detected in 85 (70%) of pernasal aspirates collected from 121 children with acute respiratory symptoms. Multiple pathogens were detected in 29 (24%) of those samples. The tandem multiplex real-time PCR was an efficient method for the rapid detection of multiple pathogens

Respiratory viral pathogens associated with lower respiratory tract disease among young children in the highlands of Papua New Guinea

Background: Acute lower respiratory tract infections (ALRI) commonly result in fatal outcomes in the young children of Papua New Guinea (PNG). However, comprehensive studies of the viral aetiology of ALRI have not been conducted in PNG for almost 30 years. Objectives: To determine the viruses associated with ALRI among children living in the PNG highlands using sensitive molecular detection techniques. Study design: Pernasal swabs were collected routinely between 1 week and 18 months of age and also during episodes of ALRI, as part of a neonatal pneumococcal conjugate vaccine trial. A tandem multiplex real-time PCR assay was used to test for a comprehensive range of respiratory viruses in samples collected from 221 young children. Picornavirus typing was supported by DNA sequence analysis. Results: Recognized pathogenic respiratory viruses were detected in 198/273 (73%) samples collected from children with no evidence of ALRI and 69/80 (86%) samples collected during ALRI episodes. Human rhinoviruses (HRV) species A, B and C were detected in 152 (56%) samples from non-ALRI children and 50 (63%) samples collected during ALRI episodes. Partial structural region sequences for two new species C rhinoviruses were added to the GenBank database. ALRI was associated with detection of adenovirus species B (p< 0.01) or C (p< 0.05), influenza A (p< 0.0001) or respiratory syncytial virus (p< 0.0001). Multiple viruses were detected more often during ALRI episodes (49%) than when children displayed no symptoms of ALRI (18%) (p< 0.0001). Conclusions: The burden of infection with respiratory viruses remains significant in young children living in the PNG highlands

Community-Acquired Pneumonia Due to Pandemic A(H1N1)2009 Influenzavirus and Methicillin Resistant Staphylococcus aureus Co-Infection

BACKGROUND: Bacterial pneumonia is a well described complication of influenza. In recent years, community-onset methicillin-resistant Staphylococcus aureus (cMRSA) infection has emerged as a contributor to morbidity and mortality in patients with influenza. Since the emergence and rapid dissemination of pandemic A(H1N1)2009 influenzavirus in April 2009, initial descriptions of the clinical features of patients hospitalized with pneumonia have contained few details of patients with bacterial co-infection. METHODOLOGY/PRINCIPAL FINDINGS: Patients with community-acquired pneumonia (CAP) caused by co-infection with pandemic A(H1N1)2009 influenzavirus and cMRSA were prospectively identified at two tertiary hospitals in one Australian city during July to September 2009, the period of intense influenza activity in our region. Detailed characterization of the cMRSA isolates was performed. 252 patients with pandemic A(H1N1)2009 influenzavirus infection were admitted at the two sites during the period of study. Three cases of CAP due to pandemic A(H1N1)2009/cMRSA co-infection were identified. The clinical features of these patients were typical of those with S. aureus co-infection or sequential infection following influenza. The 3 patients received appropriate empiric therapy for influenza, but inappropriate empiric therapy for cMRSA infection; all 3 survived. In addition, 2 fatal cases of CAP caused by pandemic A(H1N1)2009/cMRSA co-infection were identified on post-mortem examination. The cMRSA infections were caused by three different cMRSA clones, only one of which contained genes for Panton-Valentine Leukocidin (PVL). CONCLUSIONS/SIGNIFICANCE: Clinicians managing patients with pandemic A(H1N1)2009 influenzavirus infection should be alert to the possibility of co-infection or sequential infection with virulent, antimicrobial-resistant bacterial pathogens such as cMRSA. PVL toxin is not necessary for the development of cMRSA pneumonia in the setting of pandemic A( H1N1) 2009 influenzavirus co-infection

Deployable Laboratory Response to Influenza Pandemic; PCR Assay Field Trials and Comparison with Reference Methods

Background: The influenza A/H1N1/09 pandemic spread quickly during the Southern Hemisphere winter in 2009 and reached epidemic proportions within weeks of the official WHO alert. Vulnerable population groups included indigenous Australians and remote northern population centres visited by international travellers. At the height of the Australian epidemic a large number of troops converged on a training area in northern Australia for an international exercise, raising concerns about their potential exposure to the emerging influenza threat before, during and immediately after their arrival in the area. Influenza A/H1N1/09 became the dominant seasonal variant and returned to Australia during the Southern winter the following year. Methods: A duplex nucleic acid amplification assay was developed within weeks of the first WHO influenza pandemic alert, demonstrated in northwestern Australia shortly afterwards and deployed as part of the pathology support for a field hospital during a military exercise during the initial epidemic surge in June 2009. Results: The nucleic acid amplification assay was twice as sensitive as a point of care influenza immunoassay, as specific but a little less sensitive than the reference laboratory nucleic acid amplification assay. Repetition of the field assay with blinded clinical samples obtained during the 2010 winter influenza season demonstrated a 91.7% congruence with the reference laboratory method. Conclusions: Rapid in-house development of a deployable epidemic influenza assay allowed a flexible laboratory response, effective targeting of limited disease control resources in an austere military environment, and provided the public health laboratory service with a set of verification tools for resource-limited settings. The assay method was suitable for rapid deployment in time for the 2010 Northern winter

Pertussis outbreak in Papua New Guinea: the challenges of response in a remote geo-topographical setting

Introduction: A large outbreak of pertussis was detected during March 2011 in Goilala, a remote district of the Central Province in Papua New Guinea, characterized by rugged topography with no road access from the provincial headquarters. This outbreak investigation highlights the difficulties in reporting and responding to outbreaks in these settings.Method: The suspected pertussis cases, reported by health workers from the Ononge health centre area, were investigated and confirmed for the presence of Bordetella pertussis DNA using the polymerase chain reaction (PCR) method.Results: There were 205 suspected pertussis cases, with a case-fatality rate (CFR) of 3%. All cases were unvaccinated. The Central Province conducted a response vaccination programme providing 65% of children less than five years of age with diphtheria–pertussis-tetanus-HepB-Hib vaccine at a cost of US$12.62 per child.Discussion: The incurred cost of vaccination in response to this outbreak was much higher than the US$ 3.80 per child for routine outreach patrol. To prevent further outbreaks of vaccine-preventable diseases in these areas, local health centres must ensure routine vaccination is strengthened through the “Reaching Every District” initiative of the National Department of Health

Reliable quantification of rhinovirus species C using real-time PCR

AbstractBackgroundRhinovirus C (RV-C) is an important respiratory pathogen of children, but little is known about its contribution to disease severity, though viral load appears to be important. Difficulty in RV-C cultivation and target sequence variation has precluded the development of a PCR based quantification method.ObjectiveThe aim of this study was to develop and validate reverse transcription quantitative PCR (RT-qPCR) assays for a broad range of circulating RV-C genotypes in nasopharyngeal aspirates (NPAs).Study designFour assays were designed to quantify a 296bp region located within the 5′ untranslated region (UTR) of RV-C types. These assays were based on in silico analysis of available RV-C sequences. Probes were designed to provide 100% homology to the corresponding RV-C genotypes.ResultsThe linear dynamic range of each of the four assays spanned eight orders of magnitude (104–1011 copies/mL). The limit of detection for assays 1–4 was estimated to be 1147 copies/mL, 765 copies/mL, 1138 copies/mL and 1470 copies/mL respectively. Each assay demonstrated a strong linear relationship (r2=>0.995) and amplification efficiency greater than 95%. Repeatability and reproducibility of the method were shown to be high, with coefficients of variations lower than 8% and 15% respectively

The molecular epidemiology of ocular \u3cem\u3eChlamydia trachomatis\u3c/em\u3e infections in Western Australia: Implications for Trachoma control

We studied the serovar distribution of Chlamydia trachomatis in patients with clinical eye disease in Western Australia. Most disease occurred in indigenous communities and was caused by trachoma serovars Ba and C. Serovar Ba was genetically homogeneous throughout Western Australia and identical to strains previously described in indigenous communities in Northern Territory. This finding probably results from movement of these populations, and suggests that a widely coordinated, rather than local or regional, approach is needed to control trachoma in mobile populations. Serovar C strains within Western Australia were homogeneous but distinct from those in Northern Territory, possibly because of inherent differences in transmissibility or differences in population movements among communities carrying the different serovars. Genital serovars were occasional causes of eye diseases in infants, adolescents, and adults in trachoma-endemic areas. These serovars should be considered in the differential diagnosis of acute follicular conjunctivitis in these groups