The prevalence of GBS is high in pregnant women and is associated with sexual intercourse frequency, previous spontaneous abortion and the presence of candidosis or cytolytic vaginosis.

BackgroundInfections are the major cause of morbidity and mortality in children with cancer. Gaining a favorable prognosis for these patients depends on selecting the appropriate therapy, which in turn depends on rapid and accurate microbiological diagnosis. This study employed real-time PCR (qPCR) to identify the main pathogens causing bloodstream infection (BSI) in patients treated at the Pediatric Oncology Institute IOP-GRAACC-UNIFESP-Brazil. Antimicrobial resistance genes were also investigated using this methodology.MethodsA total of 248 samples from BACTEC® blood culture bottles and 99 whole-blood samples collected in tubes containing EDTA K2 Gel were isolated from 137 patients. All samples were screened by specific Gram probes for multiplex qPCR. Seventeen sequences were evaluated using gender-specific TaqMan probes and the resistance genes blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB and mecA were detected using the SYBR Green method.ResultsPositive qPCR results were obtained in 112 of the blood culture bottles (112/124), and 90 % agreement was observed between phenotypic and molecular microbial detection methods. For bacterial and fungal identification, the performance test showed: sensitivity 87 %; specificity 91 %; NPV 90 %; PPV 89 % and accuracy of 89 % when compared with the phenotypic method. The mecA gene was detected in 37 samples, extended-spectrum β-lactamases were detected in six samples and metallo-β-lactamase coding genes in four samples, with 60 % concordance between the two methods. The qPCR on whole blood detected eight samples possessing the mecA gene and one sample harboring the vanB gene. The blaKPC, blaVIM, blaIMP and blaSHV genes were not detected in this study.ConclusionReal-time PCR is a useful tool in the early identification of pathogens and antimicrobial resistance genes from bloodstream infections of pediatric oncologic patients.

Group B Streptococcus detection directly from Copan ESwab collected samples, using the BD Max GBS assay, was evaluated on receipt in the laboratory and after 24 h at room temperature. Results were compared to those using Lim broth enrichment PCR and culture. No significant difference was observed between 24 h ESwab and Lim broth PCRs.

A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens. Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium complex (MAC) are the most common slowgrowing mycobacteria isolated from respiratory infections worldwide (1, 2). Tuberculosis is still a major global public health problem and one of the leading infectious causes of death, especially in developing countries (1,3,4). In contrast, the prevalence of nontuberculous mycobacterial infection has been increasing in developed countries. MAC isolates represent the organisms most frequently associated with nontuberculous mycobacterial lung diseases in most of the world (1, 2, 5). The identification of mycobacteria responsible for diseases has important ramifications for infection control and selection of antimicrobial therapy. Identification, however, is hampered by the slow growth of most mycobacteria, which may take as long as 2 months using traditional culture methods (6, 7).Molecular methods represent a reliable and rapid alternative for laboratory diagnostics of mycobacteria in clinical samples (1,3,4). Several PCR assays have already been described for the detection of mycobacteria; however, some of them are conventional PCR requiring post-PCR processing, others use melting curve analysis and need additional interpretation, and some have not been used directly from clinical samples (8-11). The Cepheid Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is an FDAcleared assay that provides direct detection of MTC and rifampin (RIF) resistance in clinical samples, and it displays good sensitivity relative to that of culture (12). However, it does not detect MAC or of other Mycobacterium species. The BD Max system (BD Diagnostics, Sparks, MD) is an open fully integrated automated molecular platform that combines specimen processing and real-time PCR. In addition to a number of FDA-cleared assays, the BD Max also offers generic extraction kits and PCR reagents to be used on an open platform that allows users to create their own assay using their own set of primers and probes (13,14). The aim of this study was to validate a multiplex PCR test to detect Mycobacterium spp. (pan-Mycobacterium [PAN]), MTC, and MAC directly from clinical respiratory samples using a user-developed protocol (UDP) on the BD Max open-mode system. This test is for primary diagnosis only and was not designed to detect RIF resistance. The PAN target usually encompasses broad-based characterizations of gene content in a given group of organisms. The pan-Mycobacterium primers used in this study were based on the amplification of the 16S rRNA gene from Mycobacterium species.A total of 120 frozen clinical specimens previously identified...

BackgroundEarly identification of pathogens and antimicrobial resistance in bloodstream infections (BSIs) decreases morbidity and mortality, particularly in immunocompromised patients. The aim of the present study was to compare real-time polymerase chain reaction (PCR) with commercial kits for detection of 17 pathogens from blood culture (BC) and 10 antimicrobial resistance genes.MethodsA total of 160 BCs were taken from bone marrow transplant patients and screened with Gram-specific probes by multiplex real-time PCR and 17 genus-specific sequences using TaqMan probes and blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB, and mecA genes by SYBR Green.ResultsTwenty-three of 33 samples identified by phenotypic testing were concordantly positive by BC and real-time PCR. Pathogen identification was discordant in 13 cases. In 12 of 15 coagulase-negative staphylococci, the mecA gene was detected and four Enterococcus spp. were positive for vanA. Two blaCTX and three blaSHV genes were found by quantitative PCR. The blaKPC and metallo-β-lactamase genes were not detected. Five fungal species were identified only by real-time PCR.ConclusionsReal-time PCR could be a valuable complementary tool in the management of BSI in bone marrow transplants patients, allowing identification of pathogens and antimicrobial resistance genes.

CONTEXT AND OBJECTIVE: Maternal Streptococcus agalactiae colonization and early-onset neonatal sepsis have aroused interest in the worldwide literature. Streptococcal neonatal disease is associated with significant morbidity and mortality in the perinatal period, especially among premature neonates. The aim of this study was to assess the prevalence of maternal streptococcal colonization by using combined swab cultures, compared with swab collection from a single site. RESULTS:The prevalence of streptococcal colonization was 25.4%. Among the patients with positive cultures, 28.1% had this at only one collection site, 24.2% simultaneously at two sites and 47.5% at all three sites. Associating the swabs from two collection sites significantly increased streptococcal isolation, compared with a single swab (P < 0.05), except for perianal (rectal) collection. Use of combined swabs from three collection sites showed statistically higher isolation rates. CONCLUSION:In combined swab cultures collected from three collection sites, the prevalence of maternal Streptococcus agalactiae colonization was higher than in swabs collected from a single site. RESUMOCONTEXTO E OBJETIVO: Colonização materna por Streptococcus agalactiae e sepse neonatal de início precoce têm despertado interesse na literatura mundial. A doença estreptocócica neonatal está associada com significantes índices de morbidade e mortalidade perinatal, principalmente nos recém-nascidos prematuros. O objetivo deste estudo foi avaliar a prevalência de colonização estreptocócica materna por meio da coleta de swabs combinados, comparada com coleta de swab num único local. RESULTADOS: A prevalência de colonização estreptocócica foi de 25,4%. Dentre as pacientes com cultura positiva, 28,1% tiveram positividade em apenas um local de coleta, 24,2% em dois locais simultaneamente e 47,5% nos três locais avaliados. Associação de swabs de dois locais de coleta aumentou significativamente o isolamento estreptocócico, comparado a único swab (P < 0,05), exceto para coleta perianal. (retal) Utilização de swabs combinados de três locais de coleta mostrou taxas de isolamento estatisticamente superiores. TIPO DE ESTUDO E LOCAL:CONCLUSÃO: Na cultura de swabs combinados de três locais de coleta houve maior prevalência de colonização materna por Streptococcus agalactiae, comparada com coleta de swabs em um único local.

The aim of this study was to develop a multiplex PCR test to detect Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) directly from CF patient's respiratory samples using the open mode of the BD MAX™ System. A total of 402 CF respiratory samples were evaluated by culture and PCR. Specific sets of primers and probes for each target were designed in-house. Out of 402 samples tested, 196 were identified as negative and 206 as positive by culture for AX, PSA, BC and SM. Among culture positive samples, PCR detected 21/27 AX, 4/5 BC, 138/140 PSA and 29/34 SM. In addition, PCR assay identified 35 samples as positive that were initially negative by culture for those 4 targets. The CF BDM test proved to be an excellent tool to detect AX, BC, PSA and SM by real-time PCR on an automated platform.

A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform.