Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry and real-time PCR in a combined protocol for diagnosis of bloodstream infections: a turnaround time approach.

Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum ß-lactamase profiling, 77.8% for metallo-ß-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry and real-time PCR in a combined protocol for diagnosis of bloodstream infections: a turnaround time approach

ABSTRACT Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48 h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum β-lactamase profiling, 77.8% for metallo-β-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38 h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.

Tailoring antimicrobials in febrile neutropenia: using faster diagnostic and communication tools to improve treatment in the era of extensively resistant pathogens

ABSTRACT Febrile Neutropenia represents a medical emergency and the use of appropriate antimicrobial therapy is essential for a better outcome. Although being time-consuming, conventional cultures and antimicrobial susceptibility tests remain the golden standard practices for microbiology identification. Final reports are typically available within several days. Faster diagnostic tools, such as species identification trough Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) and molecular techniques might help to shorten time to diagnostic and also guide definitive therapy in this scenario. Here we present a case in which the use of a diagnostic molecular workflow combining MALDI-TOF and real-time PCR for relevant genes codifying antibiotic resistant integrated with instant communication report, led to a tailored and more appropriate treatment in a patient presenting with febrile neutropenia.

Comparison of DNA Extraction Protocols and Molecular Targets to Diagnose Tuberculous Meningitis.

Tuberculous meningitis (TBM) is a severe form of extrapulmonary tuberculosis. The aims of this study were to evaluate in-house molecular diagnostic protocols of DNA extraction directly from CSF samples and the targets amplified by qPCR as an accurate and fast diagnosis of TBM. One hundred CSF samples from 68 patients suspected of TBM were studied. Four DNA extraction techniques (phenol-chloroform-thiocyanate guanidine, silica thiocyanate guanidine, resin, and resin with ethanol) were compared and CSF samples were used to determine the best target (IS6110, MPB64, and hsp65 KDa) by qPCR. The extraction protocol using the phenol-chloroform-thiocyanate guanidine showed the best results in terms of quantification and sensitivity of PCR amplification, presenting up to 10 times more DNA than the second best protocol, the silica guanidine thiocyanate. The target that showed the best result for TBM diagnosis was the IS6110. This target showed 91% sensitivity and 97% specificity when we analyzed the results by sample and showed 100% sensitivity and 98% specificity when we analyzed the results by patient. The DNA extraction with phenol-chloroform-thiocyanate guanidine followed by IS6110 target amplification has been shown to be suitable for diagnosis of TBM in our clinical setting.

Clinical relevance of molecular identification of microorganisms and detection of antimicrobial resistance genes in bloodstream infections of paediatric cancer patients.

BACKGROUND: Bloodstream infections (BSIs) are the major cause of mortality in cancer patients. Molecular techniques are used for rapid diagnosis of BSI, allowing early therapy and improving survival. We aimed to establish whether real-time quantitative polymerase chain reaction (qPCR) could improve early diagnosis and therapy in paediatric cancer patients, and describe the predominant pathogens of BSI and their antimicrobial susceptibility. METHODS: Blood samples were processed by the BACTEC system and microbial identification and susceptibility tests were performed by the Phoenix system. All samples were screened by multiplex 16 s rDNA qPCR. Seventeen species were evaluated using sex-specific TaqMan probes and resistance genes blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB and mecA were screened by SYBR Green reactions. Therapeutic efficacy was evaluated at the time of positive blood culture and at final phenotypic identification and antimicrobial susceptibility results. RESULTS: We analyzed 69 episodes of BSI from 64 patients. Gram-positive bacteria were identified in 61 % of the samples, Gram-negative bacteria in 32 % and fungi in 7 %. There was 78.2 % of agreement between the phenotypic and molecular methods in final species identification. The mecA gene was detected in 81.4 % of Staphylococcus spp., and 91.6 % were concordant with the phenotypic method. Detection of vanA gene was 100 % concordant. The concordance for Gram-negative susceptibilities was 71.4 % for Enterobacteriaceae and 50 % for Pseudomonas aeruginosa. Therapy was more frequently inadequate in patients who died, and the molecular test was concordant with the phenotypic susceptibility test in 50 %. CONCLUSIONS: qPCR has potential indication for early identification of pathogens and antimicrobial resistance genes from BSI in paediatric cancer patients and may improve antimicrobial therapy.

Analysis of Intraocular Lens Biofilms and Fluids After Long-Term Uncomplicated Cataract Surgery.

PURPOSE: Postoperative endophthalmitis is a potentially sight-threatening complication of cataract surgery. However, the pathophysiological mechanisms are not completely understood. We sought to study and evaluate the intraocular environment (aqueous and vitreous humors), the capsular tissue, and the intraocular lens (IOL) surfaces of normal eyes after long-term uncomplicated cataract surgery. DESIGN: Experimental laboratory investigation. METHODS: We studied 69 eyes donated for transplantation that had previously undergone cataract surgery with posterior chamber IOL implantation and that had no recorded clinical history of postoperative inflammation. We assessed the intraocular environment (DNA traces and biofilm formation) by microbiological evaluation of intraocular fluids using conventional microbiology and molecular techniques, including assessment for the presence of microbes (biofilm formation) on the IOL surface by scanning electron microscopy and ultrastructural capsular remnants by transmission electron microscopy. RESULTS: Isolated or aggregated cocci were probable in 18.8% of IOL optic surfaces (n = 13) studied by scanning electron microscopy, suggesting the presence of bacterial biofilm. In 3 intraocular fluid samples for IOLs with biofilm, we identified 16S rDNA by polymerase chain reaction and sequencing. No microbial contamination was found in intraocular fluids by conventional microbiological methods. CONCLUSIONS: Our data suggest the possibility of bacterial biofilm formation on the optic surface of IOLs in normal eyes after long-term uncomplicated cataract surgery even in the absence of clinical or subclinical symptoms.

Diagnosis of bacteremia in pediatric oncologic patients by in-house real-time PCR.

BACKGROUND: Infections 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. METHODS: A 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 bla SHV, bla TEM, bla CTX, bla KPC, bla IMP, bla SPM, bla VIM, vanA, vanB and mecA were detected using the SYBR Green method. RESULTS: Positive 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 ß-lactamases were detected in six samples and metallo-ß-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 bla KPC, bla VIM, bla IMP and bla SHV genes were not detected in this study. CONCLUSION: Real-time PCR is a useful tool in the early identification of pathogens and antimicrobial resistance genes from bloodstream infections of pediatric oncologic patients.

Diagnosis by real-time polymerase chain reaction of pathogens and antimicrobial resistance genes in bone marrow transplant patients with bloodstream infections.

BACKGROUND: Early 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. METHODS: A 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. RESULTS: Twenty-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-ß-lactamase genes were not detected. Five fungal species were identified only by real-time PCR. CONCLUSIONS: Real-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.