Nested-PCR as a tool for the detection and differentiation of Gram-positive and Gram-negative bacteria in patients with sepsis-septic shock (original) (raw)
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PloS one, 2012
Treatment of septic shock relies on appropriate antimicrobial therapy. Current culture based methods deliver final results after days, which may delay potentially lifesaving adjustments in antimicrobial therapy. This study was undertaken to compare PCR with blood culture results under routine conditions regarding 1. impact on antimicrobial therapy, and 2. time to result, in patients with presumed sepsis. This was an observational study in a 50 beds ICU of a university hospital. In 245 patients with suspected sepsis, 311 concomitant blood cultures and blood for multiplex PCR (VYOO(®)) were obtained. 45 of 311 blood cultures (14.5%) and 94 of 311 PCRs (30.1%) were positive. However, blood culture or microbiological sampling from the presumed site of infection rarely confirmed PCR results and vice versa. Median time to positivity and interquartile range were 24.2 (18.0, 27.5) hours for the PCR and 68 (52.2, 88.5) hours for BC (p<0.01). PCR median time to result was dependent on tech...
The Journal of Infection in Developing Countries, 2014
Introduction: Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumanii have been found to be the most prevalent bacteremia-causing bacteria in patients with septicemia. Early detection of bloodstream infection (BSI) is crucial in the clinical setting. A multiplex PCR method for identification of these agents in clinical samples has been developed in parallel by conventional microbiological methods. Methodology: The target genes selected for each of the organisms were very specific for designing primers. Design of primers was done using Mega4, Allel ID6, Oligo6, and Oligo analyzer software. The test comprises a universal PCR from the 16S rDNA gene and multiplex PCR from the rpoB, gyrA, sss, and chromosome X (as an internal control). Results: The sensitivity and specificity for universal PCR and multiplex PCR in comparison with BC were 83.87% and 91.58%, and 74.19% and 91.58%, respectively. The positive predictive value (PPV) and the negative predictive value (NPV) for these two PCRs were 76.47% and 94.57%, and 74.19% and 91.58%, respectively. PCR failed to identify bacteria which were found conventionally in only 3.96% and 6.34% of the cases by universal and multiplex PCR (mostly bacteria not included in the PCR cassette). In 6.34% of the cases, multiplex PCR afforded identification of bacteria, but BC showed no bacteria in the sample. Conclusions: The multiplex PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Rapid detection of bacteria by multiplex PCR appears to be a valuable tool, allowing earlier pathogen-adopted antimicrobial therapy in critically ill patients.
Clinical Microbiology and Infection, 2009
Severe sepsis is increasingly a cause of death. Rapid and correct initial antimicrobial treatment reduces mortality. The aetiological agent(s) cannot always be found in blood cultures (BCs). A novel multiplex PCR test (SeptiFast (alpha version)) that allows identification of 20 bacterial and fungal species directly from blood was used, comparatively with BC, in a multicentre trial of patients with suspected bacterial or fungal sepsis. Five hundred and fifty-eight paired samples from 359 patients were evaluated. The rate of positivity was 17% for BC and 26% for SeptiFast. Ninety-six microorganisms were isolated with BC, and 186 microorganisms were identified with SeptiFast; 231 microrganisms were found by combining the two tests. Of the 96 isolates identified with BC, 22 isolates were considered to be contaminants. Of the remaining 74 non-contaminant BC isolates available for comparison with SeptiFast, 50 were identified as a species identical to the species identified with SeptiFast in the paired sample. Of the remaining 24 BC isolates for which the species, identified in the BC, could not be detected in the paired SeptiFast sample, 18 BC isolates were identified as a species included in the SeptiFast master list, and six BC isolates were identified as a species not included in the SeptiFast master list. With SeptiFast, 186 microorganisms were identified, 12 of which were considered to be contaminants. Of the 174 clinically relevant microorganisms identified with SeptiFast, 50 (29%) were detected by BC. More than half of the remaining microorganisms identified with SeptiFast (but not isolated after BC) were also found in routine cultures of other relevant samples taken from the patients. Future clinical studies should assess whether the use of SeptiFast is of significant advantage in the detection of bloodstream pathogens.
Coll Antropol, 2014
Several studies have been performed investigating the role of a real-time multiplex polymerase chain reaction assay LightCycler SeptiFast with inconsistent results. In prospective evaluation of adult patients with severe sepsis or septic shock SeptiFast assay and blood culture results were compared regarding concordance, the impact of SeptiFast assay on antimicrobial therapy adjustment, time to results and the role of SeptiFast assay as a marker of disease severity. 63 blood sample sets were collected from 57 patients. 51 (80.9%) results were concordant negative and 7 (11.1%) concordant posi- tive. In one (1.6%) sample set blood culture was positive and SeptiFast assay negative, in three (4.8%) sample sets with negative blood cultures pathogens were detected by SeptiFast assay and in one (1.6%)patient an additional pathogen was detected by SeptiFast assay. If blood culture is considered as "gold standard", 1 (1.6%) SeptiFast false negative and 4 (6.3%) false positive resul...
International Journal of Infectious Diseases
Introduction: For the identification of bacterial pathogens, blood culture is still the gold standard diagnostic method. However, several disadvantages apply to blood cultures, such as time and rather large volumes of blood sample required. We have previously established an optimised multiplex real-time PCR method in order to diagnose bloodstream infections. Material and methods: In the present study, we evaluated the diagnostic performance of this optimised multiplex RT-PCR in blood samples collected from 110 septicaemia patients enrolled at the 108 Military Central Hospital, Hanoi, Vietnam. Results: Positive results were obtained by blood culture, the Light Cylcler-based SeptiFast J assay and our multiplex RT-PCR in 35 (32%), 31 (28%), and 31 (28%) samples, respectively. Combined use of the three methods confirmed 50 (45.5%) positive cases of bloodstream infection, a rate significantly higher compared to the exclusive use of one of the three methods (P = 0.052, 0.012 and 0.012, respectively). The sensitivity, specificity and area under the curve (AUC) of our assay were higher compared to that of the SeptiFast J assay (77.4%, 86.1% and 0.8 vs. 67.7%, 82.3% and 0.73, respectively). Combined use of blood culture and multiplex RT-PCR assay showed a superior diagnostic performance, as the sensitivity, specificity, and AUC reached 83.3%, 100%, and 0.95, respectively. The concordance between blood culture and the multiplex RT-PCR assay was highest for Klebsiella pneumonia (100%), followed by Streptococcus spp. (77.8%), Escherichia coli (66.7%), Staphylococcus spp. (50%) and Salmonella spp. (50%). In addition, the use of the newly established multiplex RT-PCR assay increased the spectrum of identifiable agents (Acintobacter baumannii, 1/32; Proteus mirabilis, 1/32). Conclusion: The combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients.
Journal of Infection and Public Health, 2020
Background: Early and accurate laboratory diagnosis and appropriate management of infection improves the survival rate in sepsis. In this study we evaluated broad range 16S rRNA and 16 S-23 S intergenic spacer region (ISR) PCR assays followed by nucleotide sequencing directly from patients' serum and automated blood culture for laboratory diagnosis in admitted sepsis patients. Methods: A broad range 16S rRNA PCR and 16 S-23 S ISR PCR assay followed by nucleotide sequencing was used directly from patients' serum in hospital admitted patients in 62 sepsis and 16 suspected blood stream infection (sBSI) patients. Automated blood culture was also used in the same patients. Nucleotide sequences were analyzed against NCBI Genbank database and organisms were identified using CLSI MM18A guidelines. Results: Bacterial culture were positive in 10/62 (16.12%) sepsis and 3/16 (18.75%) suspected BSI patients along with 3 detected fungi (2 in sepsis and 1 in suspected BSI group). PCR assay was positive in 36/62 (58.06%) sepsis and 6/16 (37.5%) suspected BSI patients respectively. All but 2 bacteria (both from culture negative patients) detected by PCR assay could be identified from nucleotide sequencing. Survival in sepsis patients was 77%. PCR assay could detect bacteria in 9/14 (64.28%) of sepsis patients with death. Conclusion: Broad range PCR assay was far superior for early diagnosis of infection. The bacteria which could not be detected by culture and were not commonly reported from this centre, were detected by the broad range PCR assays. Detection of these rare bacteria/fungi had significant clinical correlation with patient's underlying clinical conditions, immune status and prognosis. The tests could provide definitive diagnosis of infection in >58% of sepsis patients, which helped in patient management and better survival.
Journal of Intensive Care, 2015
Background: Molecular amplification techniques are suggested to be a useful adjunct in early detection of pathogens in septic patients. The aim was to study the feasibility of a polymerase chain reaction (PCR) assay compared to the standard microbiological culture (MC) technique in identification of pathogenic microorganisms from blood and non-blood samples in septic patients. Methods: Samples for pathogen identification were taken during febrile septic episodes (SE) in 54 patients with sepsis and analyzed using both MC and PCR. Semi-automated multiplex PCR, provided by Philips Medical Systems, was able to detect nine different pathogens. The accuracy of pathogen identification using PCR vs. MC as well as the time-saving effect of PCR on the potential decision-making process for antimicrobial therapy was evaluated. Results: In a total of 258 samples taken during 87 SE, both methods yielded more pathogens from the non-blood than blood samples (87 % vs. 45 %; p = 0.002). PCR identified more pathogens than MC in the blood samples (98 vs. 21; p < 0.0001), but not in other body fluids. In 35 SE, the potential decision on appropriate antimicrobial therapy based on PCR results could have been made 50 (median; interquartile range 35-87) hours earlier than decisions based on standard MC. Conclusions: In septic patients, multiplex PCR identified more pathogenic microorganisms isolated from the blood samples than the standard MC technique. In the non-blood samples, PCR was comparable to that of MC.
European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology, 2018
Molecular tests may enable early adjustment of antimicrobial therapy and be complementary to blood culture (BC) which has imperfect sensitivity in critically ill patients. We evaluated a novel multiplex real-time PCR assay to diagnose bloodstream pathogens directly in whole blood samples (BSI-PCR). BSI-PCR included 11 species- and four genus-specific PCRs, a molecular Gram-stain PCR, and two antibiotic resistance markers. We collected 5 mL blood from critically ill patients simultaneously with clinically indicated BC. Microbial DNA was isolated using the Polaris method followed by automated DNA extraction. Sensitivity and specificity were calculated using BC as reference. BSI-PCR was evaluated in 347 BC-positive samples (representing up to 50 instances of each pathogen covered by the test) and 200 BC-negative samples. Bacterial species-specific PCR sensitivities ranged from 65 to 100%. Sensitivity was 26% for the Gram-positive PCR, 32% for the Gram-negative PCR, and ranged 0 to 7% f...
Journal of Clinical Microbiology, 2011
Sepsis is a major health problem in newborns and children. Early detection of pathogens allows initiation of appropriate antimicrobial therapy that strongly correlates with positive outcomes. Multiplex PCR has the potential to rapidly identify bloodstream infections, compensating for the loss of blood culture sensitivity. In an Italian pediatric hospital, multiplex PCR (the LightCycler SeptiFast test) was compared to routine blood culture with 1,673 samples obtained from 803 children with suspected sepsis; clinical and laboratory information was used to determine the patient infection status. Excluding results attributable to contaminants, SeptiFast showed a sensitivity of 85.0% (95% confidence interval [CI] ؍ 78.7 to 89.7%) and a specificity of 93.5% (95% CI ؍ 92.1 to 94.7%) compared to blood culture. The rate of positive results was significantly higher with SeptiFast (14.6%) than blood culture (10.3%) (P < 0.0001), and the overall positivity rate was 16.1% when the results of both tests were combined. Staphylococcus aureus (11.6%), coagulase-negative staphylococci (CoNS) (29.6%), Pseudomonas aeruginosa (16.5%), and Klebsiella spp. (10.1%) were the most frequently detected. SeptiFast identified 97 additional isolates that blood culture failed to detect (24.7% P. aeruginosa, 23.7% CoNS, 14.4% Klebsiella spp., 14.4% Candida spp.). Among specimens taken from patients receiving antibiotic therapy, we also observed a significantly higher rate of positivity of SeptiFast than blood culture (14.1% versus 6.5%, respectively; P < 0.0001). On the contrary, contaminants were significantly more frequent among blood cultures than SeptiFast (n ؍ 97 [5.8%] versus n ؍ 26 [1.6%]), respectively; P < 0.0001). SeptiFast served as a highly valuable adjunct to conventional blood culture in children, adding diagnostic value and shortening the time to result (TTR) to 6 h.