Conventional Blood Culture Research Articles

Usefulness of Real-Time PCR for the Diagnosis of Sepsis in ICU-Acquired Infections

Real-time PCR methods are able to rapidly detect a wide panel of microorganisms. These methods are of interest in critically ill patients to determine the presence of bacteria in the blood and other biological samples, especially in those patients with prior antimicrobial treatment. In intensive care unit (ICU), the LightCycler SeptiFast (LC-SF) Test provides 1.5 to 2 fold higher positivity rate compared with conventional blood cultures. Although identification of the bacterium by LC-SF is rapid and sensitive, susceptibility test could not be performed using this technique, except the methicillin- resistance for Staphylococci. The conventional cultures remain necessary for samples in ICU because of the high incidence of multidrug-resistant bacteria and the need for antimicrobial susceptibility of the bacterium to treat the patient correctly. A negative result for a Gram positive or negative bacterium allows deescalating the initial antimicrobial treatment, and decreasing the pressure of selection. Moreover, it is necessary to understand and interpret a DNA signal knowing that a dead bacterial material may be detected in a patient without any infection. What is the clinical relevance of bacterial DNA present in the blood and does the DNAemia found reflect true infection? Cost-effectiveness of the real-time PCR should be determined. Meanwhile, this test should be restricted to severe clinical situations, especially ICU patients with severe sepsis. In the future, real-time PCR tests should include more pathogens and antimicrobial resistant targets.

Non–Culture-Based Methods for the Diagnosis of Invasive Candidiasis

Given the limitations of current fungal diagnostics, the use of non–culture-based methods for the diagnosis of invasive candidiasis (IC) is highly warranted. The implementation of molecular diagnostic strategies could permit the timely onset of appropriate therapy and may be expected to pave the way for improved clinical outcome of IC. Polymerase chain reaction (PCR) may have higher sensitivity for the diagnosis of IC than conventional blood cultures. The detection of fungal antigens generally requires a large fungal burden, and the presence of fungus-specific antibodies may not correlate with the underlying diseases. Therefore, the combined mannan and anti-mannan antibody testing is recommended. No single test has been shown convincingly to compensate for all the limitations of culture. Real-time PCR coupled with fungal culture and/or antigen detection will likely be required to significantly ameliorate the diagnostic problems in IC.

Molecular diagnosis of bacteriemia in patients with neutropenic febrile oncohematologic.

9033 Background: The rapid microbiologic diagnosis in the course of fever of unknown origin plays an important role for the appropriate treatment in neutropenic patients. Blood culture helps identify bacterial and fungal bacteriemia. However, its efficacy can be limited due to its low sensitivity when the patient is receiving antibiotics and to the long time required to identify the pathogen (24-72 hours). Septifast (Roche Molecular Systems) is a new molecular technique which detects in 6 hours the 25 most important bacterial and fungal species reported to cause bloodstream infections. The aim of the study was to compare Septifast with conventional blood cultures in blood samples from neutropenic febrile patients. Methods: Forty-five blood samples from 19 patients were prospectively tested. All of them presented fever with neutrophil count <0.5x109/L. The patients (pts) were receiving antibiotic therapy (piperaciline-tazobactam/imipenem (19pts), vancomicine (7pts), amikacine (3pts), aciclovir (2pts) and antifungal therapy (9pts). For each sample, two blood culture vials for aerobic/anaerobic cultures and an EDTA whole-blood samples for Septifast were drawn. Results: Thirty-two percent of patients presented a positive result. Eight of the 45 (17.7%) blood samples studied were positive by PCR assay (S.aureus: 2; P.aeruginosa: 3; E.coli: 1; Coagulase-negative Staphylococci: 2). Two blood samples were positive for P.aeruginosa and Coagulase-negative Staphylococci by both Septifastâ and blood culture. The entire negative PCR test results correlated with negative blood culture samples. The negative predictive value was 100% with a sensitivity of 100% and a specificity of 86% (Table). Conclusions: Septifast is an easy and sensitive method for detecting pathogens in severely immunocompromised patients, allowing results in a shorter time and with higher sensitivity than conventional blood cultures. Comparision results from Septifast test and conventional blood culture system. Blood culture Results Positive samples (%) Negative samples (%) Total Septifast Positive samples (%) 2 (4.4) 6 (13.3) 8 (17.7) Negative samples (%) 0 (0) 37 (82.2) 37 (82.2) Total (%) 2 (4.4) 43 (95.5) 45 (100)

Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view

Polymerase chain reaction (PCR)-based techniques allow more rapid and sensitive detection of pathogens compared with conventional blood culture. Nevertheless, the climate of opinion of relevant studies is that currently PCR can supplement but not replace blood culture. In numerous studies, combined detection rate of both methods was significantly higher compared with PCR or blood culture alone. Also, complete determination of antibiotic resistance can currently be performed only by blood culture. Further increase of the panel of multiplex PCR is complicated, because the vast majority of sepsis pathogens are already included, primer interactions leading to primer heteromers limit the amount of targets detectable within one PCR tube, and an array of too many individual PCR reactions for investigation of a single specimen leads to high cost and workload. Except for diagnostics of patients in whom unusual, not culturable, or fastidious pathogens are detected more often, such as immunosuppressed patients with suspected parasitic infection, etc., it might even not be necessary to further increase the spectrum of detectable species. If the primary aim of PCR diagnostics is to decrease inappropriate empirical treatment and improve patient outcome, detection should focus on those pathogens or resistance determinants that are not covered by guideline-recommended treatment regimens and that have been identified as the major cause of inappropriate treatment according to current studies. In our opinion, such a narrower assay is more cost effective, may achieve higher accuracy due to reduced intratest interference, and would better address current and emerging clinical needs.

Multiplex PCR Allows Rapid and Accurate Diagnosis of Bloodstream Infections in Newborns and Children with Suspected Sepsis

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.

Molekularbiologische Sepsisdiagnostik mittels Multiplex-PCR in der chirurgischen Intensivmedizin als geeignete Alternative zur konventionell-mikrobiellen Kultur - ein repräsentativer Überblick

Sepsis causes a substantial rate of morbidity and mortality in intensive care patients, which is, in particular, triggered by an inadequate antimicrobial treatment from the beginning. Conventional microbiological standard procedures cannot provide valuable information on bacterial or fungal species of sepsis-relevant microbes within the first hours of a developing sepsis. However, multiplex PCR (PCR-M) focussing on the spectrum of the most relevant sepsis-associated microbes can considerably shorten the time period; the analytical tests have been standardised and, subsequently, inaugurated into clinical practice; they also have thus been available since 2005. Interestingly, in the surgical field an appropriate summary and concluding recommendation have been lacking so far. AIM, MATERIAL AND METHODS: A compact short overview based on a characteristic selection of relevant references from the literature is given on the commercially available sepsis-associated multiplex-PCR methods, reflecting critically the time point of inauguration, clinical value and future perspectives including our own experiences from clinical practice and medical studies. Multiplex PCR in adult sepsis patients yielded in a range from 13.7 to 39.9 % of positive findings, whereas conventional blood cultures only range from 8 to 29.9 %. From 8 to 16.9 % of all investigations performed prompted us to a change of the antimicrobial treatment by using a positive PCR-M finding. A prospective study (end-point, reduction of sepsis-associated mortality) has not yet been initiated. Positive PCR-M findings correlate with an increased morbidity and mortality as well as clinical and laboratory sepsis parameters. Recent studies have aimed for a comparison of PCR-M on sepsis-associated microbes with regard to specificity and sensitivity with the current "gold standard", conventional blood culture. A few studies wrongly claimed to compare the methods because of the difference in the procedures; in addition, blood culture as gold standard has been increasingly considered as very problematic from a methodological point of view. Recent publications on multiplex-PCR studies in frequently heterogenic groups of patients have been mostly performed with the Lightcycler-Septifast® test (LC-SF) with great success. The procedure has provided evidence of an improved detection rate of sepsis-associated microbes, favourable concordance of positive PCR-M findings with clinical and laboratory sepsis parameters and substantial time-saving in the microbiological analysis of the specific microbial species, which is simultaneously associated with an earlier initiation of an adequate antimicrobial treatment regimen. The available study data suggest that systrematic investigations on the molecular biological procedures should be rather related to a different standard based on the LC-SF. Positive PCR-M findings have been accepted as a sepsis marker in the mean time.

Diagnostic accuracy and potential clinical value of the LightCycler SeptiFast assay in the management of bloodstream infections occurring in neutropenic and critically ill patients

The objectives of this study were to compare the performance of the LightCycler SeptiFast Test MGRADE and conventional blood culture in the etiological diagnosis of febrile episodes occurring in neutropenic and critically ill patients (in the intensive care unit; ICU), and to assess the potential clinical value of the SeptiFast test in patient management. A total of 86 febrile episodes occurring in 33 neutropenic patients and 53 ICU patients were analyzed. Blood samples for blood culture and SeptiFast testing were obtained at the onset of fever, before the implementation of empirical antimicrobial therapy. The overall microorganism-to-isolate agreement between the SeptiFast test and blood culture was 69% (κ=0.37) in neutropenic patients and 75% (κ=0.56) in ICU patients. The sensitivity of the SeptiFast assay for clinically relevant episodes of bacteremia and fungemia was 62% in neutropenic patients and 70% in ICU patients. Based on SeptiFast results, empirical treatments were deemed adequate in all but one of the febrile episodes. Nevertheless, early antibiotic treatment readjustment was judged feasible in most of clinically significant episodes overall. The SeptiFast assay is a valuable ancillary method for the diagnosis of bloodstream infections in neutropenic and ICU patients. In these clinical settings, results of the SeptiFast assay may lead to a more targeted antibiotic therapy early after the onset of fever.

Post-natal CMV infection via mother's milk in ELBW infants born to CMV seropositive mothers

s for 2nd International Conference on Clinical Neonatology Comparison of conventional blood culture method and BacT/Alert system with respect to yield and time to positivity in neonatal sepsis Arti Maria⁎, Sumitha Phalgun, Charoo Hans, N. Dubey, Deepak Goyal PGIMER & Associated Dr RML Hospital, New Delhi, India ⁎Corresponding author. Efficient and faster ways of diagnosing neonatal sepsis than conventional blood culture are needed in clinical practice. Recently, automated systems have been developed to address this issue. We hypothesized that automated compared to conventional methods of blood culture may give better isolation performances and shorter time to yield in the diagnosis of neonatal sepsis. This was a single-center, prospective, cohort study enrolling neonates with suspected neonatal sepsis in tertiary neonatal referral unit of north India. From May 2008 to April 2009, ninety consecutive neonates with suspected sepsis were enrolled with the objective of comparing the sensitivity and the time needed to yield positivity of conventional vs. BacT/Alert blood culture. Conventional and automated cultureswere collected in standardized manner from each baby, the order being alternate in consecutive babies. Refusal to consent, being ELBW and showing hemodynamical unstability were the only exclusion criteria for the neonates screened for inclusion in the study. Statistical analysis was conducted with the SPSS software version 11.0. The results showed that positivity by BacT/Alert was 38.8% (35/90) compared to 14.4% (13/90)with conventionalmethod (p<0.05). BacT/ Alert detected 30% isolates as early as within 12 h and 100% by 48 h whereas conventional detected none before 48 h and 69.2% by 72 h (p<0.001). Staphylococcus aureus was the commonest isolated microorganism (40% of cases). We concluded that a greater yield and earlier detection of pathogens by the Bact/Alert system make it more suitable for early definitive diagnosis of neonatal sepsis. This has implications for timely rational antibiotic therapy with possible favourable outcomes in neonatal sepsis. doi:10.1016/j.earlhumdev.2010.12.021 Safety of aqueous chlorhexidine gluconate for skin antisepsis in VLBW infants Maria Grazia Capretti⁎, Marica Spinelli, Elisabetta Tridapalli, Santo Arcuri, Anna Malavolti, Emanuela Callea, Giacomo Faldella Neonatal Intensive Care Unit, St. Orsola-Malpighi General Hospital, University of Bologna, Bologna, Italy Infection Control Unit, St. Orsola-Malpighi General Hospital, University of Bologna, Bologna, Italy ⁎Corresponding author. No guidelines are available for antisepsis in infants with age <2 months. The aim of this study is to evaluate the efficacy and safety of aqueous chlorhexidine gluconate (aCG) at different concentrations for skin disinfection in VLBW (birth weight <1500 g) infants. Two different protocols for skin antisepsis were prospectively used. The rate of nosocomial infection and adverse effects were compared. In the first period (January 2007 to December 2008) we used 2%aCG solution, regardless of gestational age (GA) and birth weight (BW); in the second period (January 2009 to October 2010) 0.1%aCG was used in the first ten days of life in newborns with GA=27 weeks and/or BW=750 g and 1%aCG, in the remaining cases. Microbiological culture of CVC-tips, blood samples, urine and respiratory tract specimens were collected when infection was suspected. First period infants 15/124 vs second period infants 14/103 were found infected after 72 h of life (p=0.842); among infants with BW<750 g, 9/31were infected in the first period vs 3/21 in the second period (p=0.318). Five ELBW infants (BW<1000 g) showed burns using 2%aCG prior to insertion of umbilical venous or peripherally inserted central catheters. No burns or other adverse events were observed in the second period. Our data show that 2%aCG may not be safe when used in ELBW infants in the first days of life, suggesting that antisepsis solution in premature should be differentiated according to GA and chronologic age. doi:10.1016/j.earlhumdev.2010.12.022 Post-natal CMV infection via mother's milk in ELBW infants born to CMV seropositive mothers

Comparison of conventional blood culture method and BacT/Alert system with respect to yield and time to positivity in neonatal sepsis

s for 2nd International Conference on Clinical Neonatology Comparison of conventional blood culture method and BacT/Alert system with respect to yield and time to positivity in neonatal sepsis Arti Maria⁎, Sumitha Phalgun, Charoo Hans, N. Dubey, Deepak Goyal PGIMER & Associated Dr RML Hospital, New Delhi, India ⁎Corresponding author. Efficient and faster ways of diagnosing neonatal sepsis than conventional blood culture are needed in clinical practice. Recently, automated systems have been developed to address this issue. We hypothesized that automated compared to conventional methods of blood culture may give better isolation performances and shorter time to yield in the diagnosis of neonatal sepsis. This was a single-center, prospective, cohort study enrolling neonates with suspected neonatal sepsis in tertiary neonatal referral unit of north India. From May 2008 to April 2009, ninety consecutive neonates with suspected sepsis were enrolled with the objective of comparing the sensitivity and the time needed to yield positivity of conventional vs. BacT/Alert blood culture. Conventional and automated cultureswere collected in standardized manner from each baby, the order being alternate in consecutive babies. Refusal to consent, being ELBW and showing hemodynamical unstability were the only exclusion criteria for the neonates screened for inclusion in the study. Statistical analysis was conducted with the SPSS software version 11.0. The results showed that positivity by BacT/Alert was 38.8% (35/90) compared to 14.4% (13/90)with conventionalmethod (p<0.05). BacT/ Alert detected 30% isolates as early as within 12 h and 100% by 48 h whereas conventional detected none before 48 h and 69.2% by 72 h (p<0.001). Staphylococcus aureus was the commonest isolated microorganism (40% of cases). We concluded that a greater yield and earlier detection of pathogens by the Bact/Alert system make it more suitable for early definitive diagnosis of neonatal sepsis. This has implications for timely rational antibiotic therapy with possible favourable outcomes in neonatal sepsis. doi:10.1016/j.earlhumdev.2010.12.021 Safety of aqueous chlorhexidine gluconate for skin antisepsis in VLBW infants Maria Grazia Capretti⁎, Marica Spinelli, Elisabetta Tridapalli, Santo Arcuri, Anna Malavolti, Emanuela Callea, Giacomo Faldella Neonatal Intensive Care Unit, St. Orsola-Malpighi General Hospital, University of Bologna, Bologna, Italy Infection Control Unit, St. Orsola-Malpighi General Hospital, University of Bologna, Bologna, Italy ⁎Corresponding author. No guidelines are available for antisepsis in infants with age <2 months. The aim of this study is to evaluate the efficacy and safety of aqueous chlorhexidine gluconate (aCG) at different concentrations for skin disinfection in VLBW (birth weight <1500 g) infants. Two different protocols for skin antisepsis were prospectively used. The rate of nosocomial infection and adverse effects were compared. In the first period (January 2007 to December 2008) we used 2%aCG solution, regardless of gestational age (GA) and birth weight (BW); in the second period (January 2009 to October 2010) 0.1%aCG was used in the first ten days of life in newborns with GA=27 weeks and/or BW=750 g and 1%aCG, in the remaining cases. Microbiological culture of CVC-tips, blood samples, urine and respiratory tract specimens were collected when infection was suspected. First period infants 15/124 vs second period infants 14/103 were found infected after 72 h of life (p=0.842); among infants with BW<750 g, 9/31were infected in the first period vs 3/21 in the second period (p=0.318). Five ELBW infants (BW<1000 g) showed burns using 2%aCG prior to insertion of umbilical venous or peripherally inserted central catheters. No burns or other adverse events were observed in the second period. Our data show that 2%aCG may not be safe when used in ELBW infants in the first days of life, suggesting that antisepsis solution in premature should be differentiated according to GA and chronologic age. doi:10.1016/j.earlhumdev.2010.12.022 Post-natal CMV infection via mother's milk in ELBW infants born to CMV seropositive mothers

Comparison of Different PCR Methods for Detection of Brucella spp. in Human Blood Samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.

Comparación entre métodos convencionales, ChromAgar Candida ® y el método de la PCR para la identificación de especies de Candida en aislamientos clínicos

The increase in the incidence of yeast species causing fungemia in susceptible immunocompromised patients in the last two decades and the low sensitivity of conventional blood culture has led to the need to develop alternative approaches for the early detection and identification of causative species. The aim of this study was to compare the usefulness of molecular testing by the polymerase chain reaction (PCR) and conventional methods to identify clinical isolates of different species, using the ID32C ATB system (bioMérieux, France), chromogenic culture Chromagar Candida® (CHROMagar, France) and morphogenesis in corn meal agar. We studied 79 isolates, in which the most prevalent species using the system ID32C and PCR was C. albicans, followed by C. tropicalis, C. glabrata and C .krusei. PCR patterns obtained for the identification of clinical isolates were stable and consistent in the various independent studies and showed good reproducibility, concluding that PCR with species-specific primers that amplify genes ITS1 and ITS2 for rRNA or topoisomerase II primers is a very specific and sensitive method for the identification of C. glabrata, C. krusei, C. albicans, and with less specificity for C. tropicalis.

Efficacy of bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR for detecting neonatal sepsis: a case control study

BackgroundNeonatal sepsis is difficult to diagnose and pathogens cannot be detected from blood cultures in many cases. Development of a rapid and accurate method for detecting pathogens is thus essential. The main purpose of this study was to identify etiological agents in clinically diagnosed neonatal sepsis using bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR (BrRNA-RT-qPCR) and to conduct comparisons with the results of conventional blood culture. Since BrRNA-RT-qPCR targets bacterial ribosomal RNA, detection rates using this approach may exceed those using conventional PCR.MethodsSubjects comprised 36 patients with 39 episodes of suspected neonatal sepsis who underwent BrRNA-RT-qPCR and conventional blood culture to diagnose sepsis. Blood samples were collected aseptically for BrRNA-RT-qPCR and blood culture at the time of initial sepsis evaluation by arterial puncture. BrRNA-RT-qPCR and blood culture were undertaken using identical blood samples, and BrRNA-RT-qPCR was performed using 12 primer sets.ResultsPositive rate was significantly higher for BrRNA-RT-qPCR (15/39, 38.5%) than for blood culture (6/39, 15.4%; p = 0.0039). BrRNA-RT-qPCR was able to identify all pathogens detected by blood culture. Furthermore, this method detected pathogens from neonates with clinical sepsis in whom pathogens was not detected by culture methods.ConclusionsThis RT-PCR technique is useful for sensitive detection of pathogens causing neonatal sepsis, even in cases with negative results by blood culture.

Detection of candidaemia in patients with and without underlying haematological disease

Diagnosing candidaemia remains difficult despite the development of new diagnostics. We report a direct comparison of three different blood-culture systems and four indirect tests. One hundred and fourteen episodes either with haematological disease and fever despite antibacterials, or with documented invasive candidiasis, were enrolled prospectively. Clinical, para-clinical information and surveillance cultures were obtained. Blood culture was performed using conventional blood-culture bottles, mycosis bottles, and the Isolator 10 lysis centrifugation system. Serum D-arabinitol/L-arabinitol (DA/LA) ratios were determined by gas chromatography mass spectrometry. Antigen, mannan-antigen (Ag) and anti-mannan antibody (Ab) were detected by CandTec, Platelia Candida Ag ELISA and Candida AB/AC/AK kits, respectively. Episodes were classified as proven (n = 24), probable (n = 14), possible (n = 52) or unlikely (n = 24) invasive candidiasis. Candidaemia involved C. albicans (17), C. albicans + C. glabrata (3), C. tropicalis (1) and yeast (1). Mycosis bottles yielded two additional positives and the conventional blood culture yielded one positive not identified by other blood-culture methods. Considering proven and unlikely episodes, respectively, sensitivity and specificity were as follows: mannan-Ag and/or anti-mannan Ab: 83.3%, 78.3%; DA/LA ratio: 41.7%, 86.4%; and CandTec Candida Ag: 66.6%, 70.8. Lowering the cut-off values to mannan-Ag 0.10 ng/mL and anti-mannan Ab 4 AU/mL, the values were: 100%, 73.9. Applying the DA/LA ratio to only patients with haematological neutropenia the values were: 75%, 90.5. Fungal blood culture allowed slightly improved detection of candidaemia. The best indirect test performance was obtained from combined mannan-Ag and anti-mannan Ab detection, especially with lower cut-offs. DA/LA ratio appears to be useful in the context of haematological neutropenia.

Preliminary clinical study using a multiplex real-time PCR test for the detection of bacterial and fungal DNA directly in blood

Early diagnosis of sepsis, rapid identification of the causative pathogen(s) and prompt initiation of appropriate antibiotic treatment have a combined impact on mortality due to sepsis. In this observational study, a new DNA-based system (LightCycler SeptiFast (LC-SF) test; Roche Diagnostics) allowing detection of 16 pathogens at the species level and four groups of pathogens at the genus level has been evaluated and compared with conventional blood cultures (BCs). One hundred BC and LC-SF results were obtained for 72 patients admitted to the intensive-care unit over a 6-month period for suspected sepsis. Microbiological data were compared with other biological parameters and with clinical data. The positivity rate of BCs for bacteraemia/fungaemia was 10%, whereas the LC-SF test allowed detection of DNA in 15% of cases. The LC-SF performance, based on its clinical relevance, was as follows: sensitivity, 78%; specificity, 99%; -positive predictive value, 93%; and negative predictive value, 95%. Management was changed for four of eight (50%) of the patients because organisms were detected by the LC-SF test but not by BC. LC-SF results were obtained in 7–15 h, in contrast to the 24–72 h required for BC. According to the LC-SF results, initial therapy was inadequate in eight patients, and antibiotic treatment was changed. Our results suggest that the LC-SF test may be a valuable complementary tool in the management of patients with clinically suspected sepsis.

Comparison of generic bacterial molecular detection (16S rRNA) with C-reactive protein and blood culture in the diagnosis of late-onset sepsis preterm infants: Abstract G177 Table 1

Background Preterm infants are at great risk of bloodstream infection while receiving intensive care. The incidence of late-onset bloodstream infection varies between 10–60%. Diagnosis is currently made with a combination of clinical signs and laboratory investigations. The use of new molecular methods to improve diagnosis in a range of infections has been reported in other age groups. Objective To compare a molecular 16S rRNA assay to conventional laboratory methods (C-reactive protein (CRP) and blood culture) in the diagnosis of late-onset sepsis in preterm babies. Methods A single-centre, prospective observational study was conducted. All babies with suspicion of late-onset sepsis were included. Blood for the 16S rRNA assay was taken along with routine investigations for sepsis (blood culture, CRP). The decision to label a baby as being “septic” was based on clinical data and the assessment by a national nosocomial infection scoring system. Sepsis status was then compared to results of blood culture, CRP and the 16S rRNA assay. Diagnostic odds ratio, sensitivity, specificity, positive likelihood and negative likelihood ratios were calculated. Results Conclusion This assay, which enables generic detection of bacteria without reliance on culture, has superior sensitivity and specificity in the diagnosis of late-onset infection than conventional laboratory methods. These results indicate that molecular methods should have a place in the diagnosis of late-onset sepsis in preterm babies. Further study is required to determine the extent to which these methods could be utilised.

A fast and highly sensitive blood culture PCR method for clinical detection of Salmonella enterica serovar Typhi

BackgroundSalmonella Typhi causes an estimated 21 million new cases of typhoid fever and 216,000 deaths every year. Blood culture is currently the gold standard for diagnosis of typhoid fever, but it is time-consuming and takes several days for isolation and identification of causative organisms. It is then too late to initiate proper antibiotic therapy. Serological tests have very low sensitivity and specificity, and no practical value in endemic areas. As early diagnosis of the disease and prompt treatment are essential for optimal management, especially in children, a rapid sensitive detection method for typhoid fever is urgently needed. Although PCR is sensitive and rapid, initial research indicated similar sensitivity to blood culture and lower specificity. We developed a fast and highly sensitive blood culture PCR method for detection of Salmonella Typhi, allowing same-day initiation of treatment after accurate diagnosis of typhoid.MethodsAn ox bile tryptone soy broth was optimized for blood culture, which allows the complete lysis of blood cells to release intracellular bacteria without inhibiting the growth of Salmonella Typhi. Using the optimised broth Salmonella Typhi bacteria in artificial blood samples were enriched in blood culture and then detected by a PCR targeting the fliC-d gene of Salmonella Typhi.ResultsTests demonstrated that 2.4% ox bile in blood culture not only lyzes blood cells completely within 1.5 hours so that the intracellular bacteria could be released, but also has no inhibiting effect on the growth of Salmonella Typhi.Three hour enrichment of Salmonella Typhi in tryptone soya broth containing 2.4% ox bile could increase the bacterial number from 0.75 CFU per millilitre of blood which is similar to clinical typhoid samples to the level which regular PCR can detect. The whole blood culture PCR assay takes less than 8 hours to complete rather than several days for conventional blood culture.ConclusionsThis novel blood culture PCR method is superior in speed and sensitivity to both conventional blood culture and PCR assays. Its use in clinical diagnosis may allow early detection of the causative organism and facilitate initiation of prompt treatment among patients with typhoid fever.

Investigation of Positive Streptococcus pneumoniae Urinary Antigen Test Results in a Korean University Hospital

Background: The Streptococcus pneumoniae urinary antigen test (SPUAT) (Binax Now, USA) was developed for detecting polysaccharide C in urine samples for rapid diagnosis of pneumococcal pneumonia, the most common cause of community-acquired pneumonia (CAP). To validate positive results of these tests, we retrospectively investigated all positive results obtained from the emergency room of a Korean university hospital among patients with suspected CAP. Methods: One hundred twenty-three positive SPUAT results were abstracted and analyzed from the authors' laboratory information system among the SPUAT results performed from 1,143 pneumonic patients admitted from the emergency room of a university hospital between 2007 and 2008. Medical records, including conventional microbiologic analysis results, were reviewed in detail for all positive test results. Results: Among 123 patients with the positive SPUAT results, 24 patients were excluded due to hospitalization history during the preceding month. Nine of 99 patients (9.1%) with suspected CAP had confirmed pneumococcal pneumonia upon conventional sputum or blood culture. Thirty-five positive results (35.4%) showed other microorganisms upon conventional methods, which might be due to possible cross-reactivity. Among those, 23 positive results were considered bacterial pneumonic agents, and 12 positive results were regarded as urinary tract infection strains or contaminating agents. Fifty-five positive SPUAT results (55.6%) showed negative conventional microbiologic growth, and some positive SPUAT results might be caused by true pneumococcal infection although without cultural evidence. Conclusion: Our retrospective study demonstrated that a positive SPUAT result typically does not agree well with conventional culture methods, suggesting that the value of a positive SPUAT result in etiology determination may be limited under practical conditions in a university hospital. (Korean J Clin Microbiol 2010;13:14-18)

Utility of PCR Amplification and DNA Microarray Hybridization in 16S rDNA for Rapid Diagnosis of Bacteremia Associated with Hematological Malignancies.

Abstract 1635Poster Board I-661 IntroductionThe rapid diagnosis of bacteremia is important for patient management and the choice of antimicrobial and antifungal therapy, especially in hematological malignancies. We describe a rapid detection and identification system that uses universal PCR primers and DNA microarray hybridization compared to the conventional culture method. This new DNA-microarray method could be performed within only 24 hours. Eighty-four microorganism probes are spotted onto a microarray plate containing most causal clinical pathogens (Patent No.WO2003/106676). We can detect 93% of pathogen of blood culture by the frequency of upper 28 microorganisms. Patients and MethodsWe used universal PCR primers to amplify a variable region of bacterial 16S ribosomal DNA or fungal ITS (internal transcribed spacer region) of ribosomal DNA, followed by the reverse hybridization of products to a panel of oligonucleotides. Genomic DNA was extracted from blood with a DNA isolation kit (DNeasy; QIAGEN, CA, USA) according to the manufacturer's instructions. The 16S ribosomal ribonucleic acid gene (16S rDNA) was amplified from genomic DNA using broad-spectrum PCR primers; 5'-AGAGTTTGATCCTGGCT-CAG-3', corresponding to Escherichia coli 16S rDNA positions 8 through 27, and 5'-GTATTACCGCGGCTGCTGG-3', corresponding to Escherichia coli 16S rRNA gene positions 517 through 535. On the other hand, the PCR primers; 5'-TCCGTAGGTGAACCTGCGG-3' and 5'-GCTGCGTTCTTCATCGATGC-3' are used to amplify the ITS 1 region. Thirty-five cycles of amplification were performed in a thermal cycler (Gene Amp PCR system 9600, Perkin-Elmer Cetus, CT, USA). The PCR products from samples were labeled by Cy5- primers and applied to the microarray. The microarray was scanned using a ScanArray 4000 (Axon Instruments, CA, USA) to determine the amount of Cy5-labeled PCR products. Fluorescence intensities were calculated with DNASIS Array software (HITACHI Soft Engineering, Japan). This new identification system using DNA microarray hybridization and the conventional blood culture method (BacT/ALERT, bioMerieux and Bactec, Becton-Dickinson, USA) were applied for 336 infectious episodes from 100 patients with hematological malignancy except 2 patients with aplastic anemia retrospectively from May 2004 to February 2007. The hematological malignancies included 152 episodes of acute myeloid leukemia (45.2%), 106 of non-Hodgkin lymphoma (31.5%), 38 of acute lymphoid leukemia (11.3%), 17 of multiple myeloma (5%), 12 of myelodysplastic syndrome (3.6%), 4 of aplastic anemia (1.2%) etc. ResultsThirty-nine episodes (11.6%) were positive after a few days of culturing, and 41 episodes (12.2%) were identified by the new microarray method within 24 hours. Both methods yielded positive results 10.7% (36 episodes). Pathogens in most cases were identical at the species level (21/36 episodes 58.3%), some cases were identical at the genus level (6/36 16.7%), in the other cases, several bacteria were detected by each method. A positive DNA microarray and negative blood culture occurred at a rate of 1.5% (5 episodes), with pathogens in these cases being Bacillus cereus, Staphylococcus epidermidis, Chryseobacterium indologenes, and Enterococcus faecium. A negative DNA microarray and positive blood culture occurred at a rate of 0.9% (3 episodes), and with pathogens comprising Staphylococcus aureus, á-Streptococcus, and Pseudomonas aeruginosa. Both methods yielded negative results at a rate of 86.9% (292 episodes). The sensitivity and specificity of this new microarray method were 92.3 and 98.3%, respectively, compared with the conventional blood culture method. Regarding fungal infection, only one episode out of one patient was found to be positive microarray and negative blood culture of Yeast like organism. ConclusionThis new DNA microarray method has the potential to diagnose bacteremia quickly and detect pathogens at least at the same rate as the conventional blood culture method. Regarding the possibility of improving the sensitivity and specificity, or application for fungal infection further study should be needed. In conclusion, DNA microarray is a useful method for the management of febrile patients with hematological malignancy. DisclosuresIwasaki:The ministry of Health, Labour and Welfare of Japan: Research Funding.

Multiplex PCR To Diagnose Bloodstream Infections in Patients Admitted from the Emergency Department with Sepsis

Sepsis is caused by a heterogeneous group of infectious etiologies. Early diagnosis and the provision of appropriate antimicrobial therapy correlate with positive clinical outcomes. Current microbiological techniques are limited in their diagnostic capacities and timeliness. Multiplex PCR has the potential to rapidly identify bloodstream infections and fill this diagnostic gap. We identified patients from two large academic hospital emergency departments with suspected sepsis. The results of a multiplex PCR that could detect 25 bacterial and fungal pathogens were compared to those of blood culture. The results were analyzed with respect to the likelihood of infection, sepsis severity, the site of infection, and the effect of prior antibiotic therapy. We enrolled 306 subjects with suspected sepsis. Of these, 43 were later determined not to have infectious etiologies. Of the remaining 263 subjects, 70% had sepsis, 16% had severe sepsis, and 14% had septic shock. The majority had a definite infection (41.5%) or a probable infection (30.7%). Blood culture and PCR performed similarly with samples from patients with clinically defined infections (areas under the receiver operating characteristic curves, 0.64 and 0.60, respectively). However, blood culture identified more cases of septicemia than PCR among patients with an identified infectious etiology (66 and 46, respectively; P = 0.0004). The two tests performed similarly when the results were stratified by sepsis severity or infection site. Blood culture tended to detect infections more frequently among patients who had previously received antibiotics (P = 0.06). Conversely, PCR identified an additional 24 organisms that blood culture failed to detect. Real-time multiplex PCR has the potential to serve as an adjunct to conventional blood culture, adding diagnostic yield and shortening the time to pathogen identification.

Improved detection of blood stream pathogens by real-time PCR in severe sepsis

Evaluation of the technical and diagnostic feasibility of commercial multiplex real-time polymerase chain reaction (PCR) for detection of blood stream infections in a cohort of intensive care unit (ICU) patients with severe sepsis, performed in addition to conventional blood cultures. Dual-center cohort study. Surgical ICU of two university hospitals. One hundred eight critically ill patients fulfilling the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) severe sepsis criteria were included. None. PCR results obtained in 453 blood samples from 108 patients were compared with corresponding blood culture results. PCR resulted in a twofold higher positivity rate when compared with conventional blood culture (BC) testing (114 versus 58 positive samples). In 40 out of 58 PCR positive assays the results of the corresponding blood cultures were identical to microorganisms detected by PCR. In 18 samples PCR and BC yielded discrepant results. Compared with conventional blood culture the sensitivity and specificity of PCR was 0.69 and 0.81, respectively. Further evaluation of PCR results against a constructed gold standard including conventional microbiological test results from other significant patient specimen (such as bronchio-alveolar lavage fluid, urine, swabs) and additionally generated clinical and laboratory information yielded sensitivity of 0.83 and specificity of 0.93. Our cohort study demonstrates improved pathogen detection using PCR findings in addition to conventional blood culture testing. PCR testing provides increased sensitivity of blood stream infection. Studies addressing utility including therapeutic decision-making, outcome, and cost-benefit following diagnostic application of PCR tests are needed to further assess its value in the clinical setting.