Positive Blood Culture Samples Research Articles

Bacterial rRNA-Targeted Reverse Transcription-PCR Used To Identify Pathogens Responsible for Fever with Neutropenia

The purpose of this study was to evaluate the clinical utility of bacterial rRNA-targeted reverse transcription-quantitative PCR (BrRNA RT-qPCR) assays for identifying the bacterial pathogens that cause fever with neutropenia in pediatric cancer patients, by comparing the bacterial detection rate of this technique with that of blood culture. One milliliter of blood was collected from pediatric patients who developed fever with neutropenia following cancer chemotherapy. BrRNA RT-qPCR was performed using 16 primer sets, each designed for a specific type of bacteria. The entire BrRNA RT-qPCR procedure took less than 5 h. Blood culture was performed at the same time, following the standard institutional procedure. Blood from 13 patients was collected during 23 febrile neutropenic episodes. Of these samples, bacteria were identified in 16 by BrRNA RT-qPCR (69.6%) and in 4 by blood culture (17.4%, P<0.001). In all 4 blood culture-positive samples, BrRNA RT-qPCR detected the same type of bacteria as that identified by culture. In 9 samples, more than 4 types of bacteria were identified simultaneously by BrRNA RT-qPCR, most of which were anaerobic bacteria known to be part of the gut flora. We conclude that BrRNA RT-qPCR could be useful in the diagnosis of fever with neutropenia, given its high bacterial detection rate, short turnaround time, and the small blood sample required compared with the standard blood culture techniques. Our findings also indicate that anaerobic intestinal bacteria, which are difficult to detect by standard culture techniques, may be responsible for some cases of febrile neutropenia.

Cost-Effective Modification of a Commercial PCR Assay for Detection of Methicillin-Resistant or -SusceptibleStaphylococcus aureusin Positive Blood Cultures

Real-time detection of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in cases of clinical bacteremia may promote appropriate antimicrobial therapy and infection control. Expense inherent to molecular diagnostics may prevent laboratories from utilizing real-time PCR for this purpose. BD GeneOhm StaphSR assay master mix was reconstituted and aliquoted into SmartCycler tubes in 25-mul volumes (freshly reconstituted master mix), with a portion being frozen at -70 degrees C (frozen master mix). Incubation of 40 previously analyzed lysates from positive BacT/Alert SA and SN blood culture bottles (identified as 10 MRSA strains, 10 MSSA strains, 12 coagulase-negative Staphylococcus strains, and 8 Micrococcus strains) in freshly reconstituted master mix and master mix frozen between 1 week and 6 months generated the expected results in all PCRs. Similarly, positive- and negative-control reagents stored frozen at -70 degrees C for up to 18 weeks yielded the expected reactions. Prospective analysis of 244 positive blood culture samples utilizing 1-week-frozen master mix and freshly reconstituted master mix yielded a 1.2% discordant rate upon initial testing due to three unresolved results (two unresolved results for freshly reconstituted master mix and one unresolved result for frozen master mix). Repeat testing produced a final 100% concordance rate between the two master mix preparations. Use of master mix that was frozen up to 6 months did not compromise performance of the BD GeneOhm StaphSR assay. This modification, resulting in less reagent waste, may allow a greater number of laboratories to consider real-time PCR methodology for detection of bacteremia caused by MRSA and MSSA.

Fluoresan İn Situ Hibridizasyon (FISH) Yöntemi ile Saptanan Yüksek Oranda Çoklu Kandidemi: Klinik ve Prognostik Anlamı Nedir?

Objective: The fluorescence in situ hybridization (FISH) with rRNA-targeted probes is a molecular method that allows identification of microorganisms in mixed assemblages from direct clinical samples without cultivitation. The aim of this study was to investigate the clinical features and risk factors in patients with single and multiple-species candidemia detected with FISH method and also to evaluate the episodes of breakthrough candidemia. Material and Methods: A total of 325 positive blood culture samples were examined between January and August 2004 in medical microbiology laboratory of Mersin University Hospital. The demographic and clinical data of 45 patients with candidemia detected with conventional culture methods, FISH and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) were analyzed retrospectively, single and multiple-species candidemia were investigated among patients with candidemia and these patients were compared with respect to risk factors. Results: Thirty five (77.8%) of 45 patients with candidemia had a single Candida species and 10 (22.2%) had multiple-species detected by FISH method. Of these 45 cases, 31.1% had associated solid and hematologic malignancies. The mean duration of hospitalization was 27 (2-180) days. All patients had at least one indwelling catheter (urinary catheter: 80%, peripheric venous catheter: 55.6%, central venous catheter: 26.7%). Most of the patients (95.6%) had been treated with broad spectrum antibiotics. The candidemias were mostly caused by non-albicans Candida spp., particularly Candida parapsilosis (71.1%). The rate of breakthrough candidemia was detected in 11.1% of the patients with candidemia and all of these were in patients with single species candidemia. Surprisingly, in case of single-species candidemia, the mortality rate (77.8%) was higher than that of multiple-species candidemia (22.2%). Conclusion: According to our data, the indwelling catheters and treatment with multiple antibiotics were considered as the risk factors for single and multiple-species candidemia. Although the rate of multiple-species candidemia detected with FISH method was found to be high, clinical and prognostic significance of multiple-species candidemia are still debatable.

Gram-negative bacteremia induces greater magnitude of inflammatory response than Gram-positive bacteremia

IntroductionBacteremia is recognized as a critical condition that influences the outcome of sepsis. Although large-scale surveillance studies of bacterial species causing bacteremia have been published, the pathophysiological differences in bacteremias with different causative bacterial species remain unclear. The objective of the present study is to investigate the differences in pathophysiology and the clinical course of bacteremia caused by different bacterial species.MethodsWe reviewed the medical records of all consecutive patients admitted to the general intensive care unit (ICU) of a university teaching hospital during the eight-year period since introduction of a rapid assay for interleukin (IL)-6 blood level to routine ICU practice in May 2000. White blood cell count, C-reactive protein (CRP), IL-6 blood level, and clinical course were compared among different pathogenic bacterial species.ResultsThe 259 eligible patients, as well as 515 eligible culture-positive blood samples collected from them, were included in this study. CRP, IL-6 blood level, and mortality were significantly higher in the septic shock group (n = 57) than in the sepsis group (n = 127) (P < 0.001). The 515 eligible culture-positive blood samples harbored a total of 593 isolates of microorganisms (Gram-positive, 407; Gram-negative, 176; fungi, 10). The incidence of Gram-negative bacteremia was significantly higher in the septic shock group than in the sepsis group (P < 0.001) and in the severe sepsis group (n = 75, P < 0.01). CRP and IL-6 blood level were significantly higher in Gram-negative bacteremia (n = 176) than in Gram-positive bacteremia (n = 407) (P < 0.001, <0.0005, respectively).ConclusionsThe incidence of Gram-negative bacteremia was significantly higher in bacteremic ICU patients with septic shock than in those with sepsis or severe sepsis. Furthermore, CRP and IL-6 levels were significantly higher in Gram-negative bacteremia than in Gram-positive bacteremia. These findings suggest that differences in host responses and virulence mechanisms of different pathogenic microorganisms should be considered in treatment of bacteremic patients, and that new countermeasures beyond conventional antimicrobial medications are urgently needed.

Accurate and rapid identification of bacterial species from positive blood cultures with a DNA-based microarray platform: an observational study

New DNA-based microarray platforms enable rapid detection and species identification of many pathogens, including bacteria. We assessed the sensitivity, specificity, and turnaround time of a new molecular sepsis assay. 2107 positive blood-culture samples of 3318 blood samples from patients with clinically suspected sepsis were investigated for bacterial species by both conventional culture and Prove-it sepsis assay (Mobidiag, Helsinki, Finland) in two centres (UK and Finland). The assay is a novel PCR and microarray method that is based on amplification and detection of gyrB, parE, and mecA genes of 50 bacterial species. Operators of the test assay were not aware of culture results. We calculated sensitivity, specificity, and turnaround time according to Clinical and Laboratory Standards Institute recommendations. 1807 of 2107 (86%) positive blood-culture samples included a pathogen covered by the assay. The assay had a clinical sensitivity of 94.7% (95% CI 93.6-95.7) and a specificity of 98.8% (98.1-99.2), and 100% for both measures for meticillin-resistant Staphylococcus aureus bacteraemia. The assay was a mean 18 h faster than was the conventional culture-based method, which takes an additional 1-2 working days. 34 of 3284 (1.0%) samples were excluded because of technical and operator errors. Definitive identification of bacterial species with this microarray platform was highly sensitive, specific, and faster than was the gold-standard culture-based method. This assay could enable fast and earlier evidence-based management for clinical sepsis.

혈액배양에서 분리된 Extended Spectrum β-Lactamase 생성균의 역학적 특성 조사

본 연구는 혈액에서 분리되는 미생물의 균종과 항균제 감수성 검사 결과 양상은 환자의 일반적 특성에 따라 다르므로 혈액배양 결과를 분석한 자료는 감염증 환자의 경험적 치료를 위한 중요한 근거가 되고, 특히 ESBL을 생성하는 균주의 경우 항균제의 사용과도 밀접한 관련이 있어 이 연구를 하게 되었다. 혈액배양 결과와 항균제 내성 결과를 분석한 결과는 다음과 같다. 1. 39.305건의 혈액배양이 의뢰되어 양성 환자 수는 2,216명(20.0%)이었고, 이중에서 ESBL양성 환자 수는 40명 이었으며, 혈액배양 양성은 4,798건(12.2%)이었으며 ESBL양성 균주는 86건(중복 배양 균주 포함)이었다. 2. ESBL을 생성하는 세균 중에서 분리된 균종은 E. coli, K. pneumoniae, K. oxitoca의 순 이었다. 3. 성별로는 여자가 다소 많았고, 연령별로는 50세 이상의 군에서 E. coli, 1세 미만의 군에서는 K. pneumoniae, K. oxitoca 의 분리가 많았고, 지역별로는 충청남도 13명(32.5%)로 가장 많았다. 4. 3년간 일개대학병원의 혈액배양에서 ESBL양성인 균종 중 가장 흔히 분리되는 것은 E. coli이고, K. pneumoniae, K. oxitoca 등이 다음으로 분리 되었고, 50세 이상의 환자에서 E. coli의 분리 비율이 높았다. The data which analyze the results of blood cultures are crucial basic information of the empirical therapy for patients with infection since the patterns of the species of microorganism isolated from blood and the results of antibiotic susceptibility test vary depending upon patients general features. Especially, in case of ESBL-producing bacteria, there is a close relation with use of antibiotics. Therefore, we carried out the research with the results of blood culture and antibiotic. 1. Total 39,305 cases of blood culture samples were investigated and positive patients of 2,216 (20.0%) were found. Among those, there were 40 patients with ESBL positive, and blood culture positive samples were 4,798 (12.2%). ESBL positive bacteria were found in 86 samples (including double checked culture bacteria). 2. The majority of ESBL producing bacteria were E. coli, K. pneumoniae and K. oxitoca as ordering based on the number. 3. The research showed the results that there were more females than male with the bacterias, more E. coli in over 50 years old aged group than other bacterias, more K. pneumoniae and K.oxitoca in less 1 year old aged group than other bacterias and largest numbers of patients with 13 patients (32.5%) in Chungcheongnam-do province were found. 4. The most common ESBL producing bacteria were E. coli throughout 3 years, but K, pneumoniae and K. oxitoca were also fairly found. Interestingly, E. coli was highly found in over 50 years old patients.

RAPID DETECTION OF NON-ENTEROBACTERIACEAE DIRECTLY FROM POSITIVE BLOOD CULTURE USING FLUORESCENT IN SITU HYBRIDIZATION

Fluorescent in situ hybridization (FISH) was carried out using two different oligonucleotide probes specific for Pseudomonas spp. and Acinetobacter spp. These probes were tested against different organisms and were found to be highly specific. Sensitivity testing showed that the probes were able to detect as low as 103CFU/mL. In addition, FISH was carried out directly on positive blood culture samples and the detection of microorganisms took less than 2 h. We believe that FISH is a rapid method that can be used as a routine laboratory diagnostic technique for the detection of Acinetobacter spp. and Pseudomonas spp. in clinical samples.

Real-time broad-range PCR versus blood culture. A prospective pilot study in pediatric cancer patients with fever and neutropenia

In a pilot study, results of real-time broad-range (16S rRNA) polymerase chain reaction (PCR) performed on 45 blood samples of pediatric cancer patients with fever and neutropenia were compared with blood culture results. The PCR assay used, having proven a high sensitivity in artificially spiked blood samples, was positive in only three of ten blood culture-positive samples, and it was positive in 10 of 35 (29%) culture-negative samples. This broad-range PCR assay, which may identify not-grown bacteria potentially contributing to fever, needs improvement in sensitivity, and different reasons for positive PCR in negative blood culture samples need to be assessed before clinical application.

Rapid detection of non-enterobacteriaceae directly from positive blood culture using fluorescent &lt;i&gt;In situ &lt;/i&gt;hybridization

Fluorescent in situ hybridization (FISH) was carried out using two different oligonucleotide probes specific for Pseudomonas spp. and Acinetobacter spp. These probes were tested against different organisms and were found to be highly specific. Sensitivity testing showed that the probes were able to detect as low as 10 3 CFU/mL. In addition, FISH was carried out directly on positive blood culture samples and the detection of microorganisms took less than 2 h. We believe that FISH is a rapid method that can be used as a routine laboratory diagnostic technique for the detection of Acinetobacter spp. and Pseudomonas spp. in clinical samples.

The Bronchial Lavage of Pediatric Patients With Asthma Contains Infectious Chlamydia

Purpose of the Study. To examine the frequency of Chlamydia pneumoniae infections in pediatric patients with asthma. Study Population. Seventy pediatric patients undergoing flexible fiber-optic bronchoscopy as a part of their ongoing clinical care. Methods. Bronchoaveolar lavage (BAL) fluid and blood were examined for the presence of C pneumoniae by smear examination and culture. The BAL and blood samples were cultured on human or mouse macrophages to determine infectivity. Polymerase chain reaction (PCR) amplification of BAL samples was performed to confirm specificity of the culture technique. Blood was examined for total immunoglobulin E (IgE). Blood samples from 70 matched, nonrespiratory control patients were cultured for Chlamydia. Results. Forty-two patients undergoing bronchoscopy had asthma and 28 had various other respiratory diseases. Thirty-eight (54%) BAL samples were positive for Chlamydia by PCR and 22 (31%) samples were positive for Chlamydia by culture. Of the positive BAL samples, 28 (74%) of 38 PCR-positive and 14 (64%) of 22 culture-positive samples were from children with asthma. Culture-positive blood samples were found in 24 (34%) of 70 respiratory patients and 8 (11%) of 70 nonrespiratory controls. In the blood culture–positive respiratory group, 17 (71%) of 24 were from children with asthma. Elevated total serum IgE was associated with BAL culture–positive results, and this relationship was stronger than total IgE and asthma diagnosis. Conclusions. Viable C pneumoniae organisms are frequently present in the lung lavage in a cohort of predominately asthmatic pediatric patients. Reviewer Comments. Results from this study suggest that infectious C pneumoniae may be common in BAL fluid of children with asthma. Historically, C pneumoniae has been associated with exacerbation and increased incidence of respiratory conditions in adults, but studies to examine similar associations in children have not been performed. This is the first investigation to report viable and infectious C pneumoniae in the BAL fluid of children with asthma. These findings are intriguing and should encourage investigators to examine the clinical implications of Chlamydia infection among pediatric patients with asthma.

Quantitative detection of Salmonella enterica serovar Typhi from blood of suspected typhoid fever patients by real-time PCR

We quantified the gene copies from Salmonella enterica serovar Typhi ( S. Typhi) in the blood of patients suspected of having typhoid fever by using TaqMan-based real-time PCR (TaqMan assay) to target the S. Typhi flagellin gene in genomic DNAs isolated from blood samples. Of 55 blood samples taken from suspected typhoid fever patients, eight blood samples with a positive blood culture had S. Typhi loads ranging from 1.01×10 3 to 4.35×10 4 copies/ml blood, and from 47 blood samples with negative blood culture, there were 40 (85.1%) TaqMan assay-positive samples with loads ranging from 3.9 to 9.9×10 2 copies/ml blood. In the present study, the TaqMan assay detected more than 10 3 copies/ml blood of S. Typhi in all of the blood culture-positive samples, whereas less than 10 3 copies/ml blood of S. Typhi were detected in the blood culture-negative samples. Our findings suggest that a TaqMan assay may be useful for assessing S. Typhi loads, especially in cases of suspected typhoid fever with negative results from the standard blood culture test.

Efficient method for mycobacterial DNA extraction in blood cultures aids rapid PCR identification of Mycobacterium tuberculosis and Mycobacterium avium

The study presented here evaluated the utility of several methods of extracting mycobacterial nucleic acids from positive blood culture samples and examined the effect of each method on the performance of an in-house PCR used directly in the peripheral blood of 80 patients with AIDS to identify Mycobacterium spp. The modified Boom method for extracting DNA from blood cultures proved to be the most efficient, with subsequent PCR analysis yielding 100% positivity (7 samples positive for M. avium and 5 for M. tuberculosis). Only three of 12 patients with a positive blood culture had a PCR result positive for M. avium in peripheral blood. The identification of mycobacteria by PCR in blood culture took about 3 days, reducing the time to diagnosis by several weeks. These results demonstrate that PCR is a sensitive and quick method for identifying mycobacteria, especially when a good DNA extraction method is applied.

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.

A rapid, highly sensitive method for the detection of Francisella tularensis in clinical samples using the polymerase chain reaction.

We have developed a highly sensitive method for detection of Francisella tularensis in clinical samples based on a nested polymerase chain reaction (PCR) for the FopA gene. Mice infected with F. tularensis were killed at 24-hr intervals, and the DNA from blood and spleens was extracted by a variety of methods and analyzed by PCR. The best method, based on the ability of DNA to bind to silica in the presence of guanidine thiocyanate, yielded amplifiable DNA without dilution of the murine tissue samples. Francisella tularensis in infected murine spleens and culture-positive blood samples was reliably detected by nested PCR following this extraction procedure. We believe this technique has significant advantages over traditional methods for diagnosing F. tularensis infection in terms of speed, ease of use, reproducibility, and safety.

Rapid screening for resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture-positive blood samples

A rapid screening assay for the detection of resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture-positive blood samples was developed by using single doses of both drugs and an immediate-early antigen plaque reduction assay. Results of the rapid assay with peripheral blood leukocytes as the viral inoculum were compared with those of a conventional assay with cell-free virus from the relevant viral isolates. Both assays gave overlapping results. The rapid assay offers the following major advantages: (i) it provides results within 4 to 6 days, (ii) it can be performed with peripheral blood leukocytes, and (iii) it reliably detects drug-resistant human cytomegalovirus strains.