Protocol for the Accelerated Detection of Extended-Spectrum β-Lactamase-Producing<Emphasis Type="Italic"> Escherichia coli</Emphasis> and<Emphasis Type="Italic"> Klebsiella pneumoniae</Emphasis> Strains from Blood Cultures

The study presented here was performed to evaluate an accelerated protocol for the early detection of organisms producing extended-spectrum -lactamase (ESBL). The procedure involved testing isolates directly from positive blood-culture bottles, and a total of 40 clinical isolates (10 ESBL-producing and 10 non-ESBL-producing isolates of both Escherichia coli and Klebsiella pneumoniae) were used. The isolates were inoculated into blood cultures bottles and, upon growth signal, fluid from the bottle was cultured directly onto plates with combination discs containing cefotaxime or ceftazidime with and without clavulanate. Results were compared with those of standard methods for the detection of ESBL. High concordance between the two methods was found, and the direct test showed high sensitivity (95%) and specificity (100%). Use of this accelerated protocol may speed detection of the ESBL phenotype and thereby facilitate the early administration of appropriate antimicrobial therapy.     

Growth of nutritionally variant streptococci on common laboratory and 10 commercial blood culture media.

Nutritionally variant streptococci fail to grow on routine sheep blood agar plates. Moreover, these strains are a recognized cause of culture-negative endocarditis. We tested the ability of chocolate and brucella blood agars, sheep blood agar with a staphylococcal streak, sheep blood agar with 0.001% pyridoxal, and 10 commercial blood culture media from two manufactures to grow these bacteria. Of the original 25 strains tested, 16 were recovered on chocolate agar, 21 were recovered on brucella blood agar and 21 were recovered on sheep blood agar with a staphylococcal streak. Sheep blood agar with pyridoxal grew all 22 strains tested. Supplemented peptone, thioglycolate, and thiol broths grew all strains, but brain heart infusion and three tryptic soy broths supported five or fewer strains. The addition of 5 ml of human blood improved recovery to 100% in all media except tryptic soy broths. Unless supplemented wih pyridoxal, common laboratory agars were inadequate for recovering all strains of variant streptococci upon subculture of blood culture bottles. As used clinically, the blood culture media that we studied other than tryptic soy broths should reliably grow these bacteria.     

革兰阴性菌引起的血培养阳性标本的直接鉴定药敏试验

目的 为进一步缩短实验室菌血症诊断时间,评估联合法阳性血培养直接鉴定药敏试验的可行性.方法 将血培养瓶放人BACTEC 9000血培养系统进行培养筛选.选取65份含革兰阴性杆菌的阳性血培养瓶进行试验.抽取培养液,用BD真空分离管离心分离血细胞.在收集到足量菌液后,用Phoenix 100 NMIC/ID-4革兰阴性菌鉴定药敏卡做0.25 McF和0.5 McF直接鉴定药敏试验.用标准方法及哑培养后的鉴定药敏试验对直接鉴定药敏试验进行评估.结果 0.25 McF直接鉴定试验,65株中的63株(96.9%)准确鉴定.0.5 MeF直接鉴定试验,65株中的59株(90.8%)准确鉴定.0.25 McF直接药敏试验标准符合率97.8%以上.0.5 McF直接药敏试验标准符合率95.9%以上.KB法血标本直接药敏试验标准符合率96.4%以上,但微小错误率高于联合药敏法.结论 采用0.25 McF、0.5 McF两种菌液浓度法进行血培养阳性标本鉴定药敏试验是切实可行的.联合法0.25 McF菌液浓度的直接鉴定药敏试验具有明显优势,对临床具有很好的及时、有效地指引作用.

Abstract：

Objective To reduce the turnaround time for laboratory diagnosis of bacteremia, the feasibility of rapid identification and susceptibility testing using samples taken directly from positive blood culture bottles was evaluated. Methods The growth of microorganisms in blood culture bottles was screened by the BACTEC 9000 blood culture system. 65 positive blood culture bottles containing gram-negative bacteria were adopted to test. Culture fluid was injected into BD SST vacutainer and centrifuged to pellet blood cells. After collecting required McFarland units, they were cultured on Phoenix 100 NMIC/ID-4(identification-gram-negative bacteria and susceptibility testing) cards using 0.25 McF and 0.5 McF methods respectively. They were also evaluated by the standard method, involving subculture tests from positive blood culture bottles. Results 63 of 65 gram-negative bacteria (96. 9% ) were correctly identified with 0. 25 McF method. 59 of 65 gram-negative bacteria(90.8% ) were correctly identified with 0.5 McF method. For antimicrobial susceptibility testing, the 0.25 McF direct method had an agreement rate more than 94% , the 0.5 McF method was more than 85.7% and direct blood sample KB method was more than 93.8% compared to the standard method. But the overall minor error rate in susceptibility testing of direct blood sample KB method is higher than other methods. Conclusion Applying 0. 25 McF and 0. 5 McF rapid identification and susceptibility test was practical. During to possessing more prominent advantages, laboratory put the 0. 25 McF direct method into practice had a timely, remarkable significance.     

革兰阴性菌引起的血培养阳性标本的直接鉴定药敏试验

目的 为进一步缩短实验室菌血症诊断时间,评估联合法阳性血培养直接鉴定药敏试验的可行性.方法 将血培养瓶放人BACTEC 9000血培养系统进行培养筛选.选取65份含革兰阴性杆菌的阳性血培养瓶进行试验.抽取培养液,用BD真空分离管离心分离血细胞.在收集到足量菌液后,用Phoenix 100 NMIC/ID-4革兰阴性菌鉴定药敏卡做0.25 McF和0.5 McF直接鉴定药敏试验.用标准方法及哑培养后的鉴定药敏试验对直接鉴定药敏试验进行评估.结果 0.25 McF直接鉴定试验,65株中的63株(96.9%)准确鉴定.0.5 MeF直接鉴定试验,65株中的59株(90.8%)准确鉴定.0.25 McF直接药敏试验标准符合率97.8%以上.0.5 McF直接药敏试验标准符合率95.9%以上.KB法血标本直接药敏试验标准符合率96.4%以上,但微小错误率高于联合药敏法.结论 采用0.25 McF、0.5 McF两种菌液浓度法进行血培养阳性标本鉴定药敏试验是切实可行的.联合法0.25 McF菌液浓度的直接鉴定药敏试验具有明显优势,对临床具有很好的及时、有效地指引作用.     

革兰阴性菌引起的血培养阳性标本的直接鉴定药敏试验

目的 为进一步缩短实验室菌血症诊断时间,评估联合法阳性血培养直接鉴定药敏试验的可行性.方法 将血培养瓶放人BACTEC 9000血培养系统进行培养筛选.选取65份含革兰阴性杆菌的阳性血培养瓶进行试验.抽取培养液,用BD真空分离管离心分离血细胞.在收集到足量菌液后,用Phoenix 100 NMIC/ID-4革兰阴性菌鉴定药敏卡做0.25 McF和0.5 McF直接鉴定药敏试验.用标准方法及哑培养后的鉴定药敏试验对直接鉴定药敏试验进行评估.结果 0.25 McF直接鉴定试验,65株中的63株(96.9%)准确鉴定.0.5 MeF直接鉴定试验,65株中的59株(90.8%)准确鉴定.0.25 McF直接药敏试验标准符合率97.8%以上.0.5 McF直接药敏试验标准符合率95.9%以上.KB法血标本直接药敏试验标准符合率96.4%以上,但微小错误率高于联合药敏法.结论 采用0.25 McF、0.5 McF两种菌液浓度法进行血培养阳性标本鉴定药敏试验是切实可行的.联合法0.25 McF菌液浓度的直接鉴定药敏试验具有明显优势,对临床具有很好的及时、有效地指引作用.     

False penicillin resistance in Neisseria meningitidis following direct susceptibility tests from blood cultures

Blood cultures drawn from a patient with clinically diagnosed invasive meningococcal disease, who had been previously administered benzylpenicillin, had beta-lactamases added to increase the probability of recovery of the causative organism. The blood cultures subsequently yielded Neisseria meningitidis but direct susceptibility tests by the comparative disk diffusion method demonstrated greatly reduced zones of inhibition to penicillin (1 unit disk). Repeat testing from subcultures showed full penicillin sensitivity. Inoculation of blood culture bottles with a variety of penicillin sensitive bacteria with the addition of beta-lactamases showed the same effect of false penicillin resistance, owing to carry over of sufficient beta-lactamase from blood culture bottles during inoculation of direct susceptibility plates to inactivate the penicillin in the disks. Direct susceptibility tests to beta-lactam agents should not be carried out on positive blood cultures to which beta-lactamases have been added.     

Rapid identification of gram-negative bacteria with and without CTX-M extended-spectrum β-lactamase from positive blood culture bottles by PCR followed by microchip gel electrophoresis

We evaluated the usefulness of PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) and the CTX-M extended-spectrum β-lactamase (ESBL) followed by microchip gel electrophoresis (MGE) for direct identification and CTX-M detection of Gram-negative bacteria (GNB) from positive blood culture bottles. Of 251 GNB isolated from blood cultures containing a single bacterium, 225 (90%) were correctly identified at the species level directly from positive blood culture bottles by comparing the ITS-PCR patterns of the sample strain with those of the control strains. There were no cases of incorrect identification. Limitations encountered included the inability to detect mixed cultures (four bottles) as well as some species (Enterobacter species and Klebsiella oxytoca) demonstrating identical ITS-PCR patterns. A total of 109 ESBL-producing isolates from various clinical materials obtained between January 2005 and December 2008 were examined for bla(CTX-M), bla(SHV), and bla(TEM) genes by PCR and sequences of PCR products. CTX-M ESBL was detected in 105 isolates, and SHV ESBL was detected in two isolates. The remaining two isolates (K. oxytoca) were shown to harbor bla(OXY.) Twenty (19%) of 104 Escherichia coli isolates from blood cultures were suspected to produce ESBL by the combination disk method, and these isolates were shown to harbor CTX-M ESBL by PCR-MGE. The results were obtained within 1.5 h at a calculated cost of $6.50 per specimen. In conclusion, simultaneous detection of ITS length polymorphisms and bla(CTX)-(M) by single PCR followed by MGE is useful for rapid, cost-effective, and reliable species-level identification of CTX-M ESBL-producing GNB responsible for bloodstream infections.     

Comparative analysis of simulated candidemia using two different blood culture systems and the rapid identification of Candida albicans

The goal of this study was to determine the time to detection of Candida species isolates using the two most commonly used automated blood culture systems, and to evaluate rapid, widely available methods for the presumptive identification of C. albicans. Candidemia models of eight commonly detected Candida species were prepared using ATCC standards. The times to detection were evaluated using the BACTEC 9240 (Becton Dickinson) and BacT/Alert 3D (bioMerieux) automated blood culture systems. The presence of pseudohyphae clusters was examined by Gram staining and wet preparation. Germ tube tests were performed directly from blood culture bottles. All samples were cultured on blood agar plates and macroscopically examined for the presence of an irregular margin (spiking). Most Candida species (6/8) except C. glabrata and C. krusei grew more rapidly in aerobic than in anaerobic conditions. Clusters of pseudohyphae were observed in cultures of C. albicans and C. tropicalis. All culture bottles positive for C. albicans were positive by the germ tube test and macroscopically showed 'spiking.' Aerobic and anaerobic blood culture systems can effectively detect candidemia. Furthermore, the direct germ tube test may be the most useful available morphological presumptive identification method for C. albicans.     

Comparative evaluation of Vitek 2 identification and susceptibility testing of Gram-negative rods directly and isolated from BacT/ALERT-positive blood culture bottles

The performance of Vitek 2 was evaluated for the identification and susceptibility testing of Gram-negative bacilli directly from positive blood cultures bottles. Direct inoculation of the positive blood cultures with the Vitek cards ID-GN and AST-NO58 was compared with the standard inoculation method based on the sub-culture of the positive blood culture to agar. A total of 142 blood cultures were included in the study; of those, 119 were from patients’ clinical samples, while 23 were artificially prepared with strains showing different mechanisms of resistance. A total of 136 (95.8%) strains were correctly identified to the species level, only 2 (1.4%) were mis-identified and 4 (2.8%) were not identified. Susceptibility results were available for all isolates tested against 17 antibiotics, thus, resulting in a total of 2,414 isolate/anti-microbial combinations. The error rate was 2.8% (67/2,414) overall; 0.6% (14/2,414) very major errors, 0.1% (3/2,414) major errors and 2.1% (50/2,414) minor errors. The direct method detected 88.5% (22/25) of the strains producing extended-spectrum beta-lactamases (ESBLs). The susceptibility agreement among the added strains with ESBL, AMPc hyperproduction, resistance to ceftazidime, carbapenems and cefepime was very high. Direct identification and susceptibility testing gave rapid and reliable results, reducing by 24 h the turnaround time of the microbiology laboratory.     

Comparison of anaerobic susceptibility results obtained by two methods of inoculum preparation

We evaluated the use of inocula prepared directly from blood agar plates in agar dilution susceptibility tests of anaerobic bacteria and compared the results with susceptibility results obtained from the National Committee for Clinical Laboratory Standards proposed thioglycolate broth cultures. The objectives were to evaluate the reproducibility of each of the two methods of inoculum preparation and to compare the MICs obtained by each method. The reproducibility studies were conducted on 14 stock strains. The mode MICs obtained by the direct agar method were identical to those obtained by the reference broth method 74% of the time and within +/- 1 log2 dilution 100% of the time. The degree of reproducibility of each of the two methods was identical (93% +/- 1 log2 dilution). MIC results obtained by the direct agar method agreed with the MICs obtained by the reference broth culture method in 92.9% of 1,125 MIC data pair determinations performed on stock cultures. The reproducibility of the direct agar method within +/- 1 log2 dilution step for 115 fresh clinical isolates was 93%, including 93.4% of the results with the Bacteroides fragilis group. Only two very major discrepancies (false-susceptible by the agar method) were identified among the 708 MIC data pairs on these clinical isolates. Preparation of inocula directly from growth on agar plates provides a rapid and reproducible method for agar dilution susceptibility testing of anaerobes.     

Susceptibilities of Mycobacterium tuberculosis to isoniazid and rifampin on blood agar

In this study, blood agar was used instead of 7H10 agar for the susceptibility testing of 34 clinical isolates of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) in accordance with the NCCLS. The BACTEC 460 TB system (Becton Dickinson, Sparks, Md.) was used as a "gold standard." Results for both media were in agreement for RIF and INH at 100 and 94.1%, respectively. For INH, the specificity, sensitivity, positive predictive value, and negative predictive value were found to be 71.4, 100, 93.1, and 100%, respectively, while these values were 100% for RIF. In addition, the results of the susceptibility test performed with blood agar were obtained on day 14 of incubation. In conclusion, results were obtained much earlier with blood agar (2 weeks) than with 7H10 agar (3 weeks), and the results of this study suggest that blood agar may be used as an alternative medium for the susceptibility testing of M. tuberculosis to INH and RIF.     

Proposed quality control guidelines for antimicrobial susceptibility tests using tilmicosin.

Quality control guidelines for tilmicosin, a novel veterinary-use-only macrolide, were developed in a multi-laboratory study according to established National Committee for Clinical Laboratory Standards (NCCLS) procedures (M23-T2). Tilmicosin was incorporated into Sensititre plates for broth microdilution endpoint testing and into two lots of 15-micrograms disks for Kirby-Bauer agar disk diffusion testing. One common lot and five unique lots of Mueller-Hinton media were used. (Broth was cation adjusted, and agar was supplemented with 5% defibrinated sheep blood.) Bacteria used for reference strains included Pasteurella haemolytica 128K, Pasteurella multocida ATCC 43137, and Staphylococcus aureus ATCC 29213 (microdilution) and ATCC 25923 (disk). Replicate tests were conducted. Disk diffusion and broth microdilution quality control ranges are proposed.     

Use of positive blood cultures for direct identification and susceptibility testing with the vitek 2 system

In order to further decrease the time lapse between initial inoculation of blood culture media and the reporting of results of identification and antimicrobial susceptibility tests for microorganisms causing bacteremia, we performed a prospective study in which specially processed fluid from positive blood culture bottles from Bactec 9240 (Becton Dickinson, Cockeysville, Md.) containing aerobic media were directly inoculated into Vitek 2 system cards (bio-Mérieux, France). Organism identification and susceptibility results were compared with those obtained from cards inoculated with a standardized bacterial suspension obtained following subculture to agar; 100 consecutive positive monomicrobic blood cultures, consisting of 50 gram-negative rods and 50 gram-positive cocci, were included in the study. For gram-negative organisms, 31 of the 50 (62%) showed complete agreement with the standard method for species identification, while none of the 50 gram-positive cocci were correctly identified by the direct method. For gram-negative rods, there were 50% categorical agreements between the direct and standard methods for all drugs tested. The very major error rate was 2.4%, and the major error rate was 0.6%. The overall error rate for gram-negatives was 6.6%. Complete agreement in clinical categories of all antimicrobial agents evaluated was obtained for 19 of 50 (38%) gram-positive cocci evaluated; the overall error rate was 8.4%, with 2.8% minor errors, 2.4% major errors, and 3.2% very major errors. These findings suggest that the Vitek 2 cards inoculated directly from positive Bactec 9240 bottles do not provide acceptable bacterial identification or susceptibility testing in comparison with corresponding cards tested by a standard method.     

Extended spectrum -lactamases in urinary isolates of Escherichia coli and Klebsiella pneumoniae - prevalence and susceptibility pattern in a tertiary care hospital

A total of 411 urinary isolates (353 Escherichia coli and 58 Klebsiella pneumoniae) were studied for extended spectrum -lactamase (ESBL) production by double disk approximation test and NCCLS confirmatory test. ESBL production was found to be 41% in E. coli and 40% in K. pneumoniae. Fourteen percent and 12% of ESBL producers showed false susceptibility to ceftazidime and cefotaxime in routine susceptibility testing. The susceptibility of ESBL producers to imipenem, nitrofurantoin and amikacin was found to be 100%, 89% and 86% respectively. A high degree of associated resistance to gentamicin, co-trimoxazole and quinolones was found in ESBL producers. Majority of ESBL producers was detected among patients admitted in medical ICU and surgery ward.     

Identification and susceptibility testing of Enterobacteriaceae and Pseudomonas aeruginosa by direct inoculation from positive BACTEC blood culture bottles into Vitek 2

Inoculation of an automated system for rapid identification (ID) and antimicrobial susceptibility testing (AST) directly from positive blood culture bottles will reduce the turnaround time of laboratory diagnosis of septicemic patients, which benefits clinical outcome and decreases patient costs. Direct test results, however, must always be confirmed by testing a pure overnight culture, which is the "gold standard." We studied the accuracy of direct testing versus repeat testing in order to investigate the possibility of refraining from repeat testing. We also assessed the clinical risk of reporting results based on direct testing only. We inoculated Vitek 2 (bioMérieux) directly from 410 positive BACTEC 9240 (BD) blood culture bottles containing gram-negative rods and studied the ID and AST results. In a comparison of direct inoculation with the standard method, a total of 344 isolates of Enterobacteriaceae and Pseudomonas aeruginosa were tested, and 93.0% were correctly identified. Of the 39 (10.2%) samples that contained bacilli not identifiable by Vitek 2, only 1 gave a conclusive, correct result. The overall MIC agreement among 312 isolates was 99.2%, with 0.8% very major and 0.02% major error rates. Of only three (polymicrobial) samples, the direct susceptibility pattern would be reported to the clinician as too sensitive. Vitek 2 results obtained from direct inoculation of blood culture bottles containing gram-negative bacilli are safe enough for immediate reporting, provided that ID and AST are consistent. Repeat testing is not necessary, unless Gram stain or overnight subculture results raise doubt about the purity of the culture.     

Microbiological diagnosis of prosthetic joint infections: a prospective evaluation of four bacterial culture media in the routine laboratory

The diagnosis of prosthetic joint infection (PJI) in the routine microbiology laboratory is labour-intensive, but semi-automated methods may be appropriate. We prospectively compared four microbiological culture methods on samples taken at prosthetic joint revision surgery. Automated BACTEC blood culture bottles and cooked meat enrichment broth were the most sensitive methods (87% and 83%, respectively, as compared with fastidious anaerobic broth (57%) and direct plates (39%)); all were highly specific (97–100%). To our knowledge, this is the first prospective study aimed at comparing culture methods in routine use in UK clinical laboratories for the diagnosis of PJI.     

Laboratory detection of extended-spectrum-beta-lactamase-producing Enterobacteriaceae: evaluation of two screening agar plates and two confirmation techniques

The worldwide prevalence of extended-spectrum-beta-lactamase-producing ESBL-producing Enterobacteriaceae (ESBL-E) is increasing, making the need for optimized detection techniques more urgent. In this study we investigated the performance of two ESBL-E screening and two ESBL-E confirmation techniques. In accordance with the Dutch national guidelines (www.wip.nl), a collection of 642 highly resistant Enterobacteriaceae strains, as identified by Vitek2, was used to test the performances of two screening techniques (EbSA ESBL agar plate and ChromID ESBL agar plate) and of two confirmation techniques (MIC-strip ESBL and Vitek2 ESBL test panel). The individual test results were compared by using Etest, followed by a combination disk test if Etest results were inconclusive. Among group 1 isolates (Escherichia coli, Klebsiella spp., Proteus spp., Salmonella spp., and Shigella spp.) 291 (57.6%) were ESBL-E, versus 65 (47.4%) in group 2 (Enterobacter spp., Citrobacter spp., Morganella morganii, Serratia spp., and Providencia spp.). The sensitivities of all four tests for group 1 were comparable (EbSA, 96.6%; ChromID, 97.3%; MIC-strip, 99.6%; and Vitek2, 95.1%). The specificities of the EbSA and ChromID were the same (93.9%). However, the confirmation techniques produced many inconclusive test results, which reduces the applicability in routine laboratories. Only the two screening agar plates were validated for ESBL testing of group 2 microorganisms. They showed comparable sensitivities; however, the EbSA screening agar plate had a significantly higher specificity (78.6% versus 44.3%). In conclusion the screening agar plates performed better than the two confirmation techniques. The EbSA agar plate had the best overall performance.     

Improved detection of bacterial growth in continuous ambulatory peritoneal dialysis effluent by use of BacT/Alert FAN bottles.

Culture-negative peritonitis is a major complication for patients on continuous ambulatory peritoneal dialysis (CAPD) and precludes organism-specific therapy. The aim of the present study was to compare inoculation of 10 ml of CAPD effluent into BacT/Alert blood culture bottles (FAN [fastidious antimicrobic neutralizing], BacTAlert aerobic [BTA], and BacT/Alert anaerobic [BTAn] bottles) to our conventional method of using 50 ml of concentrated CAPD effluent to inoculate peptone broth bottles (BD bottles) and MacConkey agar and blood agar medium (BA-MAC). The FAN, BTA, and BTAn bottles were monitored automatically in the BacT/Alert blood culture instrument. A total of 207 CAPD effluents were studied, and in 97 bacteria were detected by at least one method. Compared to BTA bottles (79 of 97; 81.4%), BTAn bottles (78 of 97; 80.4%), and BD bottles (88 of 97; 90.7%), the single best broth medium for detecting bacterial growth in CAPD effluents was the FAN bottle (90 of 97 effluents; 92.8%). A total of 125 bacterial species were detected by any method, and the majority (91.8%) of CAPD effluents were infected with a single species. A combination of FAN and BTAn bottles detected 111 of 125 (88.8%) of all organisms, whereas a combination of BD bottles and BA-MAC detected 107 of 125 (85.6%) of all organisms. One or more organisms that would have been completely missed by the conventional method with BD bottles and BA-MAC were detected in 18 CAPD effluents. Of these 18 CAPD effluents, 6 showed no growth by the conventional method with BD bottles and BA-MAC. On the basis of our data, the most sensitive and least labor intensive method was direct inoculation of 10 ml of CAPD effluent into a FAN bottle and a BTAn bottle, which could be automatically monitored by the BacT/Alert blood culture instrument. On the basis of case definitions for peritonitis, the sensitivities and specificities of the methods with FAN and BTAn bottles and with BD bottles and BA-MAC were 81.1 and 98.8% and 74.5 and 96.5%, respectively.     

Evaluation of different culture methods for the diagnosis of peritonitis in patients on continuous ambulatory peritoneal dialysis

A total of 170 continuous ambulatory peritoneal dialysis (CAPD) fluids were processed by various culture methods, including direct inoculation of the centrifuged sediment, inoculation into automated blood culture bottles, water lysis, Tween-80 incorporated blood agar, and Triton-X treatment of the specimen. Of 170 CAPD fluids, 127 showed the growth of bacteria/fungi. Sixty-three fluids showed growth by all methods, the water lysis alone detected 24 additional positive cultures, while Tween-80 blood agar and Triton-X yielded 30 additional positive cultures. A combination of water lysis, Tween-80 blood agar and Triton-X treatment of the CAPD fluid is recommended for diagnosis of CAPD peritonitis in resource-limited settings.     

Isolation of Legionella pneumophila from blood with the BACTEC system: a prospective study yielding positive results.

In a prospective study of 16 patients with Legionnaires disease confirmed by cultural isolation of Legionella pneumophila from the respiratory tract, 38% (6 of 16) had positive blood cultures. Daily subcultures were made onto buffered charcoal-yeast extract plates from 6B aerobic and 7C anaerobic BACTEC blood culture bottles (Johnston Laboratories, Inc., Towson, Md.). Isolation of L. pneumophila was achieved from both aerobic and anaerobic bottles. L. pneumophila growth indices failed to exceed the BACTEC threshold limits; thus, the organism would have been overlooked despite its presence in the blood culture bottles. Bacteremic patients had statistically significant higher quantities of L. pneumophila isolated from sputum and visualized on direct fluorescent antibody stains. Thus, the potential exists for improved diagnosis of Legionella infection by a relatively noninvasive procedure (blood culture) with an instrument already in use in many hospital laboratories.