Aerobic Bottles Research Articles

Rapid Identification of Bacteria from Positive Blood Culture Bottles by Use of Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry Fingerprinting

Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

BLOOD COMPONENTS: Screening of single‐donor apheresis platelets for bacterial contamination: the PASSPORT study results

BACKGROUND: The PASSPORT study was an FDA‐mandated surveillance of outdated 7‐day apheresis platelets (APs) to assess the bacterial culture release test (RT) performance and the chance of transfusing APs containing viable bacteria compared to untested 5‐day APs.STUDY DESIGN AND METHODS: Aerobic and anaerobic culture bottles were inoculated with 4 to 5 mL from APs 24 to 36 hours postcollection. APs were released after 24 hours if no growth was observed. Released APs were recalled for RT positives, and clinical services were notified. Day 8 APs were recultured (surveillance test [ST]). Initially positive RTs and STs were confirmed by AP reculture.RESULTS: A total of 388,903 RTs were accrued September 2005 through January 2008 from 52 regional blood centers: RT‐positive APs interdicted before transfusion, 76 true positive (TP; 195/million; 95% confidence interval [CI], 154‐244/million) and 57 indeterminate (IN); and RT‐positive APs transfused, 14 TP and 242 IN. There were 14 reported septic transfusion reactions (STRs) from 13 AP collections (23 units) transfused on Days 3 through 7; three STRs were from Day 6 or 7 APs. There were two false‐negative RTs causing STRs in three patients. No deaths were reported. STs had four TPs of 6039 tested (662/million; 95% CI, 180‐1695/million).CONCLUSIONS: RT culturing prevents issuance of some bacterially contaminated APs. ST culture data and clinical reports suggest that this screening fails to detect all contaminated units. No fatalities were reported related to AP transfusion. Additional actions or testing may be required to further reduce the residual STR risk of RT APs, even with a 5‐day storage limitation.

Yields from blood cultures of patients with suspected paratyphoid fever A

The yield and speed of detection of Salmonella enterica serotype Paratyphi A from the blood of patients with suspected paratyphoid fever A in 13 500 paired aerobic and anaerobic bottles (AEB, ANB) that were each filled with 5 ml of blood by the BacT/ALERT 3D system were compared, and the blood bacterial counts of 1 000 probable patients were estimated by pour plate method. A total of 4 060 isolates were recovered, of these, 3 149 were recovered from both AEB and ANB, 461 from the AEB only, and 450 from the ANB only. The estimating median bacterial count in blood from 400 patients was 0.5 CFU/ml. The research findings demonstrate that the blood volume drawn is an important factor determining the yields from blood cultures. Growth of significantly more isolates was detected earlier in AEB.

Detection of bacterial contamination of apheresis platelets in a Chinese Blood Center

Bacterial contamination of platelets is considered as the most frequent infectious risk of transfusion. The prevalence of bacterial contamination has been reported and varied considerably in different countries, but the data for bacterial contamination of platelets in China are rarely reported. Eight thousand apheresis platelet concentrates (PCs) were analysed by aerobic and anaerobic cultures. Ten millilitres of PCs were inoculated into aerobic and anaerobic bottles (5 mL each), followed by the incubation for a maximum of 7 days. A new sample was taken from the unit for reculturing in order to confirm the initial positive reaction. All positive culture bottles were referred for bacterial isolation and identification. Twenty one cultures (0.26%) were flagged as positive in initial culture. Five cultures (0.06%) were confirmed as true positive and nine cultures (0.11%) were confirmed as indeterminate in reculture. A bacterium from skin flora (Propionibacterium spp.) was the most prevalent contaminant. Mean time to initial positive culture from start of incubation was 22.1 h for confirmed positive units and 97.3 h for indeterminate units. Most PC units had already been issued by the time of initial positive culture with a 'negative-to-date' issued strategy. There is a risk of bacterial contamination of PCs in China. Implementing bacterial screening of platelets could reduce the risk of septic reaction and fatalities due to transfusion of bacterially contaminated platelets. However, bacterial contamination PCs can still be transfused due to the delay until a positive signal in the culture system.

Frequency of bacterial contamination of platelet concentrates before and after introduction of diversion method in Japan

Bacterial contamination of platelet concentrates (PCs) is the major infectious risk in transfusion medicine. To evaluate the necessity of implementing novel strategies for the reduction of bacterial contamination, it is necessary to establish a precise contamination frequency in PCs. The frequency of bacterial contamination in PCs issued by the Japanese Red Cross was determined using expired PCs before and after the implementation of the diversion method. The culture method was designed such that it yields the least possibility of false-negative results: platelet specimens were sampled after at least 4 days of storage and the inoculum volume was 10 mL for both aerobic and anaerobic bottle cultures. Of the 21,786 PCs cultured, 36 (0.17%) were confirmed to be bacterially contaminated before the implementation of the diversion method. After its implementation, the number of contaminated PCs decreased to 11 of 21,783 (0.05%) with a reduction rate of 71% and the number of contaminations of clinical importance was 4 (0.018%) excluding PCs positive for Propionibacterium acnes. The frequency of contamination by bacteria presumed to originate from donors' blood did not decrease. The effect of the diversion method on the frequency of bacterial contamination is robust. The low incidence of septic reactions after PC transfusion in Japan in spite of the contamination frequency being comparable to those in Western countries and the noninstitution of culture screening suggests the importance of a short shelf life (72 hr) for PCs introduced in Japan.

Gordonia sputiBacteremia

To the Editor: In November 2007, a 69-year-old man with fever was hospitalized at the Northern Hospital in Marseilles, France. He also had diabetes, high blood pressure, and alcohol and tobacco addictions. In September 2007, he had received a diagnosis of laryngeal cancer, which required 2 chemotherapy treatments through a central venous catheter (CVC), the second of which he had received 6 days before his November visit. Prostatic cancer, diagnosed 1.5 years earlier, had been treated by radiotherapy. At the time of the November admission, he had leukopenia (1.77 × 109 leukocytes/L with 0.49 × 109 polymorphonuclear cells/L) and an elevated C-reactive protein level (151 mg/L). The patient was admitted with a preliminary diagnosis of drug-induced febrile granulocytosis; the origin of his fever remained unclear. Blood for culture was first collected from a peripheral vein and on the next day was collected from a peripheral vein and from the CVC. Gram-positive rods grew in the aerobic bottle from the CVC sample. The microorganism was identified by biochemical tests using API Coryne strip (bioMerieux, Marcy-l’Etoile, France) as Rhodococcus spp. (94% similarity). The day after hospital admission, the patient was empirically treated with intravenous ticarcillin-clavulanate, 5 g 3×/day; ciprofloxacin, 200 mg 2×/day; and granulocyte colony–stimulating factor. One day later, fever resolved and the polymorphonuclear cell count was within normal limits. Oral antimicrobial drug therapy was continued for 1 week. Bacterial identification of the strain was performed by 16S rRNA sequencing. We obtained a 1,464-bp sequence, which was found to differ at only 2 nt positions from that of Gordonia sputi (GenBank accession no. {type:entrez-nucleotide,attrs:{text:X80634,term_id:562305,term_text:X80634}}X80634). We concluded that our patient had catheter-related bacteremia caused by G. sputi because he was immunocompromised and had a CVC. We ruled out a contaminant because the organism did not belong to the normal flora of human skin and because fever resolved after treatment with antimicrobial drugs. The genus Gordona was first described in 1971, for coryneform bacteria isolated from sputum of patients with pulmonary disease or from soil (1). It is a member of the mycolic acid–containing group consisting of genera Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella. The genus has been revised several times by rearrangements with the genera Rhodococcus and Nocardia, and the name Gordona was changed to Gordonia in 1997. The genus belongs to suborder Corynebacterineae within the order Actinomycetales and currently contains 27 recognized species; only 7 have been described in human disease. Species are identified by molecular analysis. Gordonia spp. cause a wide spectrum of disease in humans ( 2–10; Table). Neurologic and vascular infections in immunocompromised and immunocompetent patients have been reported. Cutaneous and respiratory infections, otitis externa, osteitis, and arthritis have reportedly occurred only in immunocompetent patients. Bacteria have most often been isolated from blood samples. Bacteremia has started from underlying disease such as a sequestrated lung (4) or acute cholecystis (5) or has been related to coronary artery surgery (2) and frequently to CVCs (2,3,6). Catheter removal has been recommended for treatment of Gordonia spp. infections in children (6), but the recommendation varied for adults. Six cases of infection in children have been described (6), appearing as bacteremia, ventriculitis, and brain abscess. Table Summary of clinical reports involving Gordonia spp.* Microbiologic diagnosis of Gordonia spp. remains difficult. Biochemical profiles can lead to incorrect identification of isolates as Rhodococcus spp. (2,4–6,9) and sometimes Corynebacterium spp. (3) or Nocardia spp. (6). Identification at the genus and species levels is presently obtained by 16S rRNA sequence comparisons. No recommendation for antimicrobial drug susceptibility testing has been unanimously approved, but these microorganisms seem to be susceptible to many antimicrobial drugs (6). Previous studies suggest a combination of penicillins and aminoglycosides as a suitable therapy for Gordonia-related bacteremia (3). Carbapenem or fluoroquinolone in combination with an aminoglycoside can also be used (6). Antimicrobial drug susceptibility is similar to that of Rhodococcus spp., for which Gordonia spp. are usually incorrectly identified. However, although vancomycin is often used to treat Rhodococcus spp. infections, in a previous study 11% of Gordonia spp. isolates were resistant (6). Treatment must therefore be evaluated specifically for each patient. Gordonia spp. are environmental bacteria whose implication in human disease seems to be increasing. Phenotypic identification of bacteria included in this genus is difficult, and they are often poorly identified as Rhodococcus spp. or Corynebacterium spp. Molecular identification of Gordonia spp. by using 16S rRNA gene sequence comparison enables their characterization in human disease because the method is more accurate. The fact that these bacteria are often associated with medical devices highlights their role as nosocomial agents. Gram-positive bacilli must, therefore, not be systematically considered as contaminants, especially if associated with medical devices, and should be thoroughly identified by molecular methods in addition to biochemical tests.

Study on blood cultures and bacteria counts in the blood of paratyphoid fever A patients

Paratyphoid fever A (PA) is a systemic infection caused by Salmonella enterica serotype Paratyphi A (SPA) [1]. Although blood cultures are considered to be the ‘gold standard’ for the detection of bloodstream pathogens, there is little information on the controlled clinical comparison of the paired aerobic and anaerobic bottles in the automated continuously monitoring instrument systems, and bacterial counts in the blood of patients with bloodstream infections [2, 3]. Yuxi City is the most severe endemic area of PA in China [4]. In order to determine the factors determining the yields from blood cultures, we have performed broth blood cultures (BBC) by the BacT/ALERT 3D automated blood culture system (bioMerieux, Inc., Durham, NC) and quantitative whole-blood culture (QWBC) for a large series of patients with suspected PA between 1999 and 2008. A total of 13,500 patients admitted with suspected PA to our institutions between June 1999 and June 2008 were studied. The available blood samples were obtained with vacuum system blood collection tubes. A blood culture set included one aerobic bottle (AEB) and one anaerobic bottle (ANB) (bioMerieux), and one QWBC plate (Columbia agar, bioMerieux). A 13-ml quantity of blood from each patient for one test set was considered to be the maximum amount to obtain from a single venipuncture and was distributed as follows: 5 ml to one AEB, 5 ml to one ANB, and 3 ml to one plate (only for 1,000 probable cases of PA). The culture bottles contained 5 ml of blood sample and 40 ml of culture media. Each bottle was monitored with the BacT/ALERT 3D system at 37°C for a standard 5-day protocol or until the instrument signaled a positive result [5]. Any bottle with a positive signal was immediately removed from the instrument, and an aliquot of the blood– broth mixture was taken from the bottle with a sterile needle and swab for subculture on sheep blood nutrient agar and XLD agar (Oxoid, England); the time-to-detection of bacteria causing PA was taken as the incubation time required before the instrument gave a positive signal. All bottles were treated independently until the end of the 7-day incubation and testing period. Both BBC and QWBC were performed for 1,000 probable cases of PA. QWBC were carried out by a pour plate method and the blood bacterial counts were estimated [3]. In brief, 3 ml of blood was drawn aseptically from each patient and injected into a sterile heparinized tube. Three milliliters of heparinized blood was mixed well with 19 ml of molten (50°C) Columbia agar in a sterile Petri dish, allowed to set for solidification, and then incubated at 37°C. After 2 to 4 days, colonies were identified, counted, and recorded as colony-forming units (CFU) per milliliter. The number of SPA bacteria per milliliter of blood was estimated from the number of CFU on each pour plate. The minimum volume of whole blood used for QWBC from any one patient was 3 ml. This gives a lower limit of Eur J Clin Microbiol Infect Dis (2009) 28:1259–1261 DOI 10.1007/s10096-009-0766-9

Study on blood culture in blood of paratyphoid fever A patients

Objective To compare the yield and speed of detection of Salmonella subsp, enterica serotypo Paratyphi A from the blood of patients with suspected paratyphoid fever A. Methods With the BacT/ALERT 3D system and paired aerobic and anaerobic bottles (AEB, ANB) that were each filled with 5 ml of blood, the blood culture of 13 500 suspected paratyphoid fever A patients were performed. Results A total of 4 060 isolates were detected. About 3 149 were detected from both AEB and ANB. Four hundred and sixty-one isolates were detected only from the AEB and 450 were only from the ANB. The detection rates of the AEB and ANB were all 26.7% (χ =0.023, P=0.880). The increased detection rate attributed to the additional blood volume in the AEB and ANB were 11.3% and 11.1%, respectively. The detection speed differed between the two medium formulations. The time to detection was (23.66±15.89) h and (25.48±16.92) h for3 149 isolates, respectively (t=7.007, P<0.01).The mean time to detection was 31.80±20. 97 for 461 isolates discovered with AEB and (33.45±20.72) h for 450 isolates discovered with ANB. Conclusion The blood volume is an important factor in determining the detection rate of blood culture. Although no statistical difference for positive rate was found between the AEB and ANB, more isolates was detected earlier in AEB. Key words: Paratyphoid fever; Salmonella, paratyphi A; Colony count, microbial; Bacteriological techniques

Evaluation of MicroScan and Phoenix System for Rapid Identification and Susceptibility Testing Using Direct Inoculation from Positive BACTEC Blood Culture Bottles

Procedures for rapid identification and susceptibility testing by direct inoculation (DI) from positive blood culture bottles into an automated system have not been standardized. This study was purposed to evaluate DI from BACTEC 9240 blood culture system (BD, USA) into MicroScan (Dade Behring, USA) or Phoenix (BD, USA). From May to June 2006, bacterial pellets from positive aerobic bottles showing gram-positive cocci (GPC) or gram-negative rods (GNR) of single morphology were directly inoculated to MicroScan PosCombo1A and NegCombo32 and to Phoenix PMIC/ID-107 and NMIC/ID-53. In addition, the automated instruments were also inoculated from subcultures (standard inoculations, SI). Species identification and susceptibilities were compared between DI and SI and between MicroScan and Phoenix. A total of 108, 104, and 78 specimens were tested with MicroScan, Phoenix, and both, respectively. When DI and SI were matched, 94.8% of GPC were correctly identified with MicroScan, compared to 80.7% with Phoenix, and 93.9% of GNR were correctly identified with MicroScan, compared to 95.7% with Phoenix. DI with MicroScan and Phoenix showed correct susceptibilities in 94.6% of 1,150 and 96.5% of 660 tests (with very major error [VME] of 1.1% and 1.1%), respectively, among GPC and in 94.4% of 942 and 96.3% of 781 tests (with VME of 0.6% and 0%), respectively, of GNR. Correlation of identification/susceptibilities between MicroScan and Phoenix using DI were 81.8%/98.0% for Staphylococcus aureus and 100.0%/95.6% for Escherichia coli. DI warrants a reliable method for identification and susceptibility testing of both GPC and GNR in MicroScan, and those of only GNR in Phoenix.

Delayed insertion of blood culture bottles into automated continuously monitoring blood culture systems increases the time from blood sample collection to the detection of microorganisms in bacteremic patients

This study examined the effects on patients with bacteremia of delaying the insertion of a blood culture bottle into an automated, continuously monitoring blood culture system. We investigated the time taken from the collection of blood samples (collection) to the insertion of blood culture bottles inoculated with blood samples into the instrument (insertion), and compared the mean detection time from collection to a positive signal from the instrument with the time between collection and insertion. The study was conducted from January 2003 to December 2004 at Kyoto University Hospital. Insertion into the system on the day of blood sample collection was defined as same-day insertion, and insertion on a different day to collection was defined as delayed insertion. The 7394 aerobic and anaerobic blood culture bottle sets obtained during the study period included 4361 sets with same-day insertion and 3033 sets with delayed insertion. For same-day insertion, 458 microorganisms were isolated from 432 positive sets in which at least one blood culture bottle was positive. For delayed insertion, 405 microorganisms were isolated from 379 positive sets in which at least one blood culture bottle was positive. The mean detection time for all microorganisms was significantly earlier for same-day insertion than for delayed insertion (28.3 h vs. 45.0 h, respectively, P < 0.0001). Delays from collection to insertion affect the time from collection to the detection of microorganisms.

Detection of Bacterial and Yeast Species with the Bactec 9120 Automated System with Routine Use of Aerobic, Anaerobic, and Fungal Media

During the period 2006 and 2007, all blood cultures required by four units at high infective risk and most of those required by other units of the University Hospital of Palermo, Palermo, Italy were performed using a Bactec 9120 automated blood culture system with a complete set of Plus Aerobic/F, Plus Anaerobic/F, and Mycosis IC/F bottles. The aim of the study was to enable the authors to gain firsthand experience of the culture potentialities of the three different media, to obtain information regarding the overall and specific recovery of bacteria and yeasts from blood cultures in the hospital, and to reach a decision as to whether and when to utilize anaerobic and fungal bottles. Although very few bloodstream infections (1.8%) were associated with obligate anaerobes, the traditional routine use of anaerobic bottles was confirmed because of their usefulness, not only in the detection of anaerobes, but also in that of gram-positive cocci and fermentative gram-negative bacilli. In this study, Mycosis IC/F bottles detected 77.4% of all the yeast isolates, 87.0% of yeasts belonging to the species Candida albicans, and 45.7% of nonfermentative gram-negative bacilli resistant to chloramphenicol and tobramycin. In order to improve the diagnosis of fungemia in high-risk patients, the additional routine use of fungal bottles was suggested when, as occurred in the intensive-care unit and in the hematology unit of the University Hospital of Palermo, high percentages of bloodstream infections are associated with yeasts, and/or antibiotic-resistant bacteria and/or multiple bacterial isolates capable of inhibiting yeast growth in aerobic bottles.

Difficult Intubation Provokes Bacteremia

To evaluate the prevalence of bacteremia after mask ventilation, laryngoscopy, and endotracheal intubation before induction of general anesthesia and to discover any correlation between traumatic manipulations and bacteremia. The specific bacteria responsible, knowledge of which may guide the prophylactic use of antibiotics, also were investigated. Fifty patients were enrolled. Three 10-mL blood samples were collected from a peripheral vein 10 min before induction of anesthesia, 10 min after mask ventilation, and 10 min after intubation. All samples were placed in aerobic and anaerobic bottles for culture and bacterial identification. Cultures received 10 min after intubation were positive in 12% of patients. The following strains were isolated: Escherichia coli in two cases, Staphylococcus aureus in three cases, and Peptostreptococcus anaerobius in one case. A strong positive correlation was found between difficult intubation and bacteremia. No correlation between bacteremia and easy intubation or between bacteremia and face mask ventilation was identified. Traumatic manipulations during difficult laryngoscopy and endotracheal intubation could cause bacteremia. This finding may justify and guide prophylactic use of antibiotics.

Sterility screening of platelet concentrates: questioning the optimal test strategy

Routine bacterial monitoring of apheresis platelet concentrates (APC) and pooled platelet concentrates (PPC) was introduced in two German blood services using culture and real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). The results of testing are reviewed and used to discuss different strategies for detection of bacterial contamination of PCs. Two thousand three hundred and sixty-two APCs and 1993 PPCs have been tested by real-time RT-PCR and the BacT/Alert automated culturing system using aerobic and anaerobic culture bottles. After standard processing of PCs and storage of 22-24 h at 20-24 degrees C with agitation, samples were taken under aseptic conditions. Reactive culture bottles were confirmed as positive and bacterial isolates were identified by 16S rRNA analysis and biochemical tests. Seventeen of 2362 tested APCs were reactive in culture and one also in RT-PCR. Of these, 13 APCs were identified as initially positive as Staphylococcus warneri (n = 1, positive in aerobic and anaerobic culture), Propionibacterium acnes (n = 12, positive only in anaerobic culture) and four were initially reactive. Two of 1993 PPCs were initially reactive (anaerobic) and two more were confirmed positive (anaerobic) from a repeat culture and identified as P. acnes. All remaining specimens were tested negative. Our study demonstrates that the predominant organisms implicated in platelet bacterial contamination are part of the human skin flora. Inoculating blood culture systems and anaerobic cultivation detects these bacteria after approximately 3-7 days when blood products have been transfused. Based on the presented data different screening strategies are discussed.

Bacterial detection in apheresis platelets: blood systems experience with a two‐bottle and one‐bottle culture system

United Blood Services (UBS) began bacterial testing of platelets (PLTs) using one-bottle cultures in September 2003. Collection of 7-day PLTs using two-bottle cultures began in April 2006. This study compares our experience using both systems. PLTs from 13 UBS regional centers cultured from September 1, 2003, to September 1, 2007, were included in the analysis. Positive-signal bottles from a commercially available microbial detection system (BacT/ALERT, bioMérieux) were sent, with corresponding PLTs if available, for confirmatory testing using plate culture media. AABB definitions were used with modifications. A total of 51,265 7-day PLT collections and 191,521 5-day PLT collections were tested with bacterial cultures. The overall true-positive (TP) rate for the two-bottle system (1:8544) was comparable to the TP rate with the previous one-bottle system (1:6344). In three of six yield cases, only the anaerobic bottle was positive (two cases of Group D Streptococci, one case of Corynebacterium spp.). The false-positive (FP) and indeterminate (IND) rates in the anaerobic bottle (1:1767 and 1:1830, respectively) were significantly higher than those in the aerobic bottle (1:6408 and 1:17,088, respectively; p < 0.001). One confirmed transfusion-related septic reaction, classified as a late TP after investigation, was reported out of 242,786 tested PLT donations. The rate of TP cases by the two-bottle system was not increased over the one-bottle system, although anaerobic-bottle-only positive cases were detected. FP and IND rates were increased in the two-bottle system, attributable to the anaerobic bottle. Observation of only one documented transfusion-related septic reaction in 4 years of bacterial screening at UBS is reassuring, although limitations in passive surveillance and higher rates of reactions reported by others indicate the need for continued vigilance.

Microbial Contamination in Cord Blood (CB): A Retrospective Analysis of Three Test Protocols

Aim Since 1995, the SCBB has used three protocols to screen CB for contaminating microbes. The protocols differed in the number of culture bottles inoculated and sample volume used. The aim of this study was to compare microbial detection rates for each method. Method CB units (n = 9,618) were tested for microbial contamination using BacT/ALERT blood culture bottles (BioMerieux). Of these, 6,836 had 20 ml plasma waste fraction inoculated into adult anaerobic and aerobic bottles (Group A), 365 had 1 ml final stem cell product into an aerobic paediatric bottle (Group B), and 2,417 had 1 ml final product into a paediatric bottle and 10 ml plasma waste fraction into an adult anaerobic bottle (Group C). All bottles were incubated for a minimum of 5 days. Detection rates for microbe contamination were calculated for each group. Result Microbe detection rates were: Group A –345/6836 (5.0%), Group B – 3/365 (0.8%) and Group C – 66/2417 (2.7%)(p < 0.0001). Organisms detected by A but not by B (and to a lesser extent C) included Streptococcus, Staphylococcus, Bacteroides and Proprionibacterium. Conclusion Contamination detection rates differ significantly, depending upon the sample volume and type of culture bottle used. A protocol that uses the recommended sample volume and both anaerobic and anaerobic test bottles is likely to detect the highest number of contaminated units. Testing of a small sample from the final product using only a single paediatric aerobic bottle is likely to miss detection of the majority of anaerobic and some aerobic organisms. Prospective studies to define a standardised method for microbial detection for CB are required.

LethalMycobacterium massilienseSepsis, Italy

To the Editor: A strain of Mycobacterium massiliense was isolated from the blood of a kidney transplant patient in Italy at the same time she was diagnosed as having pulmonary tuberculosis. M. massiliense bacteremia appears to have played a role in her sudden death. The patient, a 63-year-old woman who had had a kidney transplant 10 years earlier and was receiving immunosuppressive treatment with cyclosporine, azathioprine, and prednisolone, was hospitalized in an intensive care unit because of septic shock, a stuporous condition, hypotension, respiratory insufficiency, and acute renal failure. Results of initial microbiologic investigations (cultures of blood, urine, and bronchial aspirate, and nasal and pharyngeal swabs; tests for antigens of Legionella pneumophila, pneumococcus, and Cryptococcus neoformans; and serologic analysis for pneumotropic viruses and bacteria) were negative. Hematologic analysis showed leukopenia with high neutrophil counts. Septic shock–specific therapy was given along with wide-spectrum antimicrobial drug therapy (levofloxacin, trimethoprim-sulfamethoxazole, piperacillin-tazobactam, and fluconazole). After moderate improvement, the patient’s general condition worsened and prompted a new round of microbiologic tests, including those for mycobacteria. After acid-fast bacilli were detected in a bronchial aspirate and results of nucleic acid amplification (Amplicor; Roche, Basel, Switzerland) were positive for M. tuberculosis complex, she was given standard antituberculosis treatment. She died the next day. Three blood cultures obtained 7 days before the death of the patient showed positive results in aerobic bottles but not in anaerobic bottles (BacT/ALERT SA; bioMerieux, Marcy l’Etoile, France). Subcultures spread onto blood agar yielded small white colonies of gram-positive bacilli, including branched forms, within 24 hours. One month earlier, the patient had been seen as an outpatient with intermittent fever, and unidentified gram-positive bacilli were observed in her blood culture. Genetic sequencing of the first one third of the 16S rDNA gene (1) of the strain (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU370523,term_id:169264479}}EU370523) showed 99.8% identity with M. abscessus, M. bolletii, M. chelonae, and M. massiliense. To discriminate between these 4 species, a 723-bp fragment of the RNA polymerase β subunit (rpoB) gene (2) also was sequenced (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU370524,term_id:169264486}}EU370524). Sequencing showed 100% similarity with M. massiliense; the next most closely related species was M. bolletii (98.6% similarity). An isolate of M. tuberculosis that was susceptible to all first-line antituberculosis drugs was recovered from this patient’s bronchial aspirate in both solid and liquid media. Immunocompromised patients, including those with organ transplants, are known to be prone to mycobacterial infections (3). Mixed mycobacterial infections also have been reported (4). This newly described species has been isolated from pulmonary fluids (5), blood (6), intramuscular injection sites (7), and surgical wounds (8). Because sequencing 16S rDNA does not differentiate M. abscessus, M. bolletii, M. chelonae, and M. massiliense (5), use of the rpoB sequence is crucial. As reported for isolates of M. massiliense (5–8), our isolate was characterized by high MICs (9) (broth microdilution by using the Sensititer RGMYCO; Trek Diagnostic Systems Inc., Cleveland, OH, USA) to most of the antimicrobial drugs tested. The isolate was sensitive only to clarithromycin and amikacin and showed borderline sensitivity to linezolid (Table). Table Antimicrobial drug resistance pattern of the strain of Mycobacterium massiliense isolated from the patient, Italy* Despite co-infection with M. tuberculosis, the patient’s death was likely caused by M. massiliense bacteremia. The patient died before the isolate was identified as a mycobacterium, and unfortunately, none of the drugs used empirically was active against this organism. Only speculations can be made about how this patient acquired the M. massiliense infection. However, 5 months before her hospital admission, the patient had received a coxofemoral arthroprosthesis as a result of a fall and had since complained of generalized bone pain and had remained bedridden. Although in this case no proof exists, infection caused by rapidly growing mycobacteria after surgical intervention (10) is well known and should be considered. M. massiliense has been distinguished from M. abscessus by sequencing of the rpoB gene (2). Because this technology is available in relatively few clinical laboratories, cases of infection with M. massiliense may be mistakenly attributed to M. abscessus. Although infections with M. massiliense may be underrecognized, reports of these infections are raising concern. The capacity of this bacteria to infect different body sites is further evidence for the pathogenic potential of a rapidly growing mycobacteria in human infections (10).

Ascitic Fluid Lactoferrin for Diagnosis of Spontaneous Bacterial Peritonitis

The diagnosis of spontaneous bacterial peritonitis (SBP) is based on a manual count of ascitic fluid polymorphonuclear cells (PMNs). This procedure is operator-dependent and lysis of PMNs during transport to the laboratory may lead to false-negative results. Furthermore, ascitic fluid culture is insensitive and leads to delays in diagnosis. The aim of this study was to assess the utility of ascitic fluid lactoferrin (AFLAC) for the diagnosis of SBP and to identify a cut-off level that can be used for future development of a rapid bedside test. A total of 218 consecutive ascites samples from 148 patients (1-8 samples per patient) with cirrhosis at 2 tertiary care medical centers were examined for PMN count, bedside culture, and lactoferrin concentration. AFLAC concentrations were determined using a polyclonal antibody-based enzyme-linked immunosorbent assay. An ascitic fluid PMN count of 250 cells/mL or greater with or without a positive culture was used for diagnosis of SBP. Twenty-two (10.1%) samples fulfilled diagnostic criteria for SBP. Samples with SBP had a significantly higher lactoferrin concentration (median, 3744 ng/mL; 25th-75th percentiles [P25-P75], 788-9617) compared with non-SBP samples (median, 31 ng/mL; P25-P75, 12-67; P < .001). By using a cut-off level of 242 ng/mL, the sensitivity and specificity of the assay for diagnosis of SBP were 95.5% and 97%, respectively. The area under the receiver operating characteristic curve was 0.98. AFLAC can serve as a sensitive and specific test for diagnosis of SBP. Qualitative bedside assays for the measurement of AFLAC can be developed easily and may serve as a rapid and reliable screening tool for SBP in patients with cirrhosis.

Screening platelet concentrates for bacterial contamination: low numbers of bacteria and slow growth in contaminated units mandate an alternative approach to product safety

We introduced 100% screening of platelets for bacterial contamination in 2005 to reduce the risk of clinical sepsis from platelet transfusion. We test all outdating units again at expiry to assess the sensitivity of the initial test. We test all platelet concentrates prior to release for clinical use using a large volume automated culture technique on the day after manufacture. All units that expire unused are retested. Platelets still in stock on day 4 of storage may have a repeat culture performed, and are returned to stock with two extra days of shelf life. Of 43,230 platelet units screened, 35 (0.08%) were positive; of 8282 expired unused, 18 (0.22%) were positive; and of 3310 day-4 retests, four (0.12%) were positive. Overall sensitivity of the initial screening test was 29.2% (95% confidence interval 19.4 to 39.1%). Thirteen of the 35 positive screening tests would have been expected to grow in both aerobic and anaerobic bottles; eight grew in aerobic culture only and five grew in anaerobic culture only, indicating that the likely number of bacteria in the contaminated platelet units at the time of sampling was less than 60 colony-forming unit per platelet unit. Screening platelet concentrates for bacterial contamination using the most sensitive method available has a sensitivity of less than 40% because of the low numbers of bacteria in the initial contamination. Effective resolution of this problem will require a pathogen-inactivation technique.

Nocardia asteroides peritoneal dialysis-related peritonitis: a case of successful treatment and return to peritoneal dialysis

A 66-year-old male from rural Manukau, New Zealand, presented to hospital with 2 weeks of generalized abdominal pain and cloudy PD effluent. He had been on PD for 3 years, with only one previous episode of PD-related peritonitis secondary to Streptococcus oralis a year prior. Other significant co-morbidity included well-controlled Type 2 diabetes mellitus on diet therapy, hypertension, mild chronic obstructive airways disease and quiescent gout. On initial examination, the patient was afebrile with blood pressure of 140/70mmHg. Abdominal examination showed generalized tenderness and normal bowel sounds. The PD catheter exit site was normal. Microscopy of dialysate showed 1600 leucocytes, 86% of which were neutrophils, and no organisms. After initial investigations, antibiotic treatment was commenced for PD-related peritonitis with empirical intraperitoneal cephazolin and gentamicin. His dialysate sample was processed in our microbiology laboratory; this involved inoculation of 10 mL into both BacT/ALERT FA aerobic and FN anaerobic blood culture bottles (bioMerieux, Marcy-l’Etoile, France), with the deposit from 10 mL of centrifuged sample inoculated onto two sheep blood agar plates, incubated in 5% CO2 and anaerobically, and one chocolate agar plate incubated in 5% CO2. After 5 days incubation, the BacT/ALERT FA aerobic bottle was reported as growing a fungus. Since one of the enrichment broths had become positive, the primary agar plates were held longer than the usual 5 days, but remained culture negative after 21 days. The patient was therefore treated as fungal peritonitis with a change in his antibiotic regimen to fluconazole 200 mg orally on alternate days and removal of his PD

Assessing How Many Blood Cultures Are Needed for Detecting Bloodstream Infections

In their recently published work, Lee et al. (5) have estimated the cumulative sensitivity of blood cultures in detecting bloodstream infections (BSIs) in adult inpatients using automated detection systems. They considered all instances in which at least three blood cultures per patient were taken within a 24-h period. Only cases judged to represent true infection, according to medical record review, were included. Very interesting findings were shown. For example, when only two blood cultures were considered, approximately 10% of the BSIs would be missed. In addition, important differences between microorganisms were observed. Thus, in the two blood cultures taken first, a sensitivity of 97% was reached for Staphylococcus aureus bacteremia, while just 85% sensitivity was reached for Pseudomonas aeruginosa and Candida albicans. Lee et al. (5) discussed important potential explanations for their findings. They cited an earlier study by Cockerill et al. (3), who, having found similar results, speculated that detection of low-level bacteremia by modern blood culture systems may be improved when culturing greater volumes of blood. Lee et al. mentioned that more blood cultures may also be required for patients already on antibiotic therapy. Indeed, several earlier researches have focused on increasing blood culture sensitivity by culturing greater volumes of blood (2, 6, 7, 10, 15). Lee et al. stated that their study design may have underestimated detection sensitivity (5). Disclosure of positive results obtained for each blood culture, i.e., not cumulative detection, could help clarify this issue. Assuming that every single culture had the same probability to be positive, an even distribution would be expected. Otherwise, it would be necessary to look for factors that could explain the differences. It must be noted, however, that this research design has potential additional biases. Considering only those patients who were asked for three or more blood cultures, instead of the whole population (i.e., excluding patients only asked for two blood cultures), this study may have focused on special subpopulations of patients. Health professionals could have asked for more blood cultures from patients with special clinical situations, such as previous antibiotic therapy and a wide spectrum of conditions known to produce low-level or intermittent bacteremia or fungemia. Furthermore, the inclusion of the whole population with suspected BSIs would allow better analysis of the impact of taking more blood cultures. Hopefully, this could also contribute to the identification of subpopulations where this approach would be truly effective, thus improving the cost-benefit ratio. Besides its sensitivity in detecting BSIs, its effect on contamination rates should also be assessed, since the latter clearly increases the laboratory workload and costs and could also cause clinical misinterpretation (1, 13, 16). Other issues should also be considered, such as the type of bottles used and the use of dissimilar criteria to confer clinical significance according to isolated microorganisms. More strict criteria are usually demanded for some bacteria, e.g., coagulase-negative staphylococci and viridans group streptococci, unique isolates of which tend to be considered contaminants (2, 11, 16). Thus, these bacteria are likely to be recovered from more than one blood culture considered to be significant. On the other hand, in the protocol of Lee et al. (5), each blood culture consisted of 20-ml samples equally distributed among aerobic and anaerobic bottles (10 ml each). It has been observed that the recovery rates of some microorganisms, especially Pseudomonas spp. and fungi, are significantly lower in anaerobic media than in aerobic media (4, 8-12, 17). Interestingly, in the work of Lee et al. (5), both P. aeruginosa and C. albicans showed the lowest detection rates if only one or two blood cultures were considered. As previously suggested, better recovery yields could be achieved if the aerobic/anaerobic medium ratio were increased (4, 6, 8, 14). Determining how many blood cultures are required to reach ≥99% test sensitivity is clearly of the utmost importance. Studies like those conducted by Lee et al. (5) and Cockerill et al. (3) set the basis for carrying out prospective researches specially designed to avoid all recognizable biases.