Challenges and solutions for instituting an efficient maintenance program for laboratory equipment in Central Asian, and developing world, countries.

We review the current state of quality assurance in laboratories of the five Central Asia Republics (CARs), focusing on laboratory equipment, and compare quality assurance approaches with CLSI standards. The laboratories of the CARs faced exceptional challenges including highly-structured laboratory systems that retain centralized and outmoded Soviet-era approaches to quality assurance, considerably jeopardizing the validity of laboratory tests. The relative isolation of the CARs, based on geography and almost exclusive use of the Russian language, further hamper change. CARs must make high-level government decisions to widely implement quality assurance programs within their laboratory systems, within which approaches to the management of laboratory equipment will be a prominent part.

Clinical characteristics of patients who test positive for Clostridium difficile by repeat PCR.

The high sensitivity of PCR assays for diagnosing Clostridium difficile infection (CDI) has greatly reduced the need for repeat testing after a negative result. Nevertheless, a small subset of patients do test positive within 7 days of a negative test. The aim of this study was to evaluate the clinical characteristics of these patients to determine when repeat testing may be appropriate. The results of all Xpert C. difficile PCR (Cepheid, Sunnyvale CA) tests performed in the clinical microbiology laboratory at New York-Presbyterian Hospital, Columbia University Medical Center (NYPH/CUMC) from 1 May 2011 through 6 September 2013, were reviewed. A retrospective case-control study was performed, comparing patients who tested positive within 7 days of a negative test result to a random selection of 50 controls who tested negative within 7 days of a negative test result. During the study period, a total of 14,875 tests were performed, of which 1,066 were repeat tests (7.2%). Eleven of these repeat tests results were positive (1.0%). The only risk factor independently associated with repeat testing positive was history of a prior CDI (odds ratio [OR], 19.6 [95% confidence interval {CI}, 4.0 to 19.5], P < 0.001). We found that patients who test positive for C. difficile by PCR within 7 days of a negative test are more likely to have a history of CDI than are patients who test negative with repeat PCR. This finding may be due to the high rate of disease relapse or the increased likelihood of empirical therapy leading to false-negative results in these patients.

Molecular characterization of methicillin-susceptible Staphylococcus aureus clinical isolates in the United States, 2004 to 2010.

While much is known about the geographic distribution of different clonal types of methicillin-resistant Staphylococcus aureus (MRSA), few studies have assessed the molecular epidemiology of methicillin-susceptible S. aureus (MSSA), despite its continued clinical importance. In each U.S. Census region, reference laboratories collected successive MSSA isolates from patients with invasive or superficial staphylococcal infections for use in the Tigecycline Evaluation and Surveillance Trial. All isolates from the periods of 2004 to 2005 and 2009 to 2010 underwent antimicrobial susceptibility testing and characterization of their staphylococcal protein A (spa) type. Of the 708 isolates analyzed, 274 spa types were identified and divided into 15 genetic clusters. The most common clones were spa t002 (n = 63, 8.9%) and t008 (n = 56, 7.9%). While the distribution of the predominant spa types did not differ by U.S. Census region or time period, spa t008 was nearly twice as common in community skin and soft tissue infections than in nosocomial bloodstream infections (11.1% versus 5.6%, respectively; P = 0.008). Despite such differences, both community and nosocomial settings had diverse staphylococcal clonal types representing all major spa clusters. In contrast to those of MRSA, MSSA infectious isolates show wide genetic diversity without clear geographical or temporal clustering. Notably, the prevalent MSSA strains (spa t002 and spa t008) are analogous to the predominant MRSA clones, further demonstrating the importance of both lineages.

Adenovirus infection in paediatric allogeneic stem cell transplantation recipients is a major independent factor for significantly increasing the risk of treatment related mortality.

Adenovirus infection is a significant complication in paediatric AlloSCT recipients with a mortality rate for disseminated adenovirus that may exceed 80%. We sought to determine the incidence, risk factors, and associated outcomes of adenovirus infection in 123 consecutive paediatric AlloSCT recipients. Adenovirus was diagnosed by antigen detection or viral culture, and was defined by isolation of virus with presence of correlating clinical symptoms. The probability of developing adenovirus infection was 12.3% (CI(95) 6.0-18.6). There were no statistically significant differences in probability of adenovirus infection in univariate analysis of risk factors including donor source, use of ATG/Alemtuzumab, ≥ Grade II GVHD, among others (age, diagnosis, conditioning regimen). Probability of overall survival for patients that experienced adenovirus infection was 15.4% vs. 50% for those without adenovirus (P = 0.0286). In multivariate analysis, the most important risk factor for an increased risk of death was adenovirus infection [HR 3.15 (CI(95) 1.6-6.18) P = 0.0009]. In this series of paediatric AlloSCT recipients, the development of adenovirus infection was the leading multivariate predictor of treatment-related mortality. Enhanced surveillance with adenovirus PCR and identification of the risk factors associated with poor outcomes from adenovirus infection may identify patients that may benefit from pre-emptive therapeutic interventions including adenovirus-specific cellular immunotherapies.

Multicenter evaluation of the Staphylococcus QuickFISH method for simultaneous identification of Staphylococcus aureus and coagulase-negative staphylococci directly from blood culture bottles in less than 30 minutes.

A novel rapid peptide nucleic acid fluorescence in situ hybridization (FISH) method, Staphylococcus QuickFISH, for the direct detection of Staphylococcus species from positive blood culture bottles was evaluated in a multicenter clinical study. The method utilizes a microscope slide with predeposited positive- and negative-control organisms and a self-reporting 15-min hybridization step, which eliminates the need for a wash step. Five clinical laboratories tested 722 positive blood culture bottles containing gram-positive cocci in clusters. The sensitivities for detection of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were 99.5% (217/218) and 98.8% (487/493), respectively, and the combined specificity of the assay was 89.5% (17/19). The combined positive and negative predictive values of the assay were 99.7% (696/698) and 70.8% (17/24), respectively. Studies were also performed on spiked cultures to establish the specificity and performance sensitivity of the method. Staphylococcus QuickFISH has a turnaround time (TAT) of <30 min and a hands-on time (HOT) of <5 min. The ease and speed of the method have the potential to improve the accuracy of therapeutic intervention by providing S. aureus/CoNS identification simultaneously with Gram stain results.

Transfusion of red blood cells after prolonged storage produces harmful effects that are mediated by iron and inflammation.

Although red blood cell (RBC) transfusions can be lifesaving, they are not without risk. In critically ill patients, RBC transfusions are associated with increased morbidity and mortality, which may increase with prolonged RBC storage before transfusion. The mechanisms responsible remain unknown. We hypothesized that acute clearance of a subset of damaged, stored RBCs delivers large amounts of iron to the monocyte/macrophage system, inducing inflammation. To test this in a well-controlled setting, we used a murine RBC storage and transfusion model to show that the transfusion of stored RBCs, or washed stored RBCs, increases plasma nontransferrin bound iron (NTBI), produces acute tissue iron deposition, and initiates inflammation. In contrast, the transfusion of fresh RBCs, or the infusion of stored RBC-derived supernatant, ghosts, or stroma-free lysate, does not produce these effects. Furthermore, the insult induced by transfusion of stored RBC synergizes with subclinical endotoxinemia producing clinically overt signs and symptoms. The increased plasma NTBI also enhances bacterial growth in vitro. Taken together, these results suggest that, in a mouse model, the cellular component of leukoreduced, stored RBC units contributes to the harmful effects of RBC transfusion that occur after prolonged storage. Nonetheless, these findings must be confirmed by prospective human studies.

Comparing the clinical severity of the first versus second wave of 2009 Influenza A (H1N1) in a New York City pediatric healthcare facility.

OBJECTIVE: We previously reported the epidemiology of 2009 Influenza A (H1N1) in our pediatric healthcare facility in New York City during the first wave of illness (May-July 2009). We hypothesized that compared with the first wave, the second wave would be characterized by increased severity of illness and mortality. DESIGN: : Case series conducted from May 2009 to April 2010. SETTING: Pediatric emergency departments and inpatient facilities of New York-Presbyterian Hospital. PATIENTS: All hospitalized patients ÷ 18 yrs of age with positive laboratory tests for influenza A. MEASUREMENTS AND MAIN RESULTS: We compared severity of illness during the first and second wave assessed by the number of hospitalized children, including those in the pediatric intensive care unit, bacterial superinfections, and mortality rate. Compared to the first wave, fewer children were hospitalized during the second wave (n = 115 vs. 76), but a comparable portion were admitted to the pediatric intensive care unit (30.4% vs. 19.7%; p = .10). Pediatric Risk of Mortality III scores, length of hospitalization in the pediatric intensive care unit, incidence of respiratory failure and pneumonia, and peak oxygenation indices were similar during both waves. Bacterial superinfections were comparable in the first vs. second wave (3.5% vs. 1.3%). During the first wave, no child received extracorporeal membrane oxygenation and one died, while during the second wave, one child received extracorporeal membrane oxygenation and there were no deaths. CONCLUSIONS: At our pediatric healthcare facility in New York City, fewer children were hospitalized with 2009 Influenza A (H1N1) during the second wave, but both waves had a similar spectrum of illness severity and low mortality rate.

A comparison of immune reconstitution and graft-versus-host disease following myeloablative conditioning versus reduced toxicity conditioning and umbilical cord blood transplantation in paediatric recipients.

Immune reconstitution appears to be delayed following myeloablative conditioning (MAC) and umbilical cord blood transplantation (UCBT) in paediatric recipients. Although reduced toxicity conditioning (RTC) versus MAC prior to allogeneic stem cell transplantation is associated with decreased transplant-related mortality, the effects of RTC versus MAC prior to UCBT on immune reconstitution and risk of graft-versus-host disease (GVHD) are unknown. In 88 consecutive paediatric recipients of UCBT, we assessed immune cell recovery and immunoglobulin reconstitution at days +100, 180 and 365 and analysed risk factors associated with acute and chronic GVHD. Immune cell subset recovery, immunoglobulin reconstitution, and the incidence of opportunistic infections did not differ significantly between MAC versus RTC groups. In a Cox model, MAC versus RTC recipients had significantly higher risk of grade II-IV acute GVHD [Hazard Ratio (HR) 6·1, P = 0·002] as did recipients of 4/6 vs. 5-6/6 HLA-matched UCBT (HR 3·1, P = 0·03), who also had significantly increased risk of chronic GVHD (HR 18·5, P = 0·04). In multivariate analyses, MAC versus RTC was furthermore associated with significantly increased transplant-related (Odds Ratio 26·8, P = 0·008) and overall mortality (HR = 4·1, P = 0·0001). The use of adoptive cellular immunotherapy to accelerate immune reconstitution and prevent and treat opportunistic infections and malignant relapse following UCBT warrants further investigation.

Comparison of polymyxin B, tigecycline, cefepime, and meropenem MICs for KPC-producing Klebsiella pneumoniae by broth microdilution, Vitek 2, and Etest.

We report MIC agreement and error rates between broth microdilution (BMD), Vitek 2, and Etest against 48 clinical KPC-producing Klebsiella pneumoniae isolates for polymyxin B, tigecycline, cefepime, and meropenem. Both commercial testing methods were useful for tigecycline testing; Etest provided a conservative estimate of polymyxin B susceptibility. We suggest that laboratories consider the supplemental use of reference BMD or Etest for cefepime and meropenem for susceptibility testing of KPC-producing K. pneumoniae, as Vitek 2 did not provide reliable results.

Identification of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in blood cultures: a multicenter performance evaluation of a three-color peptide nucleic acid fluorescence in situ hybridization assay.

A multicenter evaluation was undertaken to evaluate the performance of a new three-color peptide nucleic acid fluorescence in situ hybridization assay that identifies isolates directly from blood cultures positive for Gram-negative bacilli (GNB). The assay correctly identified 100% (186/186) of the Escherichia coli isolates, 99.1% (109/110) of the Klebsiella pneumoniae isolates, and 95.8% (46/48) of the Pseudomonas aeruginosa isolates in this study. Negative assay results were correctly obtained for 162 of 165 other GNB (specificity, 98.2%).

Quality monitoring of microbial contamination of cryopreserved parathyroid tissue.

Cryopreservation of parathyroid tissue (PT) provides patients undergoing parathyroidectomy with an option for delayed autologous heterotopic parathyroid transplantation. A standard protocol for quality monitoring of PT has not been established. This article describes a method for detecting the presence of bacterial contamination in PT tissue intended for autologous transplantation. PT was received in the tissue bank, processed under aseptic conditions, and placed into cryopreservation medium. Sterility testing was performed at 2 time points prior to cryopreservation. From January 2005 to October 2008, 47 PT samples were cryopreserved. The following bacteria were isolated from 11 PT specimens: Staphylococcus epidermidis, Staphylococcus capitis subspecies ureolyticus, Staphylococcus lugdunensis, Bacillus pumilus, and corynebacteria (diphtheroids). 23% of PTs were contaminated at the time of collection, predominantly with indigenous bacteria. Quality monitoring using this protocol is a useful tool to identify tissues contaminated with bacteria.

Trends in methicillin-resistant Staphylococcus aureus anovaginal colonization in pregnant women in 2005 versus 2009.

In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus. MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) (P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCCmec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) (P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.

Multicenter evaluation of a new shortened peptide nucleic acid fluorescence in situ hybridization procedure for species identification of select Gram-negative bacilli from blood cultures.

A shortened protocol for two peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assays for the detection of Gram-negative bacilli from positive blood cultures was evaluated in a multicenter trial. There was 100% concordance between the two protocols for each assay (368 of 368 and 370 of 370 results) and 99.7% (367 of 368 and 369 of 370 results) agreement with routine laboratory techniques.

The impact of delaying the initiation of appropriate antifungal treatment for Candida bloodstream infection.

We performed a retrospective analysis of the time to initiation of appropriate antifungal therapy for candidemia and in-hospital mortality. The definition of appropriate antifungal therapy was based on in vitro susceptibility results, and in the case of fluconazole, pharmacodynamic parameters. Of 123 patients, the mortality rate in the <24 h, 24-48 h, and >48 h groups was 50%, 28%, and 32%, respectively. Patients who never received antifungal treatment had a 61% mortality rate (difference between groups, P =0.06). Multivariate analysis found APACHE II score (AOR = 1.09, 95% CI: 1.02-1.17 for each point increase) to be the only independent predictor of mortality. The time to initiation of appropriate antifungal therapy did not correlate with in-hospital mortality.

Comparison of the automated Phoenix with the Vitek 2 for the identification of Streptococcus pneumoniae.

Rapid and accurate identification of Streptococcus pneumoniae is a critical component in the optimal management of infected patients. The performance of the BD Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, Md.) was evaluated for identification of S. pneumoniae (n = 311) and was compared to the Vitek 2 (bioMérieux, Marcy l'Etoile, France). Strains with discordant identification between methods were resolved with 16S rRNA gene sequencing as the gold standard. The Phoenix and the Vitek 2 correctly identified 96.8% (n = 301) and 95.2% (n = 296) of S. pneumoniae strains, respectively. Overall, there was no statistically significant difference in the performance of the 2 automated systems for the identification of S. pneumoniae in this study. The Vitek 2 mean time-to-results for all streptococcal identification was 1.5 h faster than that for the Phoenix. We conclude that the automated Phoenix and the Vitek 2 systems are comparable in their ability to identify S. pneumoniae and are preferable to the use of routine biochemical assays, which have delayed time-to-results and are not dependably accurate.

Carbapenem resistance via the blaKPC-2 gene in Enterobacter cloacae blood culture isolate.

An Enterobacter cloacae blood culture isolate expressing carbapenem resistance via the Klebsiella pneumoniae carbapenemase KPC-2 gene is reported. To our knowledge, this is the first report of a nosocomial isolate with carbapenemase-mediated resistance causing infection in a patient from Tennessee.

Gemfibrozil inhibits Legionella pneumophila and Mycobacterium tuberculosis enoyl coenzyme A reductases and blocks intracellular growth of these bacteria in macrophages.

We report here that gemfibrozil (GFZ) inhibits axenic and intracellular growth of Legionella pneumophila and of 27 strains of wild-type and multidrug-resistant Mycobacterium tuberculosis in bacteriological medium and in human and mouse macrophages, respectively. At a concentration of 0.4 mM, GFZ completely inhibited L. pneumophila fatty acid synthesis, while at 0.12 mM it promoted cytoplasmic accumulation of polyhydroxybutyrate. To assess the mechanism(s) of these effects, we cloned an L. pneumophila FabI enoyl reductase homolog that complemented for growth an Escherichia coli strain carrying a temperature-sensitive enoyl reductase and rendered the complemented E. coli strain sensitive to GFZ at the nonpermissive temperature. GFZ noncompetitively inhibited this L. pneumophila FabI homolog, as well as M. tuberculosis InhA and E. coli FabI.

Genetic organization of transposase regions surrounding blaKPC carbapenemase genes on plasmids from Klebsiella strains isolated in a New York City hospital.

Carbapenem-resistant Klebsiella strains carrying Klebsiella pneumoniae carbapenemases (KPC) are endemic to New York City and are spreading across the United States and internationally. Recent studies have indicated that the KPC structural gene is located on a 10-kb plasmid-borne element designated Tn4401. Fourteen Klebsiella pneumoniae strains and one Klebsiella oxytoca strain isolated at a New York City hospital in 2005 carrying either bla(KPC-2) or bla(KPC-3) were examined for isoforms of Tn4401. Ten of the Klebsiella strains contained a 100-bp deletion in Tn4401, corresponding to the Tn4401a isoform. The presence of this deletion adjacent to the upstream promoter region of bla(KPC) in Tn4401a resulted in a different -35 promoter sequence of TGGAGA than that of CTGATT present in isoform Tn4401b. Complete sequencing of one plasmid carrying bla(KPC) from each of three nonclonal isolates indicated the presence of genes encoding other types of antibiotic resistance determinants. The 70.6-kb plasmid from K. pneumoniae strain S9 carrying bla(KPC-2) revealed two identical copies of Tn4401b inserted in an inverse fashion, but in this case, one of the elements disrupted a group II self-splicing intron. In K. pneumoniae strain S15, the Tn4401a element carrying bla(KPC-2) was found on both a large 120-kb plasmid and a smaller 24-kb plasmid. Pulsed-field gel electrophoresis results indicate that the isolates studied represent a heterogeneous group composed of unrelated as well as closely related Klebsiella strains. Our results suggest that endemic KPC-positive Klebsiella strains constitute a generally nonclonal population comprised of various alleles of bla(KPC) on several distinct plasmid genetic backgrounds. This study increases our understanding of the genetic composition of the evolving and expanding role of KPC-producing, healthcare-associated, gram-negative pathogens.

Comparison of BD phoenix to vitek 2, microscan MICroSTREP, and Etest for antimicrobial susceptibility testing of Streptococcus pneumoniae.

The performance of the BD Phoenix Automated Microbiology System (BD Diagnostic Systems) was compared to those of the Vitek 2 (bioMérieux), the MicroScan MICroSTREP plus (Siemens), and Etest (bioMérieux) for antibiotic susceptibility tests (AST) of 311 clinical isolates of Streptococcus pneumoniae. The overall essential agreement (EA) between each test system and the reference microdilution broth reference method for S. pneumoniae AST results was >95%. For Phoenix, the EAs of individual antimicrobial agents ranged from 90.4% (clindamycin) to 100% (vancomycin and gatifloxacin). The categorical agreements (CA) of Phoenix, Vitek 2, MicroScan, and Etest for penicillin were 95.5%, 94.2%, 98.7%, and 97.7%, respectively. The overall CA for Phoenix was 99.3% (1 very major error [VME] and 29 minor errors [mEs]), that for Vitek 2 was 98.8% (7 VMEs and 28 mEs), and those for MicroScan and Etest were 99.5% each (19 and 13 mEs, respectively). The average times to results for Phoenix, Vitek 2, and the manual methods were 12.1 h, 9.8 h, and 24 h, respectively. From these data, the Phoenix AST results demonstrated a high degree of agreement with all systems evaluated, although fewer VMEs were observed with the Phoenix than with the Vitek 2. Overall, both automated systems provided reliable AST results for the S. pneumoniae-antibiotic combinations in half the time required for the manual methods, rendering them more suitable for the demands of expedited reporting in the clinical setting.

Outbreak of Mycobacterium abscessus wound infections among "lipotourists" from the United States who underwent abdominoplasty in the Dominican Republic.

BACKGROUND: Some US residents travel abroad to undergo cosmetic surgery for fat removal, a practice referred to as "lipotourism." Mycobacterium abscessus can cause postsurgical wound infection. METHODS: US residents who developed M. abscessus wound infection after undergoing cosmetic surgery in the Dominican Republic in 2003 and 2004 were identified using the Emerging Infections Network listserv. RESULTS: Twenty returning US travelers with M. abscessus infection were detected. Eight patients had matching isolates, as determined by pulsed-field gel electrophoresis and repetitive element polymerase chain reaction. All 8 patients, who had previously been healthy Hispanic women, underwent abdominoplasties at the same clinic in the Dominican Republic. Symptoms first developed 2-18 weeks after the procedure (median interval, 7 weeks). Only 2 of the 8 patients received a correct diagnosis at the initial presentation. Most patients presented with painful, erythematous, draining subcutaneous abdominal nodules. Seven patients underwent drainage procedures. Six patients received a combination of antibiotics that included a macrolide plus cefoxitin, imipenem, amikacin, and/or linezolid; 2 received clarithromycin monotherapy. All patients but 1 were cured after a median of 9 months of therapy (range, 2-12 months). Because of a lack of access to the surgical clinic, the cause of the outbreak of infection was not identified. The patients who were infected with nonmatching isolates underwent surgeries in different facilities but otherwise had demographic characteristics and clinical presentations similar to those of the 8 patients infected with matching isolates. CONCLUSIONS: This case series of M. abscessus infection in US "lipotourists" highlights the risks of traveling abroad for surgery and the potential role of the Internet in identifying and investigating outbreaks.