Description

The Journal of Clinical Microbiology publishes the most current research on the microbiological aspects of human and animal infections and infestations, with emphasis on their etiologic agents, diagnosis, and epidemiology.

Publisher details

American Society for Microbiology

Publications in this journal

• Answer to june 2012 photo quiz.

  Authors: Burke A Cunha, Jean E Hage, George K Turi

  Journal of clinical microbiology. 50(6):2184.

• Photo quiz: a 45-year-old male with rash, Fever, and diarrhea.

  Authors: Burke A Cunha, Jean E Hage, George K Turi

  Journal of clinical microbiology. 50(6):1835.

• Corynebacterium tuberculostearicum: a potentially misidentified and multiresistant Corynebacterium species isolated from clinical specimens.

  Authors: V Hinic, C Lang, M Weisser, C Straub, R Frei, D Goldenberger

  Journal of clinical microbiology.

  The species Corynebacterium tuberculostearicum is a lipophilic Corynebacterium validly characterized in 2004. We provide clinical information on 18 patients from whom this organism was isolated. TheThe species Corynebacterium tuberculostearicum is a lipophilic Corynebacterium validly characterized in 2004. We provide clinical information on 18 patients from whom this organism was isolated. The majority of the patients were hospitalized and had a history of prolonged treatment with broad-spectrum antimicrobials. In 7 out of 18 (38.9%) cases the isolates were found to be clinically relevant. The present study also includes detailed data on biochemical and molecular identification of C. tuberculostearicum, as well as identification by MALDI-TOF mass spectrometry. Our data demonstrate that routine biochemical tests do not provide reliable identification of C. tuberculostearicum. MALDI-TOF mass spectrometry represents a helpful tool for identification of this species, since all strains matched with C. tuberculostearicum as first choice and 58.3% (7/12) of the strains processed with full extraction protocol generated scores of >2.000. Nevertheless, partial 16S rRNA gene sequencing still represents the gold standard for identification of this species. Due to the challenging identification of C. tuberculostearicum, we presume that this organism is often misidentified and its clinical relevance underestimated. The antimicrobial susceptibility profile of C. tuberculostearicum presented here reveals that 14 out of 16 (87.5%) analyzed strains exhibited multiresistance.

• Performances of the MALDI-TOF Mass Spectrometry system VITEK MS for the Rapid Identification of Bacteria in Routine Clinical Microbiology.

  Authors: Damien Dubois, Marion Grare, Marie-Françoise Prere, Christine Segonds, Nicole Marty, Eric Oswald

  Journal of clinical microbiology.

  Rapid and cost-effective MALDI-TOF MS-based systems will replace conventional phenotypic methods for routine identification of bacteria. We report here the first evaluation of the new MALDI-TOFRapid and cost-effective MALDI-TOF MS-based systems will replace conventional phenotypic methods for routine identification of bacteria. We report here the first evaluation of the new MALDI-TOF MS-based VITEK MS system in a large clinical microbiology laboratory. This system used an original spectra classifier algorithm and a specific database designed for the identification of clinically relevant species.We have tested 767 routine clinical isolates representative of 50 genera and 124 species. VITEK MS-based identifications were performed by means of a single deposit on MALDI disposable target, without any prior extraction step, and compared with reference identifications, mainly obtained with the VITEK2 phenotypic system; if discordant, molecular techniques provided reference identifications.The VITEK MS system provided 96.2% of correct identifications: to the species level (86.7%), to the genus level (8.2%), or within a range of species belonging to different genera (1.3%). Conversely, 1.3% of isolates were misidentified and 2.5% unidentified, partly because the species was not included in the database; a second deposit provided a successful identification for 0.8% of isolates unidentified with the first deposit.The VITEK MS system is a simple, convenient and accurate method for routine bacterial identification with a single deposit, considering the high bacterial diversity studied as evidence by the low prevalence of species without correct identification. In addition to a second deposit in uncommon cases, expanding the spectral database is expected to further enhance performances.

• Prognostic value of indeterminate interferon-γ release assay results in HIV-1-infection.

  Authors: Maximilian C Aichelburg, Julia Tittes, Florian Breitenecker, Thomas Reiberger, Norbert Kohrgruber, Armin Rieger

  Journal of clinical microbiology.

  In this prospective, longitudinal study on 948 HIV-1-infected patients, subjects with an indeterminate Interferon-gamma release assay (IGRA) result at baseline were at significantly higher risk ofIn this prospective, longitudinal study on 948 HIV-1-infected patients, subjects with an indeterminate Interferon-gamma release assay (IGRA) result at baseline were at significantly higher risk of developing AIDS-defining manifestations other than TB irrespective of CD4+ T cell count. Thus, in HIV-1-infected patients with advanced quantitative CD4(+) T cell depletion, an indeterminate IGRA might indicate an additional loss of global T cell function warranting detailed clinical evaluation and careful follow-up.

• A Novel Extraction Method Combining Plasma With Whole Blood Fraction Shows Excellent Sensitivity and Reproducibility in Patients at High Risk for Invasive Aspergillosis.

  Authors: Jan Springer, Hannes Schloßnagel, Werner Heinz, Thomas Doedt, Rainer Soeller, Hermann Einsele, Juergen Loeffler

  Journal of clinical microbiology.

  Diagnosis of invasive aspergillosis (IA) is still a major problem in routine clinical practice. Early diagnosis is essential for a good patient prognosis. PCR is a highly sensitive method for theDiagnosis of invasive aspergillosis (IA) is still a major problem in routine clinical practice. Early diagnosis is essential for a good patient prognosis. PCR is a highly sensitive method for the detection of nucleic acids and could play an important role in improving the diagnosis of fungal infections. Therefore, a novel DNA extraction method (UCP) was developed allowing purification of both cellular and cell-free circulating fungal DNA. In this prospective study we evaluated the, commercially available, UCP extraction system and compared it to an in-house system. Sixty-three patients at high risk for IA were screened twice weekly and DNA extracted by both methods was cross-analyzed, in triplicate, by two different real-time PCR assays. The negative predictive values were high for all methods (94.3 to 100%) qualifying them as screening methods, but the sensitivity and diagnostic odds ratio was highest using the UCP extraction method. Sensitivity ranged from 33.3 to 66.7% using the in-house extracts to 100% using the UCP extraction method. Most of the unclassified patients showed no positive PCR results, however single-positive PCR replicates were observed in some cases. These can bear clinical relevance, but should be interpreted with additional clinical and laboratory data. The PCR assays from the UCP extracts showed greater reproducibility than the in-house method for probable IA patients.The standardized UCP extraction method yielded superior results, with regard to sensitivity and reproducibility, than the in-house method. This was independent of the PCR assay used to detect fungal DNA in the sample extracts.

• Population snapshot of invasive serogroup B meningococci in South Africa, 2005-2008.

  Authors: Mignon du Plessis, Chivonne Moodley, Kedibone M Mothibeli, Azola Fali, Keith P Klugman, Anne von Gottberg

  Journal of clinical microbiology.

  In South Africa, serogroup B meningococcal disease is sporadic. The aim of this study was to characterize serogroup B strains causing invasive meningococcal disease (IMD) in South Africa from 2005 toIn South Africa, serogroup B meningococcal disease is sporadic. The aim of this study was to characterize serogroup B strains causing invasive meningococcal disease (IMD) in South Africa from 2005 to 2008. Isolates, collected through a national, laboratory-based surveillance program for IMD, were characterized by multilocus sequence typing (MLST). 2235 cases were reported of which 1447 had viable isolates. Intermediate resistance to penicillin was observed in 2.8% (41/1447) of all strains. Serogroup B was the second most common serogroup (17%, 251/1447) and increased from 14% (58/414) in 2005 to 25% (72/290) in 2008 (p<0.001), however, incidence remained stable during the study period (average incidence 0.13/100,000 population) (p=0.54). Serogroup B was predominantly characterized by three clonal complexes, namely ST-41/44/lineage 3, ST-32/ET-5 and new ST-4240/6688 which accounted for 27% (65/245), 22% (55/245), and 16% (38/245) of isolates, respectively. ST-4240/6688 was more prevalent among young children (<5 years) than older children and adults (≥5 years) [28/38 (74%) vs. 109/198 (55%); p=0.03). In the most densely populated province of South Africa, Gauteng, ST-32/ET-5 increased from 8% (2/24) in 2005 to 38% (9/24) in 2008 (p=0.04). Capsular switching was observed in 8/251 (3%) strains. Newly assigned clonal complex ST-4240/6688 was more common in young children.

• The relationship between hVISA, VISA, high vancomycin MIC and outcome in serious Staphylococcus aureus infection.

  Authors: Natasha E Holmes, Paul D R Johnson, Benjamin P Howden

  Journal of clinical microbiology.

  Vancomycin has been used successfully for over 50 years for the treatment of Staphylococcus aureus infections, particularly methicillin-resistant S. aureus. It has proved remarkably resilient but itsVancomycin has been used successfully for over 50 years for the treatment of Staphylococcus aureus infections, particularly methicillin-resistant S. aureus. It has proved remarkably resilient but its efficacy is now being questioned with the emergence of strains of S. aureus that display heteroresistance, intermediate resistance and occasionally complete vancomycin resistance. More recently an association has been established between poor outcome and infections with strains of S. aureus with elevated vancomycin minimum inhibitory concentration within the susceptible range. This mini-review summarizes the definitions, mechanisms, clinical impact and laboratory identification of reduced vancomycin susceptibility in S. aureus, and discusses practical issues for the diagnostic laboratory in testing and interpreting vancomycin susceptibility for S. aureus infections.

• Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from 7 hospitals in Japan.

  Authors: Emiko Rimbara, Shigetarou Mori, Mari Matsui, Satowa Suzuki, Jun-Ichi Wachino, Yoshiaki Kawamura, Zeli Shen, James G Fox, Keigo Shibayama

  Journal of clinical microbiology.

  Helicobacter cinaedi colonizes the colons of human and animals and can cause colitis, cellulitis, and sepsis in humans, with infections in immunocompromised patients being increasingly recognized.Helicobacter cinaedi colonizes the colons of human and animals and can cause colitis, cellulitis, and sepsis in humans, with infections in immunocompromised patients being increasingly recognized. However, methods for analyzing the molecular epidemiology of H. cinaedi are not yet established. A genotyping method was developed using multilocus sequence typing (MLST) and used to analyze 50 H. cinaedi isolates from Japanese hospitals in addition to 6 reference strains. Pulse-field gel electrophoresis (PFGE) results were also compared with the MLST results. Based on the genomic information from the CCUG18818 strain, 21 housekeeping genes were selected as candidates for MLST and were observed to have high homology (96.5-100%) between isolates. Following a comparison of the 21 housekeeping genes from 8 H. cinaedi isolates, 7 genes were chosen for MLST, revealing 14 sequence types (STs). The isolates from 3 hospitals had the same STs, but the isolates from the other 4 hospitals belonged to different STs. Isolates belonging to ST6 were analyzed by PFGE and showed similar patterns, but the patterns were different between isolates. Isolates belonging to ST9, ST10, and ST11, which belonged to the same clonal complex, showed the same pattern between isolates. All isolates were found to contain mutations in GyrA and the 23S rRNA gene that confer ciprofloxacin and clarithromycin resistance, respectively, in H. cinaedi. These results raise concerns about the increase in H. cinaedi isolates resistant to clarithromycin and ciprofloxacin in Japan.

• Identification of DNA Signatures Suitable for Developing into Real-Time PCR assays by Whole Genome Sequence Approaches: Using Streptococcus pyogenes as a pilot study.

  Authors: Guo-Chiuan Hung, Kenjiro Nagamine, Bingjie Li, Shyh-Ching Lo

  Journal of clinical microbiology.

  A stepwise computational approach using three layers of publicly available software was described to effectively identify DNA signatures for Streptococcus pyogenes. PCR testing validated 9 out of 15A stepwise computational approach using three layers of publicly available software was described to effectively identify DNA signatures for Streptococcus pyogenes. PCR testing validated 9 out of 15 signature-derived primer sets could detect as low as 5 fg of target DNA with high specificity. The selected signature-derived primer sets were successfully evaluated against all 23 clinical isolates. The approach is readily applicable for designing molecular assays for rapid detection and characterization of various pathogenic bacteria.

• Haemophilus haemolyticus causing clinical disease.

  Authors: Raydel Anderson, Xin Wang, Elizabeth C Briere, Lee S Katz, Amanda C Cohn, Thomas A Clark, Nancy E Messonnier, Leonard W Mayer

  Journal of clinical microbiology.

  We report seven cases of Haemophilus haemolyticus invasive disease detected in the United States, which were previously misidentified as non-typeable Haemophilus influenzae (Hi). All cases hadWe report seven cases of Haemophilus haemolyticus invasive disease detected in the United States, which were previously misidentified as non-typeable Haemophilus influenzae (Hi). All cases had different symptoms and presentations. Our study suggests that a testing scheme that includes reliable PCR assays and standard microbiological methods should be used in order to improve H. haemolyticus identification.

• Resistance to voriconazole due to a G448S substitution in Aspergillus fumigatus in a patient with cerebral aspergillosis.

  Authors: Teresa Pelaez, Paloma Gijón, Eleonora Bunsow, Emilio Bouza, Waldo Sánchez-Yebra, Maricela Valerio, Beatriz Gama, Manuel Cuenca-Estrella, Emilia Mellado

  Journal of clinical microbiology.

  A voriconazole-resistant isolate of Aspergillus fumigatus was recovered from an immunocompetent patient receiving long-term antifungal therapy for cerebral aspergillosis. A G448S amino acidA voriconazole-resistant isolate of Aspergillus fumigatus was recovered from an immunocompetent patient receiving long-term antifungal therapy for cerebral aspergillosis. A G448S amino acid substitution in the azole target (Cyp51A) was identified as the cause accounting for the resistant phenotype. This article describes the first isolation of a voriconazole-resistant A. fumigatus isolate from an immunocompetent patient in Spain.

• Microfluidic chip-based Multiple-Locus Variable-Number Tandem Repeat Fingerprinting (MLVF) with new primer sets for Methicillin-Resistant Staphylococcus aureus.

  Authors: Artur J Sabat, Monika A Chlebowicz, Hajo Grundmann, Jan P Arends, Greetje Kampinga, Nico E L Meessen, Alexander W Friedrich, Jan Maarten van Dijl

  Journal of clinical microbiology.

  The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settingsThe detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study we investigated the application potential of a new approach based on Multiple-Locus Variable number tandem repeat Fingerprinting (MLVF) and microfluidic technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 non-repetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF with newly designed primers using microcapillary electrophoresis were compared to those obtained by spa typing and Multiple-Locus Variable number tandem repeat Analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles) and 0.959 (66 types) for MLVF, MLVA and spa typing, respectively. All methods tested showed a good concordance of results calculated by adjusted Rand's coefficient. Comparison of data obtained by the three approaches allowed us to propose an 88% cut-off value of the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible and highly discriminating tool to determine the clonality of MRSA isolates, and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added-value because it is more discriminatory.

• Optimising out-patient serial sputum colony counting for studies of tuberculosis treatment in resource-poor settings.

  Authors: Derek J Sloan, Liz Corbett, Anthony Butterworth, Henry Mwandumba, Saye Khoo, Aaron Mdolo, Doris Shani, Mercy Kamdolozi, Jenny Allen, Denis Mitchison, David Coleman, Gerry Davies

  Journal of clinical microbiology.

  Serial Sputum Colony Counting (SSCC) is an important technique in clinical trials of new treatments for tuberculosis (TB). Quantitative cultures on selective Middlebrook agar are used to calculateSerial Sputum Colony Counting (SSCC) is an important technique in clinical trials of new treatments for tuberculosis (TB). Quantitative cultures on selective Middlebrook agar are used to calculate the rate of bacillary elimination from sputum collected from patients at different time points during the first two months of therapy. However, the procedure can be complicated by high sample contamination rates. This study, conducted in a resource poor setting in Malawi, assessed the ability of different anti-fungal drugs in selective agar to reduce contamination. Overall, 229 samples were studied and 15-27% were contaminated. Fungal organisms were particularly implicated and samples collected later in treatment were at particular risk (p<0.001). Amphotericin B (AmB) is the standard anti-fungal drug used on SSCC plates at a concentration of 10mg/ml. On selective Middlebrook 7H10 plates AmB (30mg/ml) reduced sample contamination by 17% compared with AmB (10mg/ml). The relative risk of contamination using AmB (10mg/ml) was 1.79 (95% CI: 1.25-3.55). On Middlebrook 7H11 plates, a combination of AmB (10mg/ml) and carbendazim (50mg/ml) was associated with 10% less contamination than AmB (30mg/ml). The relative risk of contamination with AmB (30mg/ml) was 1.79 (95% CI: 1.01-3.17). Improved anti-fungal activity was accompanied by a small reduction in bacillary counts but this did not affect modelling of bacillary elimination. In conclusion, a combination of AmB and carbendazim optimised the anti-fungal activity of selective media for growth of TB. We recommend this method to reduce contamination rates and improve SSCC studies in African countries where the burden of TB is highest.

• A matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method for discrimination between molecular types of Cryptococcus neoformans and Cryptococcus gattii.

  Authors: Brunella Posteraro, Antonietta Vella, Massimo Cogliati, Elena De Carolis, Ada Rita Florio, Patrizia Posteraro, Maurizio Sanguinetti, Anna Maria Tortorano

  Journal of clinical microbiology.

  We evaluated the usefulness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels, byWe evaluated the usefulness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels, by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcus isolates (72 C. neoformans and 10 C. gattii) tested were correctly identified in respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.

• Enzootic enteropathogenic Escherichia coli infection in laboratory rabbits.

  Authors: Alton G Swennes, Ellen M Buckley, Nicola M A Parry, Carolyn M Madden, Alexis García, Peter B Morgan, Keith M Astrofsky, James G Fox

  Journal of clinical microbiology.

  Enteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, andEnteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, and compelling evidence for cross-species EPEC transmission exists. In this report, enzootic EPEC infection associated with up to 10.5% diarrhea-associated morbidity in a large laboratory Dutch Belted rabbit colony was investigated. These rabbits were obtained from a commercial vendor and had acute diarrhea following shipment. Fecal culture of 20 rabbits yielded 48 E. coli isolates, 83% of which were eae-positive. REP-PCR and serologic analysis identified a single disease-associated EPEC O145:H2 strain. In sampled rabbits, EPEC-positive culture and the presence of diarrhea were significantly associated. This strain displayed a localized adherence-like HEp-2 cell adherence pattern as seen in diarrheic human infant EPEC isolates. Treatment was instituted with the fluoroquinolone antibiotic enrofloxacin, to which all isolates were susceptible. Pre-shipment parenteral enrofloxacin administration reduced diarrhea-associated morbidity 22-fold and mortality 12-fold in subsequent deliveries. This report emphasizes the zoonotic potential of animal EPEC strains and the need for virulence determinant-based screening of E. coli isolates from diarrheic animals.

• Detection of a knockdown resistance (kdr) mutation associated with permethrin resistance in the body louse Pediculus humanus corporis using melting curve analysis genotyping.

  Authors: Rezak Drali, Samir Benkouiten, Sékéné Badiaga, Idir Bitam, Jean Marc Rolain, Philippe Brouqui

  Journal of clinical microbiology.

  Louse-borne diseases are prevalent in the homeless and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in largeLouse-borne diseases are prevalent in the homeless and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in large field-based clinical studies to monitor permethrin resistance in the human body louse Pediculus humanus corporis. We assessed a melting curve analysis genotyping method based on real-time PCR using hybridization probes to detect the M815I-T917I-L920F kdr mutation in the para-orthologous voltage-sensitive sodium channel (VSSC) α-subunit gene which is associated with permethrin resistance.The 908 bp DNA fragment of the VSSC gene, encoding the α-subunit of the sodium channel and encompassing the three mutation sites, was PCR-sequenced from 65 lice collected from a homeless population. We noted a high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F mutations and 56.73% for the T917I mutation). These results were confirmed by melting curve analysis genotyping which had a calculated sensitivity of 100% for the M815I and T917I mutations and of 98% for the L920F mutation. The specificity was 100% for M815I and L920F and 96% for T917I. Melting curve analysis genotyping is a fast, sensitive and specific tool that is fully compatible with the analysis of a large number of samples in epidemiological surveys, allowing the simultaneous genotyping of 96 samples in just over an hour (75 minutes). Thereby, it is perfectly suited for the epidemiological monitoring of permethrin resistance in human body lice in large-scale clinical studies.

• First Report of Spontaneous Intrapartum Atopobium vaginae Bacteremia.

  Authors: Jasper F W Chan, Susanna K P Lau, Shirly O T Curreem, Kelvin K W To, Sally S M Leung, Vincent C C Cheng, Kwok-Yung Yuen, Patrick C Y Woo

  Journal of clinical microbiology.

  We report the first case of spontaneous intrapartum Atopobium vaginae bacteremia identified by 16S rRNA gene sequencing. The bacterium was misidentified by RapID ANA II, API RAPID 32A, andWe report the first case of spontaneous intrapartum Atopobium vaginae bacteremia identified by 16S rRNA gene sequencing. The bacterium was misidentified by RapID ANA II, API RAPID 32A, and MALDI-TOF-MS. The likely source of bacteremia was the female genital tract. In invasive infections caused by A. vaginae, β-lactams and clindamycin are the antibiotics of choice as most strains are resistant to metronidazole.

• Promise versus reality: optimism bias in package inserts of tuberculosis diagnostics.

  Authors: Claudia M Denkinger, Jasmine Grenier, Jessica Minion, Madhukar Pai

  Journal of clinical microbiology.

  Laboratorians and clinicians often rely on package inserts of diagnostic tests to assess their accuracy. We compared test accuracy for tuberculosis diagnostics reported in 19 package inserts againstLaboratorians and clinicians often rely on package inserts of diagnostic tests to assess their accuracy. We compared test accuracy for tuberculosis diagnostics reported in 19 package inserts against estimates in published meta-analyses, and found that package inserts generally report over-optimistic accuracy estimates. However, package inserts of most tests approved by the FDA or endorsed by the WHO provide more realistic estimates that agree with meta-analyses.

• Identification of Bacterial Pathogens in Ascitic Fluids from Patients with Suspected Spontaneous Bacterial Peritonitis by Use of Broad-Based PCR (16S PCR) Coupled with High-Resolution Melt Analysis (HRMA).

  Authors: Justin Hardick, Helen Won, Kevin Jeng, Yu-Hsiang Hsieh, Charlotte A Gaydos, Richard E Rothman, Samuel Yang

  Journal of clinical microbiology.

  Spontaneous bacterial peritonitis (SBP) can be a severe complication occurring in patients with cirrhosis and ascites, with associated mortality often as high as 40%. Traditional diagnostics for SBPSpontaneous bacterial peritonitis (SBP) can be a severe complication occurring in patients with cirrhosis and ascites, with associated mortality often as high as 40%. Traditional diagnostics for SBP rely on culture techniques for proper diagnosis, although recent reports suggest that the presence of bacterial DNA in peritoneal fluid in patients with cirrhosis and ascites is an indicator of SBP.A previously published broad-based PCR (16S PCR) coupled with high resolution melt analysis (HRMA) was compared with standard culture techniques for diagnosis of SBP in 106 peritoneal fluid samples from patients with suspected SBP. Sensitivity and specificity for 16S PCR for detecting eubacterial DNA when compared with standard culture techniques were 100% (17/17) and 91.5% (85/89), respectively. Overall, HRMA concordance with species identification was 70.6% (12/17), although the 5 discordant samples at the species level were SBP resulting from a polymicrobial infection, and species level identification for polymicrobial infections is outside the capability of HRMA.Both the broad-based 16S PCR and HRMA analysis provide useful diagnostic adjunctive assays for clinicians in detecting and identifying pathogens responsible for SBP.

• Case-report: Molecular diagnosis of Plasmodium knowlesi in a Dutch traveler by real-time PCR.

  Authors: Lonneke Link, Aldert Bart, Nienke Verhaar, Tom van Gool, Marjolijn Pronk, Volkher Scharnhorst

  Journal of clinical microbiology.

  P. knowlesi infection with low parasitemia forms a diagnostic challenge as rapid diagnostic tests often are negative, and species determination by microscopy is difficult. P. knowlesi malaria in aP. knowlesi infection with low parasitemia forms a diagnostic challenge as rapid diagnostic tests often are negative, and species determination by microscopy is difficult. P. knowlesi malaria in a traveler is described and real-time PCR is demonstrated to support fast and reliable diagnosis and species determination.

• Multilocus sequence typing scheme for Staphylococcus aureus: revision of the gmk locus.

  Authors: Jesper Larsen, Mark C Enright, Daniel Godoy, Brian G Spratt, Anders R Larsen, Robert L Skov

  Journal of clinical microbiology.

  Multilocus sequence 18 typing (MLST) is a sequence-based genotyping method based on polymorphisms (each variant is termed an allele) in seven housekeeping genes (loci) (arcC, aroE, glpF, gmk, pta,Multilocus sequence 18 typing (MLST) is a sequence-based genotyping method based on polymorphisms (each variant is termed an allele) in seven housekeeping genes (loci) (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) in Staphylococcus aureus, providing unique allelic profiles known as sequence types (STs) (1).

• Inter-laboratory reproducibility of Etest amphotericin-B and caspofungin yeast susceptibility testing and comparison with the CLSI method.

  Authors: S Ranque, L Lachaud, M Gari-Toussaint, A Michel-Nguyen, M Mallié, J Gaudart, S Bertout

  Journal of clinical microbiology.

  This study aimed to assess inter-laboratory reproducibility in four university hospital laboratories of the southeast region of France of the Etest technique for caspofungin (CAS) and amphotericin-BThis study aimed to assess inter-laboratory reproducibility in four university hospital laboratories of the southeast region of France of the Etest technique for caspofungin (CAS) and amphotericin-B (AMB) MICs determination and to compare it to the CLSI broth microdilution reference method.Consecutive clinical yeasts isolates (n=198) were included in the study. AMB and CAS MICs were read at 24 and 48h. Inter-laboratory reproducibility of was estimated using: i) intra-class correlation coefficient (ICC); ii) essential agreement (EA); and iii) categorical agreement (CA).Etest inter-laboratory reproducibility. For CAS, ICC were 0.80 [0.76-0.84] and 0.81 [0.77-0.85] at 24 and 48h, respectively. For AMB, the ICC were 0.51 [0.43-0.58] and 0.69 [0.63-0.74] at 24 and 48h, respectively. At 48, the between-centers EA ranged from 94.4 to 99.0% for both antifungals.CLSI and Etest Comparison. The between-technique ICCs were 0.69 [0.63-0.74] and 0.62 [0.55-0.68] for CAS and AMB, respectively. EA ranged from 76.5 to 98.5% for CAS and from 90.3 to 97.4% for AMB, according to the centers. CAs ranged from 87.9% to 91.4%, with four very major errors for 2 strains (1 C. albicans and 1 C. krusei) for CAS; and from 97.5 to 99.5%, with four major errors, for AMB.In conclusion, Etest showed a good inter-laboratory reproducibility and correlation with the CLSI technique. It is well suited to the routine clinical laboratory and can thus be used to monitor clinical yeast isolates' in vitro susceptibly in this setting.

• Early Serum Galactomannan Trend as a Predictor of Outcome in Invasive Aspergillosis.

  Authors: Louis Y A Chai, Bart-Jan Kullberg, Elizabeth M Johnson, Steven Teerenstra, Lay Wai Khin, Alieke G Vonk, Johan Maertens, Olivier Lortholary, Peter J Donnelly, Haran T Schlamm, Peter F Troke, Mihai G Netea, Raoul Herbrecht

  Journal of clinical microbiology.

  Monitoring and prediction of treatment response in invasive aspergillosis (IA) is difficult. We determined whether serum galactomannan index (GMI) trends early in the course of disease may be usefulMonitoring and prediction of treatment response in invasive aspergillosis (IA) is difficult. We determined whether serum galactomannan index (GMI) trends early in the course of disease may be useful in predicting eventual clinical outcome. From the subjects recruited into the multicentre Global Aspergillosis Study, serial GMI were measured at baseline, and at Weeks 1, 2 and 4 following anti-fungal treatment. Clinical response and survival at 12 weeks were the outcome measures. GMI trends were analyzed using generalized estimating equations approach. GMI cut-offs were evaluated using receiver operating curve analysis incorporating pre- and post-test probabilities. From the 202 study patients diagnosed with IA, 71 (35.1%) had baseline GMI ≥0.5. Week 1 GMI was significantly lower in eventual responders to treatment at Week 12 as compared to the non-responders (GMI 0.62±0.12 versus 1.15±0.22 respectively, p=0.035). A GMI reduction >35% between baseline and Week 1 predicted probability of satisfactory clinical response. In IA patients with pre-treatment GMI <0.5 (n=131, 64.9%), GMI ought to remain low during treatment and a rising absolute GMI to >0.5 at Week 2 despite anti-fungals heralded poor clinical outcome. Here, every 0.1 unit increase in GMI between baseline and Week 2 increased the likelihood of unsatisfactory clinical response by 21.6% (p=0.018). In summary, clinical outcomes may be anticipated by charting early GMI trends during the first two weeks of anti-fungal therapy. These findings have significant implications for the management of IA.

• Can a simple flotation method lower the limit of detection of M. tuberculosis in extrapulmonary samples by the GeneXpert MTB/RIF assay?

  Authors: Nathan Taylor, Rajiv L Gaur, Ellen J Baron, Niaz Banaei

  Journal of clinical microbiology.

  Background. The rapid and accurate diagnosis of tuberculosis (TB) in children and extrapulmonary TB in adults continues to be a challenge. In this study we determined the lower limit of detectionBackground. The rapid and accurate diagnosis of tuberculosis (TB) in children and extrapulmonary TB in adults continues to be a challenge. In this study we determined the lower limit of detection (LOD) of the GeneXpert MTB/RIF assay with non-respiratory specimens and investigated the utility of flotation procedures for concentrating the bacilli.Methods. Clinical specimens (9 CSF, 13 gastric aspirate, 8 tissue, and 17 stool) were spiked with single-celled M. tuberculosis and the LOD of the GeneXpert was determined. Flotation studies were conducted with sucrose and NaCl and the cycle thresholds of the MTB/RIF assay were compared between treated and untreated samples.Results. There was no significant difference between the LOD of the GeneXpert with saline (median 33 CFU/ml) and CSF (median 25 CFU/ml) (P > 0.05) or gastric aspirate samples (median 58 CFU/ml) (P > 0.05). The LOD with spiked tissue (median 1,525 CFU/ml) and stool samples (median 6,800 CFU/ml) was significantly elevated compared to saline (P ≤ 0.05 and ≤ 0.0005, respectively). Flotation studies with sucrose or NaCl did not consistently result in lowered cycle thresholds in stool or gastric aspirates but >10 cycle reduction was achieved in two of the three pooled CSF samples.Conclusions. Unlike with tissue and stool samples, there was no significant PCR inhibition in the MTB/RIF assay with CSF and gastric aspirates. Although preconcentration of CSF samples with sucrose and NaCl may enhance detection of M. tuberculosis by PCR, further advances are needed to concentrate the bacilli and eliminate PCR inhibitors in paucibacillary non-respiratory samples.

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