Mise à jour le : 15-10-2014
Les derniers abstracts de la revue JCM Current Issue : |
Date de mise en ligne : Mardi 16 septembre 2014
Editorial Board [Masthead]
Date de mise en ligne : Mardi 16 septembre 2014
Farfour, E., Badell, E., Jacques Natali, L., Dommergues, M. A., Blot, S., Guiso, N., Pangon, B., Foucaud, P.
Photo Quiz: Multiple Skin Lesions on a 9-Year-Old Boy Returning from Mali [Photo Quiz]
Date de mise en ligne : Mardi 16 septembre 2014 Species of Sarcocystis are Apicomplexan parasites requiring intermediate and definitive hosts to complete their life cycle. Humans are one of many natural host species and may serve as both intermediate and definitive hosts. However, the extent and public health significance of human Sarcocystis infection are incompletely known. In this minireview, we provide an update on the epidemiology and diagnosis of human sarcocystosis and propose some tools that could contribute to a better understanding of the clinical significance and epidemiology of Sarcocystis infections.
Poulsen, C. S., Stensvold, C. R.
Current Status of Epidemiology and Diagnosis of Human Sarcocystosis [Minireviews]
Date de mise en ligne : Mardi 16 septembre 2014 In this study, we developed rapid and sensitive assays for the detection of Cladophialophora carrionii, a common agent of human chromoblastomycosis. The isothermal techniques evaluated were rolling-circle amplification (RCA), multiplex ligation-dependent probe amplification (MLPA), and loop-mediated isothermal amplification (LAMP). The probes for RCA and MLPA were designed with target sequences in the rDNA internal transcribed spacer gene (ITS) region, and LAMP primers were designed using the elongation factor 1α gene (EF1); these probes and primers specifically amplified DNA of isolates of the species. The three techniques were sufficiently specific and sensitive for discriminating target DNA of C. carrionii from that of related Cladophialophora species and other agents of chromoblastomycosis. RCA, MLPA, and LAMP are advantageous in their reliability and ease of operation compared to standard PCR and conventional methods.
Deng, S., de Hoog, G. S., Pan, W., Chen, M., van den Ende, A. H. G. G., Yang, L., Sun, J., Najafzadeh, M. J., Liao, W., Li, R.
Three Isothermal Amplification Techniques for Rapid Identification of Cladophialophora carrionii, an Agent of Human Chromoblastomycosis [Mycology]
Date de mise en ligne : Mardi 16 septembre 2014 Nonculture-based tests are gaining popularity in the diagnosis of invasive fungal disease (IFD), but PCR is excluded from disease-defining criteria because of limited standardization and a lack of commercial assays. Commercial PCR assays may have a standardized methodology while providing quality assurance. The detection of PCR products by a surface-enhanced Raman scattering (SERS) assay potentially provides superior analytical sensitivity and multiplexing capacity compared to that of real-time PCR. Using this approach, the RenDx Fungiplex assay was developed to detect Candida and Aspergillus. Analytical and clinical evaluations of the assay were undertaken using extraction methods according to European Aspergillus PCR Initiative (EAPCRI) recommendations. A total of 195 previously extracted samples (133 plasma, 49 serum, and 13 whole blood) from 112 patients (29 with proven/probable IFD) were tested. The 95% limit of detection of Candida and Aspergillus was 200 copies per reaction, with an overall reproducibility of 92.1% for detecting 20 input copies per PCR, and 89.8% for the nucleic acid extraction–PCR-SERS process for detecting fungal burdens of <20 genome equivalents per sample. A clinical evaluation showed that assay positivity significantly correlated with IFD (P < 0.0001). The sensitivity of the assay was 82.8% and was similar for both Candida (80.0%) and Aspergillus (85.7%). The specificity was 87.5% and was increased (97.5%) by using a multiple (≥2 samples) PCR-positive threshold. In summary, the RenDx Fungiplex assay is a PCR-SERS assay for diagnosing IFD and demonstrates promising clinical performance on a variety of samples. This was a retrospective clinical evaluation, and performance is likely to be enhanced through a prospective analysis of clinical validity and by determining clinical utility.
White, P. L., Hibbitts, S. J., Perry, M. D., Green, J., Stirling, E., Woodford, L., McNay, G., Stevenson, R., Barnes, R. A.
Evaluation of a Commercially Developed Semiautomated PCR-Surface-Enhanced Raman Scattering Assay for Diagnosis of Invasive Fungal Disease [Mycology]
Date de mise en ligne : Mardi 16 septembre 2014 Simplified HIV testing based on oral fluid (OF) may allow the expansion of HIV infection counseling and testing (CT) while reducing the risk due to exposure to needles and blood collection. This study evaluated the performance and acceptability of two OF tests (the OraQuick Advance Rapid HIV-1/2 and the Chembio DPP HIV-1/2) from May to September 2009 in two CT sites in Maputo City, Mozambique, compared with results for the national testing algorithm. OF testing was conducted in parallel with whole blood-based testing according to the national HIV algorithm. Blood samples were collected as dried blood spot (DBS) specimens from all participants for quality assurance. HIV infection results were delivered according to the national algorithm. According to the national HIV algorithm, 512 (30.5%) samples were reactive, 1,151 (68.7%) were nonreactive, and 13 (0.8%) were discordant. All discordant cases were retested with an enzyme immunoassay followed by Western blotting, and five (38.5%) were confirmed as HIV positive. The OraQuick OF test showed 518 (30.9%) reactive samples and 1,158 (69.1%) nonreactive samples, with a sensitivity and specificity of 99.8% and 99.8%, respectively. The Chembio DPP OF test showed 519 (31.0%) reactive samples and 1,157 (69.0%) nonreactive samples with a sensitivity and specificity of 100% and 99.8%, respectively. The participants perceived blood testing (49.9%) to be more accurate than OF testing (46.8%). The OF tests showed high performance for the diagnosis of HIV infection when examined individually and in an algorithm, compared with results according to the national testing algorithm.
Sema Baltazar, C., Raposo, C., Jani, I. V., Shodell, D., Correia, D., Goncalves da Silva, C., Kalou, M., Patel, H., Parekh, B.
Evaluation of Performance and Acceptability of Two Rapid Oral Fluid Tests for HIV Detection in Mozambique [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 Although pertussis disease is vaccine preventable, Washington State experienced a substantial rise in pertussis incidence beginning in 2011. By June 2012, the reported cases reached 2,520 (37.5 cases per 100,000 residents), a 1,300% increase compared with the same period in 2011. We assessed the molecular epidemiology of this statewide epidemic using 240 isolates collected from case patients reported from 19 of 39 Washington counties during 2012 to 2013. The typing methods included pulsed-field gel electrophoresis (PFGE), multilocus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), and pertactin gene (prn) mutational analysis. Using the scheme PFGE-MLVA-MLST-prn mutations-Prn deficiency, the 240 isolates comprised 65 distinct typing profiles. Thirty-one PFGE types were found, with the most common types, CDC013 (n = 51), CDC237 (n = 44), and CDC002 (n = 42), accounting for 57% of them. Eleven MLVA types were observed, mainly comprising type 27 (n = 183, 76%). Seven MLST types were identified, with the majority of the isolates typing as prn2-ptxP3-ptxA1-fim3-1 (n = 157, 65%). Four different prn mutations accounted for the 76% of isolates exhibiting pertactin deficiency. PFGE provided the highest discriminatory power (D = 0.87) and was found to be a more powerful typing method than MLVA and MLST combined (D = 0.67). This study provides evidence for the continued predominance of MLVA 27 and prn2-ptxP3-ptxA1 alleles, along with the reemergence of the fim3-1 allele. Our results indicate that the Bordetella pertussis population causing this epidemic was diverse, with a few molecular types predominating. The PFGE, MLVA, and MLST profiles were consistent with the predominate types circulating in the United States and other countries. For prn, several mutations were present in multiple molecular types.
Bowden, K. E., Williams, M. M., Cassiday, P. K., Milton, A., Pawloski, L., Harrison, M., Martin, S. W., Meyer, S., Qin, X., DeBolt, C., Tasslimi, A., Syed, N., Sorrell, R., Tran, M., Hiatt, B., Tondella, M. L.
Molecular Epidemiology of the Pertussis Epidemic in Washington State in 2012 [Epidemiology]
Date de mise en ligne : Mardi 16 septembre 2014 We compared the clinical performances of the BacT/Alert Plus (bioMérieux) and Bactec Plus (Becton Dickinson) aerobic and anaerobic blood culture (BC) media with adsorbent polymeric beads. Patients ≥16 years old with suspected bloodstream infections (BSIs) were enrolled in intensive care units and infectious disease wards. A single 40-ml blood sample was collected from each and used to inoculate (10 ml/bottle) one set of BacT/Alert Plus cultures and one set of Bactec Plus cultures, each set consisting of one aerobic and one anaerobic bottle. Cultures were incubated ≤5 days in the BacT/Alert 3D and Bactec FX instruments, respectively. A total of 128 unique BSI episodes were identified based on the recovery of clinically significant growth in 212 aerobic cultures (106 BacT/Alert and 106 Bactec) and 151 anaerobic cultures (82 BacT/Alert and 69 Bactec). The BacT/Alert aerobic medium had higher recovery rates for Gram-positive cocci (P = 0.024), whereas the Bactec aerobic medium was superior for recovery of Gram-negative bacilli (P = 0.006). BacT/Alert anaerobic medium recovery rates exceeded those of the Bactec anaerobic medium for total organisms (P = 0.003), Gram-positive cocci (P = 0.013), and Escherichia coli (P = 0.030). In terms of capacity for diagnosing the 128 septic episodes, the BacT/Alert and Bactec sets were comparable, although the former sets diagnosed more BSIs caused by Gram-positive cocci (P = 0.008). They also allowed earlier identification of coagulase-negative staphylococcal growth (mean, 2.8 h; P = 0.003) and growth in samples from patients not on antimicrobial therapy that yielded positive results (mean, 1.3 h; P < 0.001). Similarly high percentages of microorganisms in BacT/Alert and Bactec cultures (93.8% and 93.3%, respectively) were identified by direct matrix-assisted laser desorption ionization–time of flight mass spectrometry assay of BC broths. The BacT/Alert Plus media line appears to be a reliable, timesaving tool for routine detection of BSIs in the population we studied, although further studies are needed to evaluate their performance in other settings.
Fiori, B., D'Inzeo, T., Di Florio, V., De Maio, F., De Angelis, G., Giaquinto, A., Campana, L., Tanzarella, E., Tumbarello, M., Antonelli, M., Sanguinetti, M., Spanu, T.
Performance of Two Resin-Containing Blood Culture Media in Detection of Bloodstream Infections and in Direct Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Broth Assays for Isolate Identification: Clinical Comparison of the BacT/Alert Plus and Bactec Plus Systems [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Streptococcus suis, an important zoonotic pathogen, is a highly diverse species with only a subset of strains that cause disease in humans. Our previous study proposed a minimum core genome (MCG) sequence typing method and defined seven MCG groups, with MCG group 1 as the prevalent group causing human infections. In this study, we identified a set of 10 single nucleotide polymorphisms (SNPs) distributed in six genes that were used to identify the seven MCG groups. The 10 SNPs were typed for 179 S. suis isolates collected from slaughtered pigs. The most prevalent groups among the tested isolates were MCG groups 6 and 7. Most of the isolates (147/179) were genotyped as mrp negative, epf negative, sly negative, and CDS2157 positive. The 179 isolates were also typed by multilocus sequence typing (MLST) and divided into 115 sequence types (STs), 111 of which were new. The 6 serotypes (29, 11, 5, 12, 30, and 2) represented 72.3% of the serotyped isolates. Our data show that the typing assay facilitates the application of genome data to the surveillance of S. suis.
Zheng, H., Ji, S., Lan, R., Liu, Z., Bai, X., Zhang, W., Gottschalk, M., Xu, J.
Population Analysis of Streptococcus suis Isolates from Slaughtered Swine by Use of Minimum Core Genome Sequence Typing [Clinical Veterinary Microbiology]
Date de mise en ligne : Mardi 16 septembre 2014 Nontuberculous mycobacterial infections caused by Mycobacterium abscessus are responsible for a range of disease manifestations from pulmonary to skin infections and are notoriously difficult to treat, due to innate resistance to many antibiotics. Previous population studies of clinical M. abscessus isolates utilized multilocus sequence typing or pulsed-field gel electrophoresis, but high-resolution examinations of genetic diversity at the whole-genome level have not been well characterized, particularly among clinical isolates derived in the United States. We performed whole-genome sequencing of 11 clinical M. abscessus isolates derived from eight U.S. patients with pulmonary nontuberculous mycobacterial infections, compared them to 30 globally diverse clinical isolates, and investigated intrapatient genomic diversity and evolution. Phylogenomic analyses revealed a cluster of closely related U.S. and Western European M. abscessus subsp. abscessus isolates that are genetically distinct from other European isolates and all Asian isolates. Large-scale variation analyses suggested genome content differences of 0.3 to 8.3%, relative to the reference strain ATCC 19977T. Longitudinally sampled isolates showed very few single-nucleotide polymorphisms and correlated genomic deletion patterns, suggesting homogeneous infection populations. Our study explores the genomic diversity of clinical M. abscessus strains from multiple continents and provides insight into the genome plasticity of an opportunistic pathogen.
Davidson, R. M., Hasan, N. A., Reynolds, P. R., Totten, S., Garcia, B., Levin, A., Ramamoorthy, P., Heifets, L., Daley, C. L., Strong, M.
Genome Sequencing of Mycobacterium abscessus Isolates from Patients in the United States and Comparisons to Globally Diverse Clinical Strains [Mycobacteriology and Aerobic Actinomycetes]
Date de mise en ligne : Mardi 16 septembre 2014 There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥1 positive sample for strict pathogens and ≥2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis.
Bemer, P., Plouzeau, C., Tande, D., Leger, J., Giraudeau, B., Valentin, A. S., Jolivet-Gougeon, A., Vincent, P., Corvec, S., Gibaud, S., Juvin, M. E., Hery-Arnaud, G., Lemarie, C., Kempf, M., Bret, L., Quentin, R., Coffre, C., de Pinieux, G., Bernard, L., Burucoa, C., the Centre de Reference des Infections Osteo-articulaires du Grand Ouest (CRIOGO) Study Team
Evaluation of 16S rRNA Gene PCR Sensitivity and Specificity for Diagnosis of Prosthetic Joint Infection: a Prospective Multicenter Cross-Sectional Study [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral reverse transcriptase PCR (RT-PCR) of nose and throat swabs (NTS) is useful for patient care by informing antiviral use and appropriate isolation. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscosity. We evaluated a simple method of processing sputum samples in a fully automated respiratory viral panel RT-PCR assay (FilmArray). Archived sputum and NTS samples collected in 2008-2012 from hospitalized adults with RTI were evaluated. A subset of sputum samples positive for 10 common viruses by a uniplex RT-PCR was selected. A sterile cotton-tip swab was dunked in sputum, swirled in 700 μL of sterile water (dunk and swirl method) and tested by the FilmArray assay. Quantitative RT-PCR was performed on "dunked" sputum and NTS samples for influenza A (Flu A), respiratory syncytial virus (RSV), coronavirus OC43 (OC43), and human metapneumovirus (HMPV). Viruses were identified in 31% of 965 illnesses using a uniplex RT-PCR. The sputum sample was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases and significantly increased the diagnostic yield of NTS alone (302/965 [31%] versus 197/965 [20%]; P = 0.0001). Of 108 sputum samples evaluated by the FilmArray assay using the dunk and swirl method, 99 (92%) were positive. Quantitative RT-PCR revealed higher mean viral loads in dunked sputum samples compared to NTS samples for Flu A, RSV, and HMPV (P = 0.0001, P = 0.006, and P = 0.011, respectively). The dunk and swirl method is a simple and practical method for reliably processing sputum samples in a fully automated PCR system. The higher viral loads in sputa may increase detection over NTS testing alone.
Branche, A. R., Walsh, E. E., Formica, M. A., Falsey, A. R.
Detection of Respiratory Viruses in Sputum from Adults by Use of Automated Multiplex PCR [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 Authoritative guidelines state that the diagnosis of ventilator-associated pneumonia (VAP) can be established using either endotracheal aspirate (ETA) or bronchoalveolar lavage fluid (BALF) analysis, thereby suggesting that their results are considered to be in accordance. Therefore, the results of ETA Gram staining and semiquantitative cultures were compared to the results from a paired ETA-BALF analysis. Different thresholds for the positivity of ETAs were assessed. This was a prospective study of all patients who underwent bronchoalveolar lavage for suspected VAP in a 27-bed university intensive care unit during an 8-year period. VAP was diagnosed when ≥2% of the BALF cells contained intracellular organisms and/or when BALF quantitative culture revealed ≥104 CFU/ml of potentially pathogenic microorganisms. ETA Gram staining and semiquantitative cultures were compared to the results from paired BALF analysis by Cohen's kappa coefficients. VAP was suspected in 311 patients and diagnosed in 122 (39%) patients. In 288 (93%) patients, the results from the ETA analysis were available for comparison. Depending on the threshold used and the diagnostic modality, VAP incidences varied from 15% to 68%. For the diagnosis of VAP, the most accurate threshold for positivity of ETA semiquantitative cultures was moderate or heavy growth, whereas the optimal threshold for BALF Gram staining was ≥1 microorganisms per high power field. The Cohen's kappa coefficients were 0.22, 0.31, and 0.60 for ETA and paired BALF Gram stains, cultures, and BALF Gram stains, respectively. Since the ETA and BALF Gram stains and cultures agreed only fairly, they are probably not interchangeable for diagnosing VAP.
Scholte, J. B. J., van Dessel, H. A., Linssen, C. F. M., Bergmans, D. C. J. J., Savelkoul, P. H. M., Roekaerts, P. M. H. J., van Mook, W. N. K. A.
Endotracheal Aspirate and Bronchoalveolar Lavage Fluid Analysis: Interchangeable Diagnostic Modalities in Suspected Ventilator-Associated Pneumonia? [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 The diagnosis and management of pneumonia are limited by the use of culture-based techniques of microbial identification, which may fail to identify unculturable, fastidious, and metabolically active viable but unculturable bacteria. Novel high-throughput culture-independent techniques hold promise but have not been systematically compared to conventional culture. We analyzed 46 clinically obtained bronchoalveolar lavage (BAL) fluid specimens from symptomatic and asymptomatic lung transplant recipients both by culture (using a clinical microbiology laboratory protocol) and by bacterial 16S rRNA gene pyrosequencing. Bacteria were identified in 44 of 46 (95.7%) BAL fluid specimens by culture-independent sequencing, significantly more than the number of specimens in which bacteria were detected (37 of 46, 80.4%, P ≤ 0.05) or "pathogen" species reported (18 of 46, 39.1%, P ≤ 0.0001) via culture. Identification of bacteria by culture was positively associated with culture-independent indices of infection (total bacterial DNA burden and low bacterial community diversity) (P ≤ 0.01). In BAL fluid specimens with no culture growth, the amount of bacterial DNA was greater than that in reagent and rinse controls, and communities were markedly dominated by select Gammaproteobacteria, notably Escherichia species and Pseudomonas fluorescens. Culture growth above the threshold of 104 CFU/ml was correlated with increased bacterial DNA burden (P < 0.01), decreased community diversity (P < 0.05), and increased relative abundance of Pseudomonas aeruginosa (P < 0.001). We present two case studies in which culture-independent techniques identified a respiratory pathogen missed by culture and clarified whether a cultured "oral flora" species represented a state of acute infection. In summary, we found that bacterial culture of BAL fluid is largely effective in discriminating acute infection from its absence and identified some specific limitations of BAL fluid culture in the diagnosis of pneumonia. We report the first correlation of quantitative BAL fluid culture results with culture-independent evidence of infection.
Dickson, R. P., Erb-Downward, J. R., Prescott, H. C., Martinez, F. J., Curtis, J. L., Lama, V. N., Huffnagle, G. B.
Analysis of Culture-Dependent versus Culture-Independent Techniques for Identification of Bacteria in Clinically Obtained Bronchoalveolar Lavage Fluid [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Beginning in July 2011, 31 green anaconda (Eunectes murinus) juveniles from an oceanarium in Hong Kong died over a 12-month period. Necropsy revealed at least two of the following features in 23 necropsies: dermatitis, severe pan-nephritis, and/or severe systemic multiorgan necrotizing inflammation. Histopathological examination revealed severe necrotizing inflammation in various organs, most prominently the kidneys. Electron microscopic examination of primary tissues revealed intralesional accumulations of viral nucleocapsids with diameters of 10 to 14 nm, typical of paramyxoviruses. Reverse transcription (RT)-PCR results were positive for paramyxovirus (viral loads of 2.33 x 104 to 1.05 x 108 copies/mg tissue) in specimens from anaconda juveniles that died but negative in specimens from the two anaconda juveniles and anaconda mother that survived. None of the other snakes in the park was moribund, and RT-PCR results for surveillance samples collected from other snakes were negative. The virus was isolated from BHK21 cells, causing cytopathic effects with syncytial formation. The virus could also replicate in 25 of 27 cell lines of various origins, in line with its capability for infecting various organs. Electron microscopy with cell culture material revealed enveloped virus with the typical "herringbone" appearance of helical nucleocapsids in paramyxoviruses. Complete genome sequencing of five isolates confirmed that the infections originated from the same clone. Comparative genomic and phylogenetic analyses and mRNA editing experiments revealed a novel paramyxovirus in the genus Ferlavirus, named anaconda paramyxovirus, with a typical Ferlavirus genomic organization of 3'-N-U-P/V/I-M-F-HN-L-5'. Epidemiological and genomic analyses suggested that the anaconda juveniles acquired the virus perinatally from the anaconda mother rather than from other reptiles in the park, with subsequent interanaconda juvenile transmission.
Woo, P. C. Y., Lau, S. K. P., Martelli, P., Hui, S.-W., Lau, C. C. Y., Fan, R. Y. Y., Groff, J. M., Tam, E. W. T., Chan, K.-H., Yuen, K.-Y.
Fatal Systemic Necrotizing Infections Associated with a Novel Paramyxovirus, Anaconda Paramyxovirus, in Green Anaconda Juveniles [Clinical Veterinary Microbiology]
Date de mise en ligne : Mardi 16 septembre 2014 Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis.
Didi, J., Lemee, L., Gibert, L., Pons, J.-L., Pestel-Caron, M.
Multi-Virulence-Locus Sequence Typing of Staphylococcus lugdunensis Generates Results Consistent with a Clonal Population Structure and Is Reliable for Epidemiological Typing [Epidemiology]
Date de mise en ligne : Mardi 16 septembre 2014 Aspergillus spp. are among the most common causes of opportunistic invasive fungal infections in tertiary care hospitals. Little is known about the prevalence and in vitro susceptibility of Aspergillus species in Latin America, because there are few medical centers able to perform accurate identification at the species level. The purpose of this study was to analyze the distribution of cryptic and rare Aspergillus species among clinical samples from 133 patients with suspected aspergillosis admitted in 12 medical centers in Brazil and to analyze the in vitro activity of different antifungal drugs. The identification of Aspergillus species was performed based on a polyphasic approach, as well as sequencing analysis of the internal transcribed spacer (ITS) region, calmodulin, and β-tubulin genes and phylogenetic analysis when necessary. The in vitro susceptibility tests with voriconazole, posaconazole, and itraconazole were performed according to the CLSI M38-A2 document (2008). We demonstrated a high prevalence of cryptic species causing human infection. Only three isolates, representing the species Aspergillus thermomutatus, A. ochraceus, and A. calidoustus, showed less in vitro susceptibility to at least one of the triazoles tested. Accurate identifications of Aspergillus at the species level and with in vitro susceptibility tests are important because some species may present unique resistance patterns against specific antifungal drugs.
Negri, C. E., Goncalves, S. S., Xafranski, H., Bergamasco, M. D., Aquino, V. R., Castro, P. T. O., Colombo, A. L.
Cryptic and Rare Aspergillus Species in Brazil: Prevalence in Clinical Samples and In Vitro Susceptibility to Triazoles [Mycology]
Date de mise en ligne : Mardi 16 septembre 2014 Staphylococcus pseudintermedius is the most common microorganism isolated from canine pyoderma and postoperative wound infections. The prevalence of methicillin-resistant S. pseudintermedius (MRSP) has increased, and recently, isolates that are resistant not only to methicillin but also to other classes of antibiotic drugs, including aminoglycosides, have become common. A total of 422 S. pseudintermedius isolates collected from 413 dogs were analyzed for amikacin and methicillin resistance using broth microdilution and disk diffusion testing. Methicillin-resistant isolates were significantly (P < 0.0001) more likely to be resistant to amikacin (37%, 31/84) than were methicillin-susceptible isolates (7%, 22/338). Additionally, resistance to non-β-lactam antibiotics was significantly associated with resistance to amikacin irrespective of methicillin resistance. Among the 422 isolates, 32 that tested positive for amikacin resistance by broth microdilution or disk diffusion testing were investigated further for the presence of aminoglycoside-modifying enzyme genes using multiplex PCR. Of these isolates, 66% (21/32) were methicillin resistant. In contrast to previous studies of Staphylococcus aureus, the most prevalent gene detected was aph(3')-IIIa found in 75% (24/32) of isolates followed by aac(6')/aph(2'') and ant(4')-Ia in 12% (4/32) and 3% (1/32), respectively. Understanding the differences in antimicrobial resistance gene carriage between different species of Staphylococcus may improve antimicrobial drug selection for clinical therapy and provide insights into how resistance develops in S. pseudintermedius.
Gold, R. M., Cohen, N. D., Lawhon, S. D.
Amikacin Resistance in Staphylococcus pseudintermedius Isolated from Dogs [Clinical Veterinary Microbiology]
Date de mise en ligne : Mardi 16 septembre 2014 The aim of this study was to identify the best practices for the detection of Shiga toxin-producing Escherichia coli (STEC) in children with diarrheal illness treated at a tertiary care center, i.e., sorbitol-MacConkey (SMAC) agar culture, enzyme immunoassay (EIA) for Shiga toxin, or the simultaneous use of both methods. STEC was detected in 100 of 14,997 stool specimens submitted for enteric culture (0.7%), with 65 cases of E. coli O157. Among E. coli O157 isolates, 57 (88%) were identified by both SMAC agar culture and EIA, 6 (9%) by SMAC agar culture alone, and 2 (3%) by EIA alone. Of the 62 individuals with diarrheal hemolytic uremic syndrome (HUS) seen at our institution during the study period, 16 (26%) had STEC isolated from cultures at our institution and 15 (24%) had STEC isolated at other institutions. No STEC was recovered in 31 cases (50%). Of the HUS cases in which STEC was isolated, 28 (90%) were attributable to E. coli O157 and 3 (10%) were attributable to non-O157 STEC. Consistent with previous studies, we have determined that a subset of E. coli O157 infections will not be detected if an agar-based method is excluded from the enteric culture workup; this has both clinical and public health implications. The best practice would be concomitant use of an agar-based method and a Shiga toxin EIA, but a Shiga toxin EIA should not be considered to be an adequate stand-alone test for detection of E. coli O157 in clinical samples.
Schindler, E. I., Sellenriek, P., Storch, G. A., Tarr, P. I., Burnham, C.-A. D.
Shiga Toxin-Producing Escherichia coli: a Single-Center, 11-Year Pediatric Experience [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 The identification of mycobacteria outside biocontainment facilities requires that the organisms first be rendered inactive. Exposure to 70% ethanol (EtOH) either before or after mechanical disruption was evaluated in order to establish a safe, effective, and rapid inactivation protocol that is compatible with identification of Mycobacterium and Nocardia species using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A combination of 5 min of bead beating in 70% EtOH followed by a 10-min room temperature incubation period was found to be rapidly bactericidal and provided high-quality spectra compared to spectra obtained directly from growth on solid media. The age of the culture, the stability of the refrigerated or frozen lysates, and freeze-thaw cycles did not adversely impact the quality of the spectra or the identification obtained.
Dunne, W. M., Doing, K., Miller, E., Miller, E., Moreno, E., Baghli, M., Mailler, S., Girard, V., van Belkum, A., Deol, P.
Rapid Inactivation of Mycobacterium and Nocardia Species before Identification Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry [Mycobacteriology and Aerobic Actinomycetes]
Date de mise en ligne : Mardi 16 septembre 2014 The prevalence of Mycoplasma genitalium is high in vulnerable populations of women in low-resource settings. However, the epidemiology of infection in these populations is not well established. To determine the prevalence of Mycoplasma genitalium and its association with cervical cytology and other correlates, we recruited 350 female sex workers (FSW) who were 18 to 50 years old in Nairobi, Kenya, for a cross-sectional study. A questionnaire was administered at baseline to obtain information on sociodemographics and sexual behaviors. Women underwent a pelvic exam, during which a physician collected cervical-exfoliation samples for conventional cytology and sexually transmitted infection (STI) testing. Samples were tested for M. genitalium and other STI organisms (Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis) and the E6/E7 mRNA of human papillomavirus (HPV) by Aptima nucleic amplification assays. The prevalence of M. genitalium was 12.9%. FSW who engaged in sexual intercourse during menses were less likely to have M. genitalium infection than those who did not (odds ratio [OR], 0.3; 95% confidence interval [95% CI], 0.1, 0.9). M. genitalium was also less prevalent among FSW who had worked in prostitution for >5 years (6.2%) than among those who had worked for <3 years (17.6%) (OR, 0.3; 95% CI, 0.1, 0.8). FSW who reported more frequent condom use were more likely to be infected with M. genitalium than those who reported less frequent use (OR, 3.8; 95% CI, 1.2, 11.6). These correlates differ from those found in M. genitalium studies conducted with FSW from West Africa and China. Further longitudinal analyses assessing associations with persistent M. genitalium infection are needed.
Gomih-Alakija, A., Ting, J., Mugo, N., Kwatampora, J., Getman, D., Chitwa, M., Patel, S., Gokhale, M., Kimani, J., Behets, F. S., Smith, J. S.
Clinical Characteristics Associated with Mycoplasma genitalium among Female Sex Workers in Nairobi, Kenya [Epidemiology]
Date de mise en ligne : Mardi 16 septembre 2014 The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 μl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 μl raw stool; however, 100 μl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.
Khare, R., Espy, M. J., Cebelinski, E., Boxrud, D., Sloan, L. M., Cunningham, S. A., Pritt, B. S., Patel, R., Binnicker, M. J.
Comparative Evaluation of Two Commercial Multiplex Panels for Detection of Gastrointestinal Pathogens by Use of Clinical Stool Specimens [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Antimicrobial susceptibility testing (AST) assigns pathogens to "susceptible" or "resistant" clinical categories based on clinical breakpoints (CBPs) derived from MICs or inhibition zone diameters and indicates the likelihood for therapeutic success. AST reports do not provide quantitative measures for the reliability of such categorization. Thus, it is currently impossible for clinicians to estimate the technical forecast uncertainty of an AST result regarding clinical categorization. AST error rates depend on the localization of pathogen populations in relation to CBPs. Bacterial species are, however, not homogeneous, and subpopulations behave differently with respect to AST results. We addressed how AST reporting errors differ between isolates with and without acquired drug resistance determinants. Using as an example the beta-lactams and their most important resistance mechanisms, we analyzed different pathogen populations for their individual reporting error probabilities. Categorization error rates were significantly higher for bacterial populations harboring resistance mechanisms than for the wild-type population. Reporting errors for amoxicillin-clavulanic acid and piperacillin-tazobactam in Escherichia coli infection cases were almost exclusively due to the presence of broad-spectrum- and extended-spectrum-beta-lactamase (ESBL)-producing microorganisms (79% and 20% of all errors, respectively). Clinicians should be aware of the significantly increased risk of erroneous AST reports for isolates producing beta-lactamases, particularly ESBL and AmpC. Including probability indicators for interpretation would improve AST reports.
Maurer, F. P., Courvalin, P., Bottger, E. C., Hombach, M.
Integrating Forecast Probabilities in Antibiograms: a Way To Guide Antimicrobial Prescriptions More Reliably? [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5' untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1.
Yang, R., Cong, X., Du, S., Fei, R., Rao, H., Wei, L.
Performance Comparison of the Versant HCV Genotype 2.0 Assay (LiPA) and the Abbott Realtime HCV Genotype II Assay for Detecting Hepatitis C Virus Genotype 6 [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 From January 2011 to December 2013, a total of 262 samples, from 188 patients suspected of having syphilis were tested for the presence of treponemal DNA by PCR amplification of five chromosomal loci, including the polA (TP0105), tmpC (TP0319), TP0136, TP0548, and 23S rRNA genes. Altogether, 146 samples from 103 patients were PCR positive for treponemal DNA. A set of 81 samples from 62 PCR-positive patients were typeable, and among them, nine different genotypes were identified. Compared to a previous study in the Czech Republic during 2004 to 2010, the number of genotypes detected among syphilis patients in a particular year increased to six in both 2012 and 2013, although they were not the same six. The proportion of macrolide-resistant clinical isolates in this 3-year study was 66.7%.
Grillova, L., Petrošova, H., Mikalova, L., Strnadel, R., Dastychova, E., Kuklova, I., Kojanova, M., Kreidlova, M., Vaňousova, D., Hercogova, J., Prochazka, P., Zakoucka, H., Krchňakova, A., Vašků, V., Šmajs, D.
Molecular Typing of Treponema pallidum in the Czech Republic during 2011 to 2013: Increased Prevalence of Identified Genotypes and of Isolates with Macrolide Resistance [Epidemiology]
Date de mise en ligne : Mardi 16 septembre 2014 From June to September 2012, 500 urine samples were recovered from patients with urinary tract infections (UTI) due to Gram-negative bacilli (≥104 leukocytes/ml and ≥105 Gram-negative isolates/ml) who visited the University hospital Bicêtre (France). They were challenged with extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) using the rapid diagnostic ESBL NDP test. Results of the ESBL NDP test were compared to the results of the double-disc susceptibility test (DDST) performed on solid-agar plates and molecular identification of the β-lactamase genes. Among the 450 nonduplicate urine samples, 11.3% were positive for ESBL-E by using the DDST, the ESBL determinants being mostly of the CTX-M type (CTX-M-15) according to molecular testing. Results of the ESBL NDP test were obtained within 15 min. The sensitivity and specificity of the ESBL NDP test were 98% and 99.8%, respectively, whereas the positive and negative predictive values of this test were 98% and 99.8%, respectively. A perfect correlation between cefotaxime resistance and positivity of the ESBL NDP test was observed. Therefore, the ESBL NDP test offers a powerful tool for a rapid identification of ESBL-E and associated resistance to expanded-spectrum cephalosporins. It may be useful in particular for guiding first-line antibiotic therapy.
Dortet, L., Poirel, L., Nordmann, P.
Rapid Detection of Extended-Spectrum-{beta}-Lactamase-Producing Enterobacteriaceae from Urine Samples by Use of the ESBL NDP Test [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Aspergillus section Fumigati contains 12 clinically relevant species. Among these Aspergillus species, A. fumigatus is the most frequent agent of invasive aspergillosis, followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigati revealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelis and Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2 isolates of both A. parafelis and A. pseudofelis. The MAT1-2 isolate of A. parafelis was fertile when paired with the MAT1-1 isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensis but was not fertile with the MAT1-1 isolate of A. lentulus. The MAT1-2 isolates of A. pseudofelis were fertile when paired with the MAT1-1 isolate of A. felis but not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2 isolates of A. parafelis and A. pseudofelis being the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management.
Sugui, J. A., Peterson, S. W., Figat, A., Hansen, B., Samson, R. A., Mellado, E., Cuenca-Estrella, M., Kwon-Chung, K. J.
Genetic Relatedness versus Biological Compatibility between Aspergillus fumigatus and Related Species [Mycology]
Date de mise en ligne : Mardi 16 septembre 2014 Efficient detection of human respiratory viral pathogens is crucial in the management of patients with acute respiratory tract infection. Sequence-independent amplification of nucleic acids combined with next-generation sequencing technology and bioinformatics analyses is a promising strategy for identifying pathogens in clinical and public health settings. It allows the characterization of hundreds of different known pathogens simultaneously and of novel pathogens that elude conventional testing. However, major hurdles for its routine use exist, including cost, turnaround time, and especially sensitivity of the assay, as the detection limit is dependent on viral load, host genetic material, and sequencing depth. To obtain insights into these aspects, we analyzed nasopharyngeal aspirates from a cohort of 81 Thai children with respiratory disease for the presence of respiratory viruses using a sequence-independent next-generation sequencing approach and routinely used diagnostic real-time reverse transcriptase PCR (real-time RT-PCR) assays. With respect to the detection of rhinovirus and human metapneumovirus, the next-generation sequencing approach was at least as sensitive as diagnostic real-time RT-PCR in this small cohort, whereas for bocavirus and enterovirus, next-generation sequencing was less sensitive than real-time RT-PCR. The advantage of the sequencing approach over real-time RT-PCR was the immediate availability of virus-typing information. Considering the development of platforms capable of generating more output data at declining costs, next-generation sequencing remains of interest for future virus diagnosis in clinical and public health settings and certainly as an additional tool when screening results from real-time RT-PCR are negative.
Prachayangprecha, S., Schapendonk, C. M. E., Koopmans, M. P., Osterhaus, A. D. M. E., Schurch, A. C., Pas, S. D., van der Eijk, A. A., Poovorawan, Y., Haagmans, B. L., Smits, S. L.
Exploring the Potential of Next-Generation Sequencing in Detection of Respiratory Viruses [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 Invasive aspergillosis is a difficult-to-diagnose infection with a high mortality rate that affects high-risk groups such as patients with neutropenia and hematologic malignancies. We performed a bivariate meta-analysis of diagnostic data for an Aspergillus sp. PCR assay with blood specimens from high-risk hematology patients. We included all studies involving human subjects that assessed the performance of any PCR assay for invasive aspergillosis in whole blood or serum and that used the European Organization for the treatment of Cancer/Mycoses Study Group criteria as a reference standard. Three investigators independently searched the literature for eligible studies and extracted the data. Out of a total of 37 studies, 25 met strict quality criteria and were included in our evidence synthesis. Twenty-five studies with 2,595 patients were analyzed. The pooled diagnostic performance of whole-blood and serum PCR assays was moderate, with a sensitivity and specificity of 84% (95% confidence interval [CI], 75 to 91%) and 76% (95% CI, 65 to 84%), respectively, suggesting that a positive or negative result is unable, on its own, to confirm or exclude a suspected infection. The performance of a PCR assay of serum was not significantly different from that of whole blood. Notably, at least two positive PCR test results were found to have a specificity of 95% and a sensitivity of 64% for invasive infection, achieving a high positive likelihood ratio of 12.8. Importantly, the European Aspergillus PCR Initiative (EAPCRI) recommendations improved the performance of the PCR even further when at least two positive specimens were used to define PCR positivity. In conclusion, two positive PCR results should be considered highly indicative of an active Aspergillus sp. infection. Use of the EAPCRI recommendations by clinical laboratories can further enhance PCR performance.
Arvanitis, M., Ziakas, P. D., Zacharioudakis, I. M., Zervou, F. N., Caliendo, A. M., Mylonakis, E.
PCR in Diagnosis of Invasive Aspergillosis: a Meta-Analysis of Diagnostic Performance [Mycology]
Date de mise en ligne : Mardi 16 septembre 2014 Fourth-generation HIV rapid tests (RTs) claim to detect both p24 antigen (Ag) and HIV antibodies (Ab) for early identification of acute infections, important for targeting prevention and reducing HIV transmission. In a nationally representative household survey in Swaziland, 18,172 adults, age 18 to 49 years, received home-based HIV rapid testing in 2010 and 2011. Of the 18,172 individuals, 5,822 (32.0%) were Ab positive (Ab+) by the Determine HIV-1/2 Ab/Ab combo test, and 5,789 (99.4%) of those were confirmed to be reactive in the Uni-Gold test. Determine combo identified 12 individuals as having acute infections (Ag+/Ab negative [Ab–]); however, none had detectable HIV-1 RNA and 8 of 12 remained HIV negative at their 6-week follow-up visit (4 were lost to follow-up). All RT-nonreactive samples were pooled and tested by nucleic acid amplification testing (NAAT) to identify acute infections. NAAT identified 13 (0.1%) of the 12,338 HIV antibody-negative specimens as HIV RNA positive, with RNA levels ranging from 300 to >10,000,000 copies/ml. However, none of them were Ag+ by Determine combo. Follow-up testing of 12 of the 13 NAAT-positive individuals at 6 months demonstrated 12 seroconversions (1 individual was lost to follow-up). Therefore, the Determine combo test had a sensitivity of 0% (95% confidence interval, 0 to 28) and positive predictive value of 0% for the detection of acute infections. The ability of the 4th-generation Determine combo to detect antigen was very poor in Swaziland. Thus, the Determine combo test does not add any value to the current testing algorithm; rather, it adds additional costs and complexity to HIV diagnosis. The detection of acute HIV infections may need to rely on other testing strategies.
Duong, Y. T., Mavengere, Y., Patel, H., Moore, C., Manjengwa, J., Sibandze, D., Rasberry, C., Mlambo, C., Li, Z., Emel, L., Bock, N., Moore, J., Nkambule, R., Justman, J., Reed, J., Bicego, G., Ellenberger, D. L., Nkengasong, J. N., Parekh, B. S.
Poor Performance of the Determine HIV-1/2 Ag/Ab Combo Fourth-Generation Rapid Test for Detection of Acute Infections in a National Household Survey in Swaziland [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 Topical mupirocin is widely used for the decolonization of methicillin-resistant Staphylococcus aureus (MRSA) carriers. We evaluated the capacity of various MRSA clonotypes to develop mutations in the ileS gene associated with low-level mupirocin resistance. Twenty-four mupirocin-sensitive MRSA isolates from a variety of genotypes (determined by a multilocus variable-number tandem-repeat assay) were selected. Mupirocin MICs were determined by Etest. The isolates were then incubated in subinhibitory concentrations of mupirocin for 7 to 14 days. Repeat MIC determinations and sequencing of the ileS gene were then performed. Doubling times of isolates exposed to mupirocin and of unexposed isolates were compared. We found that exposure to mupirocin led to rapid induction of low-level resistance (MICs of 8 to 24 μg/ml) in 11 of 24 (46%) MRSA isolates. This phenomenon was observed in strains with diverse genetic backgrounds. Various mutations were detected in 18 of 24 (75%) MRSA isolates. Acquisition of mutations appeared to be a stepwise process during prolonged incubation with the drug. Among the five isolates exhibiting low-level resistance and the highest MICs, four tested sensitive after incubation in the absence of mupirocin but there was no reversion to the susceptible wild-type primary sequence. Resistance was not associated with significant fitness cost, suggesting that MRSA strains with low-level mupirocin resistance may have a selective advantage in facilities where mupirocin is commonly used. Our findings emphasize the importance of the judicious use of this topical agent and the need to closely monitor for the emergence of resistance.
Lee, A. S., Gizard, Y., Empel, J., Bonetti, E.-J., Harbarth, S., Francois, P.
Mupirocin-Induced Mutations in ileS in Various Genetic Backgrounds of Methicillin-Resistant Staphylococcus aureus [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Serological assays and a two-tiered test algorithm are recommended for laboratory confirmation of Lyme disease. In the United States, the sensitivity of two-tiered testing using commercially available serology-based assays is dependent on the stage of infection and ranges from 30% in the early localized disease stage to near 100% in late-stage disease. Other variables, including subjectivity in reading Western blots, compliance with two-tiered recommendations, use of different first- and second-tier test combinations, and use of different test samples, all contribute to variation in two-tiered test performance. The availability and use of sample sets from well-characterized Lyme disease patients and controls are needed to better assess the performance of existing tests and for development of improved assays. To address this need, the Centers for Disease Control and Prevention and the National Institutes of Health prospectively collected sera from patients at all stages of Lyme disease, as well as healthy donors and patients with look-alike diseases. Patients and healthy controls were recruited using strict inclusion and exclusion criteria. Samples from all included patients were retrospectively characterized by two-tiered testing. The results from two-tiered testing corroborated the need for novel and improved diagnostics, particularly for laboratory diagnosis of earlier stages of infection. Furthermore, the two-tiered results provide a baseline with samples from well-characterized patients that can be used in comparing the sensitivity and specificity of novel diagnostics. Panels of sera and accompanying clinical and laboratory testing results are now available to Lyme disease serological test users and researchers developing novel tests.
Molins, C. R., Sexton, C., Young, J. W., Ashton, L. V., Pappert, R., Beard, C. B., Schriefer, M. E.
Collection and Characterization of Samples for Establishment of a Serum Repository for Lyme Disease Diagnostic Test Development and Evaluation [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Genital human papillomavirus (HPV) is the etiologic agent of more than 99% of all cervical cancers worldwide, with 14 genotypes being considered oncogenic or "high risk" because of their association with severe dysplasia and cervical carcinoma. Among these 14 high-risk types, HPV-16 and -18 account for approximately 70% of cervical cancers. The aim of this study was to evaluate three FDA-approved HPV nucleic acid-based tests for the ability to predict high-grade cervical intraepithelial neoplasias (CIN2 or worse) in corresponding tissue biopsy specimens. Residual specimens (total n = 793, cervical n = 743, vaginal n = 50) collected in ThinPrep PreservCyt medium with a cytologic result of ≥atypical squamous cells of undetermined significance were tested by the Hybrid Capture 2 (HC2) assay (Qiagen, Gaithersburg, MD), the cobas HPV test (Roche Diagnostics, Indianapolis, IN), and the APTIMA HPV assay (Hologic, San Diego, CA). Genotyping for HPV-16 and HPV-18 was simultaneously performed by the cobas HPV test. Results were compared to cervical or vaginal biopsy findings, when they were available (n = 350). Among the 350 patients with corresponding biopsy results, 81 (23.1%) showed ≥CIN2 by histopathology. The ≥CIN2 detection sensitivity was 91.4% by the cobas and APTIMA assays and 97.5% by HC2 assay. The specificities of the cobas, APTIMA, and HC2 assays were 31.2, 42.0, and 27.1%, respectively. When considering only positive HPV-16 and/or HPV-18 genotype results, the cobas test showed a sensitivity and a specificity of 51.9 and 86.6%, respectively. While the HC2, cobas, and APTIMA assays showed similar sensitivities for the detection of ≥CIN2 lesions, the specificities of the three tests varied, with the greatest specificity (86.6%) observed when the HPV-16 and/or HPV-18 genotypes were detected.
Binnicker, M. J., Pritt, B. S., Duresko, B. J., Espy, M. J., Grys, T. E., Zarka, M. A., Kerr, S. E., Henry, M. R.
Comparative Evaluation of Three Commercial Systems for Detection of High-Risk Human Papillomavirus in Cervical and Vaginal ThinPrep PreservCyt Samples and Correlation with Biopsy Results [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 At the Islet Isolation Laboratory of the Scottish National Blood Transfusion Service, manual sterility testing data show that contamination rates are 57.7% for pancreas transport fluid, 4.3% for postpurification islet samples, and 0% for pretransplant islet samples. This pilot study presents the BacT/Alert System as an alternative to manual testing to provide more rapid and sensitive sterility results for islet cell products.
Murray, L., McGowan, N., Fleming, J., Bailey, L.
Use of the BacT/Alert System for Rapid Detection of Microbial Contamination in a Pilot Study Using Pancreatic Islet Cell Products [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Using crude whole-genome assemblies, we analyzed 25 isolates of Neisseria gonorrhoeae by using a high-resolution single nucleotide polymorphism (SNP) approach for nine housekeeping genes, characterizing penA alleles, and antimicrobial susceptibility phenotypes coupled with population structure analysis. Two clonal complexes, characterized by their spatial and geographical persistence, were identified. In addition, the clonal spread of penicillin-resistant/intermediate phenotypes and a novel introduction of the azithromycin resistance phenotype in Saskatchewan, Canada, were ascertained using this method.
Vidovic, S., Caron, C., Taheri, A., Thakur, S. D., Read, T. D., Kusalik, A., Dillon, J.-A. R.
Using Crude Whole-Genome Assemblies of Neisseria gonorrhoeae as a Platform for Strain Analysis: Clonal Spread of Gonorrhea Infection in Saskatchewan, Canada [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 There are no data about the comparative accuracy of commercially available nucleic acid amplification tests (GeneXpert MTB/RIF and Roche Amplicor) for the diagnosis of tuberculous meningitis (TBM). A total of 148 patients with suspected TBM were evaluated, and cultures served as the reference standard. The sensitivities and specificities (95% confidence interval [CI]) for the Amplicor and Xpert MTB/RIF tests were similar: 46 (31–60) versus 50 (33–67) and 99 (93–100) and 94 (84–99), respectively.
Patel, V. B., Connolly, C., Singh, R., Lenders, L., Matinyenya, B., Theron, G., Ndung'u, T., Dheda, K.
Comparison of Amplicor and GeneXpert MTB/RIF Tests for Diagnosis of Tuberculous Meningitis [Mycobacteriology and Aerobic Actinomycetes]
Date de mise en ligne : Mardi 16 septembre 2014 This study evaluated and compared the performance of two real-time PCR assays using nested reverse transcription (RT)-PCR as the reference method. Among 117 nested RT-PCR-positive cases, the abTES DEN 5 qPCR kit detected 97.4% of dengue virus (DENV) infections, while the innuDETECT Dengue TwoStep assay did so for 44.4%. Sensitivity varied by infecting serotype and the stage of infection. The abTES kit has the potential to replace nested RT-PCR for the rapid diagnosis of DENV infection.
Saengsawang, J., Nathalang, O., Kamonsil, M., Watanaveeradej, V.
Comparison of Two Commercial Real-Time PCR Assays for Detection of Dengue Virus in Patient Serum Samples [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 Four different rapid diagnostic tests (RDTs) for malaria were evaluated by testing 82 healthy control patients, 89 Plasmodium vivax-infected patients, and 92 rheumatoid factor (RF)-positive nonmalaria patients. The false-positive rate ranged from 2.2% to 13% in RF-positive patients. High RF levels are associated with malaria RDT false positivity.
Lee, J.-H., Jang, J. W., Cho, C. H., Kim, J. Y., Han, E. T., Yun, S. G., Lim, C. S.
False-Positive Results for Rapid Diagnostic Tests for Malaria in Patients with Rheumatoid Factor [Parasitology]
Date de mise en ligne : Mardi 16 septembre 2014 Fifteen bacterial isolates from spotted fever group rickettsiosis in Brazil were genetically identified as Rickettsia rickettsii. In a phylogenetic analysis with other R. rickettsii isolates from GenBank, the Central/South American isolates showed low polymorphism and formed a clade distinct from two North American clades, with the North American clades having greater in-branch polymorphism.
Labruna, M. B., Santos, F. C. P., Ogrzewalska, M., Nascimento, E. M. M., Colombo, S., Marcili, A., Angerami, R. N.
Genetic Identification of Rickettsial Isolates from Fatal Cases of Brazilian Spotted Fever and Comparison with Rickettsia rickettsii Isolates from the American Continents [Chlamydiology and Rickettsiology]
Date de mise en ligne : Mardi 16 septembre 2014 With the β-Lacta test, production of extended-spectrum β-lactamases (ESBLs) was assayed in 200 urine samples showing Gram-negative bacilli during direct microscopic examination. While 168 samples tested negative, all samples yielding ESBL-producing Enterobacteriaceae after culture gave positive (n = 30) or uninterpretable (n = 2) results. The sensitivity and specificity of ESBL detection were 94% and 100%, respectively.
Gallah, S., Decre, D., Genel, N., Arlet, G.
The {beta}-Lacta Test for Direct Detection of Extended-Spectrum-{beta}-Lactamase-Producing Enterobacteriaceae in Urine [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Mycobacterium tuberculosis isolates of the Manila sublineage are genetically homogeneous. In this study, we used whole-genome sequencing (WGS) to type a collection of 36 M. tuberculosis isolates of the Manila family. WGS enabled the subtyping of these 36 isolates into at least 10 distinct clusters. Our results indicate that WGS is a powerful approach to determining the relatedness of Manila family M. tuberculosis isolates.
Jamieson, F. B., Teatero, S., Guthrie, J. L., Neemuchwala, A., Fittipaldi, N., Mehaffy, C.
Whole-Genome Sequencing of the Mycobacterium tuberculosis Manila Sublineage Results in Less Clustering and Better Resolution than Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat (MIRU-VNTR) Typing and Spoligotyping [Mycobacteriology and Aerobic Actinomycetes]
Date de mise en ligne : Mardi 16 septembre 2014 We evaluated the Lyra Direct HSV 1+2/VZV multiplex real-time PCR assay for the detection and differentiation of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) on 695 consecutive cutaneous and mucocutaneous lesion specimens. The intra-assay and interassay coefficient of variation values for the Lyra assay were 0.29 to 1.30% and 2.33 to 2.61%, respectively. The sensitivities, specificities, and positive and negative predictive values were 93.4 to 95.0%, 96.1 to 96.8%, 78.0 to 80.3%, and 99.0 to 99.1%, respectively, in comparison to those of viral culture. The values were further improved when a resolution analysis was performed with a laboratory-developed PCR assay.
Fan, F., Stiles, J., Mikhlina, A., Lu, X., Babady, N. E., Tang, Y.-W.
Clinical Validation of the Lyra Direct HSV 1+2/VZV Assay for Simultaneous Detection and Differentiation of Three Herpesviruses in Cutaneous and Mucocutaneous Lesions [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 The commutability of international reference standards is critical for ensuring quantitative agreement across different viral load assays. Here, we demonstrate the commutability of the Epstein-Barr virus (EBV) WHO international standard for the BamHI-W and artus EBV assays.
Abeynayake, J., Johnson, R., Libiran, P., Sahoo, M. K., Cao, H., Bowen, R., Chan, K. C. A., Le, Q.-T., Pinsky, B. A.
Commutability of the Epstein-Barr Virus WHO International Standard across Two Quantitative PCR Methods [Virology]
Date de mise en ligne : Mardi 16 septembre 2014 The Verigene BC-GN assay correctly identified all 51 Gram-negative bacilli (GNB) from positive blood cultures and all 14 carbapenemase enzymes tested. The assay gave organism identification (ID) results an average of 24 h faster compared to conventional identifications. Medical management could have been modified for 31.8% of patients an average 33 h sooner. In conclusion, the BC-GN assay is a very accurate, rapid assay which would allow for more-immediate medical management decisions in patients with bacteremia from GNB.
Hill, J. T., Tran, K.-D. T., Barton, K. L., Labreche, M. J., Sharp, S. E.
Evaluation of the Nanosphere Verigene BC-GN Assay for Direct Identification of Gram-Negative Bacilli and Antibiotic Resistance Markers from Positive Blood Cultures and Potential Impact for More-Rapid Antibiotic Interventions [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 Self-collected vaginal Aptima swabs and flocked swabs in Aptima specimen transport medium and ESwabs in ESwab medium detected all 37 Chlamydia trachomatis-infected patients from 287 women tested by the Aptima Combo assay. Prevalence rates of C. trachomatis, Neisseria gonorrhoeae, and dual infection were 12.8%, 3.1%, and 2.4%, respectively.
Li, J., Jang, D., Gilchrist, J., Smieja, M., Ewert, R., MacRitchie, C., Chernesky, M.
Comparison of Flocked and Aptima Swabs and Two Specimen Transport Media in the Aptima Combo 2 Assay [Chlamydiology and Rickettsiology]
Date de mise en ligne : Mardi 16 septembre 2014 Colistin adherence to plastics can be diminished by adding a surfactant, i.e., polysorbate 80. Incorporating polysorbate 80 resulted in 3 twofold and 2 twofold modal MIC decreases for Enterobacteriaceae and Pseudomonas aeruginosa, respectively. The reproducibility of the quality controls (QCs) with and without polysorbate 80 supports the use of Escherichia coli strain 25922 and P. aeruginosa strain 27853.
Sutherland, C. A., Nicolau, D. P.
To Add or Not To Add Polysorbate 80: Impact on Colistin MICs for Clinical Strains of Enterobacteriaceae and Pseudomonas aeruginosa and Quality Controls [Bacteriology]
Date de mise en ligne : Mardi 16 septembre 2014 A case of fever, sepsis, and chest lesions evident on a computed tomography scan of an indigenous man in northern Australia following burns to the feet is described. Sputum PCR testing revealed Mycobacterium leprae, and a fine-needle aspirate of the chest lesions demonstrated Cryptococcus coinfection.
Edwards, L. J., Price, R. N., Krause, V. L., Huffam, S. E., Globan, M., Fyfe, J., Hajkowicz, K. M.
Detection of Mycobacterium leprae by PCR Testing of Sputa from a Patient with Pulmonary Cryptococcus Coinfection in Northern Australia [Case Reports]
Date de mise en ligne : Mardi 16 septembre 2014 We report here a rare case of chronic lumbar discitis caused by Clostridium perfringens in an elderly patient that was treated with a combination of β-lactams and clindamycin. Molecular analysis performed on the strain revealed an unusual toxin gene pattern.
Lotte, R., Popoff, M. R., Degand, N., Lotte, L., Bouvet, P., Baudin, G., Cua, E., Roger, P.-M., Ruimy, R.
Lumbar Discitis Caused by Clostridium perfringens [Case Reports]
Date de mise en ligne : Mardi 16 septembre 2014 A patient was colonized by IMP-4-producing Enterobacter cloacae and Escherichia coli strains for 7 months. IMP-4-producing E. cloacae strains were first and last isolated at day 33 and at 8 months after admission, respectively. IMP-4-producing E. coli strains were first and last isolated at days 88 and 181 after admission, respectively. The E. cloacae and E. coli isolates shared identical genetic features in terms of blaIMP-4, blaTEM-1, qnrB2, aacA4, HI2 plasmids, and ISCR1. This study shows the first prolonged colonization with in vivo interspecies transfer of blaIMP-4.
Sidjabat, H. E., Heney, C., George, N. M., Nimmo, G. R., Paterson, D. L.
Interspecies Transfer of blaIMP-4 in a Patient with Prolonged Colonization by IMP-4-Producing Enterobacteriaceae [Case Reports]
Date de mise en ligne : Mardi 16 septembre 2014 The toxigenic bacterium Vibrio cholerae belonging to the O1 and O139 serogroups is commonly associated with epidemic diarrhea in tropical settings; other diseases caused by this environmental pathogen are seldom identified. Here we report two unassociated cases of nonfatal, nontoxigenic V. cholerae non-O1, non-O139 bacteremia in patients with comorbidities in Ho Chi Minh City, Vietnam, that occurred within a 4-week period.
Lan, N. P. H., Nga, T. V. T., Yen, N. T. T., Dung, L. T., Tuyen, H. T., Campbell, J. I., Whitehorn, J., Thwaites, G., Chau, N. V. V., Baker, S.
Two Cases of Bacteriemia Caused by Nontoxigenic, Non-O1, Non-O139 Vibrio cholerae Isolates in Ho Chi Minh City, Vietnam [Case Reports]
Date de mise en ligne : Mardi 16 septembre 2014
La, M.-V., Jureen, R., Lin, R. T. P., Teo, J. W. P.
Unusual Detection of an Acinetobacter Class D Carbapenemase Gene, blaOXA-23, in a Clinical Escherichia coli Isolate [Letters To The Editor]
Date de mise en ligne : Mardi 16 septembre 2014
Read, T. D., Satola, S. W.
Using Genomics To Standardize Population Analysis Profile-Area under the Curve Ratio for Vancomycin-Intermediate Staphylococcus aureus [Letters To The Editor]
Date de mise en ligne : Mardi 16 septembre 2014
Wachino, J.-i., Kimura, K., Yamada, K., Jin, W., Arakawa, Y.
Evaluation of Disk Potentiation Test Using Kirby-Bauer Disks Containing High-Dosage Fosfomycin and Glucose-6-Phosphate To Detect Production of Glutathione S-Transferase Responsible for Fosfomycin Resistance [Letters To The Editor]
Date de mise en ligne : Mardi 16 septembre 2014
Sun, T., Liu, Y., Zhang, Y., Zhou, L.
Molecular Phylogeny of Coxsackievirus A16 [Letters To The Editor]
Date de mise en ligne : Mardi 16 septembre 2014
Barker, A. P., Simmon, K. E., Cohen, S., Schuetz, A., Slechta, E. S., Fisher, M. A., Schlaberg, R.
Correction for Barker et al., Isolation and Identification of Kroppenstedtia eburnea Isolates from Multiple Patient Samples [Author Correction]
Date de mise en ligne : Mardi 16 septembre 2014
Farfour, E., Badell, E., Jacques Natali, L., Dommergues, M. A., Blot, S., Guiso, N., Pangon, B., Foucaud, P.
Answer to October 2014 Photo Quiz [Photo Quiz]