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Journal: Diagnostic microbiology and infectious disease

166

Blastomyces dermatitides is increasingly found in Canada along the St. Lawrence River. However, there are only rare reports of this disease in New England. We describe a case of disseminated blastomycosis in a patient from northern Vermont who acquired the fungus while gardening.

Concepts: United States, Canada, Quebec, Appalachian Mountains, Saint Lawrence River, Wisconsin, Lake Champlain, Samuel de Champlain

154

Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level.

Concepts: DNA, Gene expression, Evolution, Species, Cellular differentiation, Brucellosis, Brucella melitensis, Brucella

28

In the present study, the antifungal effects of phenylmercuric nitrate and benzalkonium chloride versus those of natamycin and ketoconazole were assessed against 216 filamentous fungi isolates from cases of fungal keratitis. They included 112 Fusarium isolates, 94 Aspergillus isolates, and 10 Alternaria alternata isolates. The strains were tested by broth dilution antifungal susceptibility testing of filamentous fungi approved by the Clinical and Laboratory Standards Institute M38-A document. The results showed that the MIC(50) values of phenylmercuric nitrate were 0.0156, 0.0156, and 0.0313 μg/mL for Fusarium spp., Aspergillus spp., and A. alternata, respectively. The MIC(90) values of phenylmercuric nitrate were 0.0313, 0.0313, and 0.0313 μg/mL for Fusarium spp., Aspergillus spp., and A. alternata, respectively. The MIC(50) values of benzalkonium chloride were 16, 32, and 8 μg/mL for Fusarium spp., Aspergillus spp., and A. alternata, respectively. The MIC(90) values of benzalkonium chloride were 32, 32, and 16 μg/mL for Fusarium spp., Aspergillus spp., and A. alternata, respectively. The study indicates that phenylmercuric nitrate has considerable antifungal activity and its effect is significantly superior to those of benzalkonium chloride, natamycin, and ketoconazole against ocular pathogenic filamentous fungi in vitro, deserving further investigation for treating fungal keratitis as a main drug.

Concepts: Fungus, Mycotoxin, Aspergillus, Ascomycota, Aspergillus oryzae, Fungi, Alternaria alternata, Chlorides

28

We evaluated the limits of detection of 3 rapid influenza diagnostic tests-BD Veritor™ System for Flu A+B, Binax NOW® Influenza A+B, and QuickVue® Influenza-for influenza strains circulating in 2010-2012. Limits of detection varied by influenza strain, with Veritor™ Flu A+B test showing the lowest limit of detection for all strains.

Concepts: Virus, Antiviral drug, Influenza, Avian influenza, Strain, Orthomyxoviridae, Influenzavirus B, Reassortment

28

A better understanding of virulence gene profiling and molecular characterization of Staphylococcus aureus isolates associated with bloodstream infection (BSI) may provide further insights related to clinical outcomes with these infections. We analyzed 89 S. aureus isolates including 37 MRSA isolates (41.6%) recovered from 89 adult patients with BSI from 4 hospitals in Zhejiang province, eastern China. Thirty-five (94.6%) of MRSA isolates and 4 (7.7%) of methicillin-sensitive S. aureus (MSSA) isolates were resistant to multiple antimicrobials. All isolates harbored at least 2 of 22 possible virulence genes, including sdrC (92.1%), icaA (89.9%), hla (80.9%), clf (69.7%), sea (68.5%), sdrD (67.4%), hlb (67.4%), sdrE (65.2%), sei (51.7%), seg (50.6%), and cna (50.6%). Forty-four (49.4%) of all S. aureus BSI isolates, including 23 (62.2%) of MRSA isolates, harbored ≥10 of the virulence genes evaluated in this study. Sixteen (43.2%) MRSA isolates and 5 (9.6%) MSSA isolates harbored the gene encoding Panton-Valentine leukocidin (PVL). Collective genes for pvl, sdrE, sed, seg, and sei among MRSA isolates were significantly more frequent relative to MSSA isolates (P < 0.05). A total of 22 sequence types (STs), including novel ST2184, ST2199, and ST2200, and 33 spa types, including novel spa types t9530 and t9532, were identified among S. aureus BSI isolates, among which ST188 (15.7%) and ST7 (15.7%), and t091 (12.4%) and t189 (12.4%), seldom noted for Chinese isolates previously, were major STs and spa types, respectively. In contrast to previous reports, no predominant clones were found in the present study. Among the MRSA isolates, although ST239-MRSA-SCCmecIII, predominant clone in China, still represented the most common clone, it only accounted for 18.9%. However, ST188-MRSA- SCCmecIV seldom reported before accounted for 10.8%. Among the MSSA isolates, ST7-MSSA represented the most common clone (23.1%), followed by ST188-MSSA and ST630-MSSA (9.6% each). In conclusion, simultaneous carriage of multiple virulence genes and genetically considerable diversity were common among S. aureus BSI isolates. Furthermore, MRSA isolates exhibited more frequent carriage of superantigen genes and pvl relative to MSSA isolates. Taken together, there are distinctive virulence gene profiling and molecular characteristic among S. aureus isolates associated with bloodstream infection in China.

Concepts: Pneumonia, Staphylococcus aureus, Infection, Antibiotic resistance, Staphylococcus, Methicillin-resistant Staphylococcus aureus, Vancomycin, Panton-Valentine leukocidin

28

We have evaluated 696 samples (488 swabs and 208 urine specimens) with the cobas 4800 (c4800) CT/NG Test for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA in swab and urine specimens. c4800 results were compared with those obtained from COBAS AMPLICOR (CAM) CT/NG Test. Discordant results were reanalyzed with the MultiNA system and compared with clinical data. For C. trachomatis detection by both methods, we obtained 93.8%, 100%, 100%, and 99.1% for sensitivity, specificity, and positive and negative predictive values, respectively. For urine specimens analyzed in c4800, our results were 96.6%, 100%, 100%, and 99.4%, respectively. For N. gonorrhoeae detection, swab results were:88.0%, 100%, 100%, and 99.4%. For urine specimen, results obtained were 100%, 100%, 100%, and 100%. Reanalyses were all concordant between both methods. c4800 results were comparable with those obtained with the CAM system. We had an excellent correlation between swab and urine specimens analyzed by c4800.

Concepts: Neisseria gonorrhoeae, Chlamydia trachomatis

28

We quantified vaginal lactobacilli and determined their relationship with genital HIV-1 shedding and found a significant negative association between reduced quantity of lactobacilli and cervical HIV-1 viral load (r(2) = - 0.8900, P < 0.01), which may have implications of increased chances of sexual transmission of HIV-1 and genital infections.

Concepts: HIV, HIV/AIDS, Viral load, Sexual intercourse, Bacteria, Female reproductive system, Cytomegalovirus, Clitoris

27

We describe a real-time PCR-based assay capable of simultaneously detecting femA (Staphylococcus aureus-specific), mecA (methicillin resistance), qacA/B (chlorhexidine tolerance), and mupA (high-level mupirocin resistance) from bacterial cells in less than 90 minutes. The assay was validated with 1968 clinical MRSA submitted to a surveillance network.

Concepts: Bacteria, Pneumonia, Staphylococcus aureus, Antibiotic resistance, Methicillin-resistant Staphylococcus aureus, Penicillin, Staphylococcaceae, Mupirocin

27

Even though community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) was described a decade ago, reports from Brazil are scarce and cases occurred in large urban centers. We report MRSA sepsis in a 16-year-old male from a small town and who had no history of exposure to healthcare or recent travel. After trauma during a soccer match, he presented swelling in the right thigh, which evolved in a month to cellulitis complicated by local abscess, orchitis and pneumonia. The patient presented severe sepsis, with fever and respiratory failure. Laboratory findings included blood leukocyte counts above 40,000/mm(3) and thrombocytopenia. He was submitted to mechanical ventilation and therapy with vancomycin and imipenem. He had a slow but favorable response to therapy and was discharged after six weeks of hospitalization. MRSA grew from blood cultures and respiratory aspirates obtained before antimicrobial therapy. The isolate belonged to sequence type 5, spa type t311, harbored SCCmec type IV and genes for Panton-Valentine leukocidin and Enterotoxin A. The pulsed-field gel electrophoresis pattern was distinct from North American classic CA-MRSA clones. However, the sequence type and the spa type revealed that the clone belong to the same clonal complex isolated in Argentina. This is the first CA-MRSA infection reported in that region, with significant epidemiologic and clinical implications.

Concepts: Inflammation, Pneumonia, Staphylococcus aureus, Antibiotic resistance, Methicillin-resistant Staphylococcus aureus, Vancomycin, Panton-Valentine leukocidin, Sepsis

26

A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10(5) CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.

Concepts: DNA, Gene expression, Polymerase chain reaction, Molecular biology, 16S ribosomal RNA, Vibrio cholerae, Vibrio, Vibrionales