Differential lipase virulence in Malassezia furfur dimorphism isolated from pityriasis versicolor patients and healthy individuals.

BACKGROUND: Malassezia furfur is a member of the human skin microbiomes that can cause various skin diseases. Dimorphism plays a role as the yeast phase predominates during skin colonisation whereas mycelial forms are observed in the scales of patients with pityriasis versicolor (PV). However, due to their condition-dependence for growth, it is difficult to culture M. furfur and this is an additional challenge for studying the pathogenicity of this fungus. OBJECTIVE: To describe different media suitable for culturing Malassezia from the yeast phase into mycelial forms, with a particular focus on nutritional supplements and pH conditions. METHODS: Clinical M. furfur isolates from patients with PV and healthy individuals were used to investigate Malassezia dimorphism as well as the activity and expression of lipase enzymes. RESULTS: Our experimental media were significantly more likely to promote mycelial growth in strains from healthy individuals compared to those from patients with PV. Lipase activity was increased in the mycelial phase cells compared to yeast forms for all strains tested. Assessment of the relative transcriptional expression of lipase within M. furfur revealed that LIP-coding genes were upregulated in mycelium relative to yeast forms for the strains tested. However, the increases in LIP3, LIP5 and LIP6 gene expressions were significantly greater in strains from healthy individuals compared to those from patients with PV. CONCLUSION: Overall, this study validated effective growth conditions to study M. furfur virulence factors and demonstrated that lipase is associated with M. furfur dimorphism.

Epidemiology and identification of potential fungal pathogens causing invasive fungal infections in a tertiary care hospital in northeast Thailand.

Invasive fungal infections (IFIs) are life threatening and associated with a high mortality rate. Here, we describe the distribution of pathogens, host risk factors, and significance of fungi isolated from patients with IFIs. The study included 861 fungal isolates recovered between 2006 and 2011 from 802 patients at Srinagarind Hospital, Thailand. Based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group 2008 criteria, 28.5% (245/861 isolates) of the fungal isolates were considered to be causative agents of IFIs. The most common fungus was Candida albicans (46%, 396/861 isolates). However, the most common yeast causing IFIs was Cryptococcus neoformans (34.7%, 85/245 isolates), while the most common mould was Penicillium marneffei (10.6%, 26/245 isolates). Cryptococcosis was significantly associated with human immunodeficiency virus infections (P < 0.001). Trend analysis revealed that there was no significant increase in IFI cases (P = 0.34) from 2006 to 2011 or from 2007 to 2011 (P = 0.05), but there was a trend toward significant increases in candidiasis (P = 0.04). The fungal isolates were categorized according to the positive predictive value of their recovery in cultures as being true (>95%), moderate (5%-95%), and rare (<5%) pathogens. This classification system could facilitate the prediction of the likelihood of diseases caused by the isolated fungi.

Evaluation of an immunochromatographic test kit for detecting Mycobacterium tuberculosis complex in sputum samples and on solid and in liquid cultures.

A rapid, cheap and effective method for diagnosing tuberculosis (TB) is essential for TB control. We evaluated the performance of an immunochromatographic assay (ICA) kit (SD Bioline TB Ag MPT64 rapid test) designed for detecting Mycobacterium tuberculosis complex (MTC) in liquid and solid cultures to determine its ability to detect and differentiate MTC directly in sputum samples. We attempted to optimize antigen extraction using several sputum solvents under various conditions. Adding the sputum solvent prior to using the ICA kit gave a sensitivity of 71.7% (43/60) for all acid-fast bacillus (AFB) stain positive specimens and a 100% specificity (20/20) among AFB negative specimens. Without sputum solvent, the ICA kit had 0% sensitivity for detecting MTC in sputum. We also evaluated the ICA test kit for its designed purpose of detecting MTC in 80 solid and liquid culture specimens positive for MTC using the niacin accumulation test or polymerase chain reaction (PCR) (16s-23s ITS). The ICA kit gave 100% sensitivity and specificity. We also evaluated the ICA test kit on 3 reference specimens of MTC, 15 nontuberculous mycobacteria (NTM) species, 7 bacterial species and 5 Candida albicans specimens. The tested ICA kit gave 100% specificity. The tested ICA kit was useful for detecting and differentiating MTC in solid and liquid cultures, but not useful for detecting MTC in sputum samples even treated with sputum solvent. The tested ICA kit should be used only for liquid and solid culture specimens. Therefore, the tested kit is inappropriate for use in evaluating sputum samples.

GENOTYPE AND DRUG RESISTANCE OF CLINICAL AND ENVIRONMENTAL VIBRIO CHOLERAE NON-O1/NON-O139 IN NORTHEASTERN THAILAND.

A total of 124 V cholerae non-O1/non-O139 isolates were collected in Khon Kaen, Thailand from diarrheal patients, asymptomatic carriers and environmental water. The presence of virulence-associated and regulatory genes including ctxA, tcpA, zot, ace, ompU, stn, hlyA and toxR) were examined using multiplex PCR. The genomic diversity of the various V. cholerae isolates were differentiated using the random amplified polymorphic DNA (RAPD) method. Antimicrobial susceptibility was tested using disk diffusion. All of V. cholerae non-O/non-O139 isolates carried hlyA and toxR and none carried ctxA and tcpA. The zot, ace and both genes together were found in 1.6%, 4.7% and 4.7% of 64 clinical V. cholerae non-O1 isolates, respectively, while the environmental ones did not. The stn gene was found in 3.1% (2/64) of the clinical and 3.3% (2/60) of the environmental isolates. The RAPD patterns were differentiated into 45 types (A to 2S). RAPD type A (32.3%) was the most frequently found in both clinical and environmental V cholerae non-O1 strains (34.4% and 30.0%, respectively); indicating that there was a clonal relationship between some clinical and environmental isolates whereas almost all of the environmental isolates belonged to different clones. All strains were sensitive to ciprofloxacin and norfloxacin. The environmental isolates (30%) were more resistant than the clinical ones (21.9%). Resistance to sulfamethoxazole/trimethoprim and tetracycline among the clinical isolates occurred in 9.4% (6/64) in 2007, during which period the prevalence of V cholerae O1 increased. We conclude that V. cholerae non-O1/non-O139 from the aquatic environment are potentially pathogenic and this same aquatic environment may be a source of antimicrobial resistance in V. cholerae.

Geographically structured populations of Cryptococcus neoformans Variety grubii in Asia correlate with HIV status and show a clonal population structure.

Cryptococcosis is an important fungal disease in Asia with an estimated 140,000 new infections annually the majority of which occurs in patients suffering from HIV/AIDS. Cryptococcus neoformans variety grubii (serotype A) is the major causative agent of this disease. In the present study, multilocus sequence typing (MLST) using the ISHAM MLST consensus scheme for the C. neoformans/C. gattii species complex was used to analyse nucleotide polymorphisms among 476 isolates of this pathogen obtained from 8 Asian countries. Population genetic analysis showed that the Asian C. neoformans var. grubii population shows limited genetic diversity and demonstrates a largely clonal mode of reproduction when compared with the global MLST dataset. HIV-status, sequence types and geography were found to be confounded. However, a correlation between sequence types and isolates from HIV-negative patients was observed among the Asian isolates. Observations of high gene flow between the Middle Eastern and the Southeastern Asian populations suggest that immigrant workers in the Middle East were originally infected in Southeastern Asia.

PCR-RFLP and antimicrobial susceptibility profiles of Helicobacter pylori isolated from antrum and corpus of dyspeptic patients in Thailand.

The study determined the genetic heterogeneity of Helicobacter pylori isolates from antrum and corpus of the same dyspeptic patients in a Thai population and determined the relationship between the antimicrobial susceptibility (AS) profile (antibiogram) and PCR-restriction fragment length polymorphism (PCR-RFLP) pattern. One hundred and nineteen H. pylori isolates comprising 7 single and 56 paired antrum and corpus isolates obtained by gastric biopsy from 160 dyspeptic patients were analyzed. For PCR-RFLP, the 820 bp amplicon of ureC was digested with Sau3AI and HhaI, which revealed 16 (A-Q) and 19 (a- s) different PCR-RFLP patterns after Sau3AI and HhaI digestion, respectively. Combination of the restriction enzyme digestion patterns resulted in 35 distinct RFLP types. Among the 56 paired isolates, 47 were infected with H. pylori having the same AS and PCR-RFLP profiles, 7 with different AS profiles but the same PCR-RFLP profiles and 2 with different PCR-RFLP profiles but the same AS profiles. No patient was infected with H. pylori different in both PCR-RFLP and AS profiles. The results indicate that the majority of the paired H. pylori isolates displayed identical AS profile and PCR-RFLP patterns suggesting that most patients were infected with a single strain. Some patients could have been infected with single strains that were different in the AS profiles.

Resistance of Asian Cryptococcus neoformans serotype A is confined to few microsatellite genotypes.

BACKGROUND: Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries. METHODOLOGY/PRINCIPAL FINDINGS: Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17. CONCLUSIONS: The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine.

Nested polymerase chain reaction for detection of Helicobacter pylori in gastric biopsy specimens.

Sensitivity and specificity are important for tests used to defect Helicobacter pylori infection from gastric biopsy specimens. Molecular methods, such as PCR and nested PCR, are sensitive methods for H. pylori detection. The objective of this study was to evaluate the performance of PCR and nested PCR compared to culture, the rapid urease test (RUT) and histology for the diagnosis of H. pylori in 130 gastric biopsy specimens from symptomatic dyspeptic patients. Sensitivity and specificity with PCR were 91 and 100% and with nested PCR were 95 and 97%, respectively. H. pylori was detected by PCR and nested PCR at levels as low as 125 fg (70 cells) and 25 fg (14 cells), respectively. These results suggest nested PCR is a highly sensitive direct method to detect H. pylori infection from biopsy specimens.

Antimicrobial susceptibility of Helicobacter pylori isolated from gastric biopsies in dyspeptic patients.

The objective of this study was to evaluate the prevalence of antimicrobial resistance in Helicobacter pylori isolated from the antrum and corpus of dyspeptic patients in Khon Kaen, Thailand, and to compare the antimicrobial susceptibility patterns of H. pylori isolated from the antrum and corpus in individual patients. Antimicrobial susceptibility was determined by disk diffusion, studying susceptibility to metronidazole, clarithromycin, amoxicillin, erythromycin, ciprofloxacin, and tetracycline. The H. pylori resistant rate to at least one of the six antimicrobial agents tested was 37%. The resistance rates were 30.2% for metronidazole, 9.2% for ciprofloxacin, 5% for clarithromycin, 2.4% for amoxicillin, and 1.7% for erythromycin and tetracycline. Single, double, and more than double antimicrobial resistances were found in 27.7, 6.7 and 2.5%, respectively. Antimicrobial susceptibility testing revealed 11 antibiotypes. The most common antimicrobial susceptibility pattern found was sensitivity to 6 antimicrobial agents (63%). H. pylori antimicrobial resistance in specimens isolated from the antrum and corpus were nearly equivalent, 37.3% (22/59) and 36.7% (22/60), respectively. Most of the H. pylori specimens isolated from the antrum and corpus in individual patients were identical (87.7%).

Association of ompU gene in Vibrio cholerae from patients and environment with bile resistance.

The objective of this study was to determine whether Vibrio cholerae, possessing ompU isolated from patients and the environment, conferred bile resistance and whether other virulence genes were also related to bile resistance. Fifty-two V cholerae O1 and non-O1 isolates were examined by PCR for the presence of the virulence-associated and regulatory genes, ctxA, tcpA, zot, ace, ompU, toxR, hlyA and stn/sto. V. cholerae possessing ompU resistant to equal or greater than 10% sodium deoxycholate were found in 93% of isolates but only in 9% of V. cholerae isolates not possessing ompU. The effects of other virulence genes on bile resistance could not be ascertained in this study. Thus V cholerae non-O1 with ompU and possibly other virulence genes isolated from the environment have the potential of affecting public health.

Prevalence of Helicobacter pylori vacA, cagA, cagE, iceA and babA2 genotypes in Thai dyspeptic patients.

OBJECTIVES: To investigate the prevalence of the vacA, cagA, cagE, iceA, and babA2 genotypes in Helicobacter pylori strains isolated from Thai dyspeptic patients, and to determine whether any correlation exists between these genotypes and clinical manifestations. METHODS: Helicobacter pylori was examined in 112 patients (62 with non-ulcer dyspepsia (gastritis), 34 with peptic ulcer disease, and 16 with gastric cancer (GCA)), detected by culture or direct detection from gastric biopsies. Allelic variants of the vacA, cagA, cagE, iceA, and babA2 genotypes were identified by using the polymerase chain reaction. RESULTS: The positive rates for the vacAs1, vacAs2, cagA, cagE, iceA1, iceA2, and babA2 genes in H. pylori of dyspeptic patients were 100%, 0%, 98.2%, 88.4%, 45.5%, 33.1%, and 92%, respectively. The allelic variant vacAs1m1 was more prevalent (58%) than vacAs1m2 (42%). The cagA and cagE genes were commonly found together (87.5%). The most predominant genotypes were vacAs1m1, cagA, cagE, iceA1, and babA2. The various genes alone or in combination had no statistically significant association with the clinical outcomes (p>0.05). CONCLUSION: Neither single gene nor combination of vacA, cagA, cagE, iceA, and babA2 genes was significantly helpful in predicting the clinical outcome of H. pylori infection in Thai patients. The high prevalence of these genes in H. pylori isolated from Thai patient groups suggests that H. pylori strains are geographically dependent.

Enteropathogenic bacteria and enterotoxin-producing Staphylococcus aureus isolated from ready-to-eat foods in Khon Kaen, Thailand.

The objective of this study was to investigate the microbiological quality of ready-to-eat food in the Municipality of Khon Kaen, Thailand. Four categories of 186 food samples were collected: (1) high heat food; (2) low heat food; (3) no heat food; and, 4) on-site prepared fruit juices and beverages. Of the food samples, 145 (78%) failed to meet acceptable microbiological standards, including fruit juice and beverages (100%), no heat food (91.7%), low heat food (81.7%) and high heat food (57.9%). The most frequent bacterial indexes indicating unacceptability were the most probable number (MPN) of coliforms (78%), the bacterial colony count (58%), and the MPN of E. coli (46%). Pathogenic bacteria were found in 6.5% of food samples. Salmonella, Vibrio cholerae non O1 and Aeromonas hydrophila were found in 4.3, 1.6 and 0.5% of the total food samples, respectively. The serovars of Salmonella found in food were S. Derby, S. Give, S. Krefield, S. Paratyphi B, S. Verchow, S. Lexington and S. Senftenberg. Staphylococcus aureus concentrations of >10(2) CFU/g and >10(5) CFU/g were found in 10.8% and 1.1% of the food samples. Enterotoxin types AB and A of S. aureus were found in 2.7% of the food samples. These results indicate that more than half of the ready-to-eat foods tested in Khon Kaen municipality did not meet microbiological national standards and many kinds of enteropathogenic bacteria were found, suggesting food stalls may be a source of foodborne disease.

Characterization of diarrheagenic Escherichia coli isolated from food in Khon Kaen, Thailand.

Four categories of 186 ready-to-eat food samples in Khon Kaen municipality, Thailand, were collected and investigated for fecal contamination by enumeration of Escherichia coli using the most probable number (MPN) method. Then, the E. coli isolates were presumptively identified as diarrheagenic E. coil by agglutinating with polyvalent O-antisera and monovalent O-antisera commonly found in diarrheagenic strains and were subsequently investigated for the presence of the recognized virulence genes for enteroaggregative (EAEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), and shiga toxin-producing E. coli (STEC or EHEC) by multiplex PCR assays. All E, coli isolates were examined for antimicrobial susceptibilities by the agar disc diffusion method, and the results were compared with those obtained from clinical samples. The percentage of each type of food with E. coli, including no heat food, low heat food, high heat food, and fruit juices and beverages, was higher than accepted standards at 60.4, 46.5, 38.6 and 20%, respectively. Of 140 E. coli isolates obtained from food samples, 11 isolates (7.9%) agglutinated with 6 monovalent O-antisera, including one isolate each of O6, O8, O114 and O159, two isolates of O1, and five isolates of O157. None of the 11 isolates harbored the virulence genes for EPEC, ETEC, EAEC, EIEC and STEC. Although O157 E. coli isolates were found, the most frequent, E. coli O157:H7, was not found in this study. The astA gene, however, was found in 1 E. coli isolate that showed weakly positive agglutination against the polyvalent antisera. Approximately 50% of the 140 E. coli isolates were resistance to at least one antimicrobial agent. The resistant strains showed high resistance to tetracycline (43%), co-trimoxazole (36%), ampicillin (26%) and chloramphenicol (23%), respectively. The resistance of E. coli was high for nearly all antimicrobial agents, particularly ampicillin (76%), tetracycline (70%), co-trimoxazole (69%) and nalidixic acid (44%). The results show that nearly half of the ready-to-eat food samples evaluated in Khon Kaen Municipality had levels of E. coli higher than acceptable standards. Of the diarrheagenic E. coli classified by serogroup, almost none of the isolates had virulence genes. These results indicate the disadvantage of relying on serogrouping alone for the recognition of diarrheagenic E. coli. E. coli isolated from food may not be an enteropathogenic strain. We also found that E. coli antimicrobial resistant strains are widespread in both food and humans.

Analysis of three phenotyping methods and pulsed-field gel electrophoresis for differentiation of methicillin resistant Staphylococcus aureus isolated from two hospitals in Thailand.

Methicillin resistant Staphylococcus aureus (MRSA) is an important hospital and community-acquired pathogen. Rapid and reliable epidemiologic typing is necessary for controlling the spread of MRSA outbreak. The objective of this study was to compare the phenotyping with the genotyping method to differentiate MRSA isolates obtained from the two hospitals in Thailand (central and northeastern). Seventy-four MRSA isolates were randomly collected and confirmed by the presence of mecA gene. Antibiogram, phage typing and enterotoxin production were used for the phenotyping analysis. Pulsed-field gel electrophoresis (PFGE) with Smal digestion of chromosomal DNA was used for the genotyping analysis. We found 17 distinct profiles by the 3 phenotypic typing methods and 18 PFGE types designated as 5 major types (A-E) and 13 subtypes. The most frequent PFGE types and their related subtypes found in both hospitals were A and C, comprising 54 and 27%, respectively. The antibiogram could differentiate 6 different types. All isolates were resistant to the majority of antimicrobial agents tested, but were susceptible to vancomycin and fosfomycin. Ten (13.5%) MRSA isolates produced enterotoxin A. Nontypable phage and phage type 77 were found predominantly in MRSA isolated from the northeast and central hospital, respectively. A significant correlation was found between the phenotyping and the genotyping methods and there was a good correlation between antibiogram and PFGE. Antibiogram typing alone can be used as a useful epidemiological marker for practical purposes. PFGE types A and C were the common endemic MRSA clones in both hospitals in Thailand.

Evaluation of polymerase chain reaction, conventional and MRSA screen latex agglutination methods for detection of methicillin-resistant, -borderline and -susceptible Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), is difficult and expensive to treat, therefore early screening is essential. Several phenotypic and genotypic methods are used to detect MRSA; however, the method of choice remains problematic. We have evaluated four phenotypic methods, broth microdilution (MIC), oxacillin disk agar diffusion (ODD), oxacillin screening salt agar (OSS), and a new rapid phenotypic (MRSA screen latex agglutination, MSLA) with the genotypic gold standard of PCR mecA detection to determine the most appropriate method for routine laboratory use. We randomly collected 203 S. aureus isolates from patients and carriers at two hospitals in Thailand. Using MIC method, three sub-groups were differentiated from among these isolates, namely MRSA (106 isolates), borderline-resistant S. aureus (BRSA) (65 isolates), and methicillin-susceptible S. aureus (MSSA)(32 isolates). A total of 10 methicillin-resistant S. epidermidis (MRSE) isolates were also included. The sensitivity and specificity of MIC, ODD, OSS, and MSLA were 99 and 96, 100 and 97, 100 and 97, and 100 and 100%, respectively. Our study indicated that ODD is still appropriate for routine laboratory. MSLA had the highest sensitivity and specificity and is rapid but expensive, so is the most appropriate method for emergency cases. MIC method was better for BRSA detection and OSS method was more appropriate for screening clinical specimens and carriers.

Evaluation of different primers for detecting mecA gene by PCR in comparison with phenotypic methods for discrimination of methicillin-resistant Staphylococcus aureus.

Detection of the mecA gene by polymerase chain reaction (PCR) is the gold standard for identifying methicillin-resistant Staphylococcus aureus (MRSA). PCR assays, employing MR1-MR2 primers (primer set 1) and MR3-MR4 primers (primer set 2) to generate 154 and 533 bp fragment, respectively, are most widely used for amplification of mecA gene. The purpose of this study was to evaluate the presence of mecA gene in 100 clinical isolates of S. aureus using PCR with the two pairs of primers. The results were compared to the broth dilution MIC method, oxacillin salt screening method (OSS) and oxacillin disk agar diffusion method (ODD). Fifteen of the 100 isolates showed a discrepancy between the mecA primer sets 1 and 2. Three isolates (3%) without the mecA gene showed discrepancies with phenotypic methods. The sensitivity, specificity and positive and negative predictive values for the 154 and 533 bp products of mecA were 79, 85, 83, 81 and 94, 100, 100, 94%, respectively. The results indicated that primer set 2 was more appropriate than primer set 1 for the detection of mecA gene in MRSA. There was a good correlation among the mecA gene detection, ODD and OSS methods. The discrepancy of three isolates between PCR and phenotypic methods should be clarified for other resistant mechanisms.