Investigation of fluconazole susceptibility to Candida albicans by MALDI-TOF MS and real-time PCR for CDR1, CDR2, MDR1 and ERG11.

BACKGROUND: C. albicans is a pathogenic yeast that is the most common cause of fungal infections in humans. Unfortunately, the yeast's resistance to the antifungal medication fluconazole (FLC) is increasing; furthermore, testing its susceptibility to FLC by conventional methods takes time, resulting in treatment failure. The susceptibility of C. albicans to FLC was investigated using MALDI-TOF Mass Spectrometry and Real-time PCR tests for CDR1, CDR2, MDR1 and ERG11. Overall, 32 C. albicans strains made up of four reference strains (three FLC susceptible [S] and one FLC resistant [R], one spontaneous mutant strain [FLC susceptible-dose-dependent (SDD)] and 27 clinical strains obtained from two Thai University Hospitals) were tested for susceptibility to FLC. The following tests were performed: SensititreYeastOne and broth microdilution method, FLC resistant expression mechanism by Real-time PCR, and the major peak determination by MALDI-TOF MS. RESULTS: The change of CDR1 and CDR2 mRNA expression was only significantly observed in SDD and R strains. MALDI-TOF MS was performed after incubation for six hours; the change of mass spectral intensity at range 3376-3382 m/z (major peak) was significantly related to FLC susceptibility as SDD (decreased at 4 µg/mL and increased at 8 µg/mL), S (all increased), and R (all slightly decreased or no change). All 27 clinical strains showed FLC minimum inhibitory concentrations (MIC range 0.25-2 µg/mL), no change in CDR1 and CDR2 expression and S major peak type. The FLC resistant C. albicans with CDR1and CDR2 expression may possibly affect the change of mass spectral intensity at range 3376-3382 m/z. CONCLUSIONS: The MALDI-TOF MS may be used to simultaneously classify and predict FLC resistant C. albicans strains associated with CDR1 and CDR2 expression. Further studies are essential to clarify the methodology and improve the reliability of this assay for routine diagnosis.

Characterization of katG, inhA, rpoB and pncA in Mycobacterium tuberculosis isolates from MDR-TB risk patients in Thailand.

INTRODUCTION: Multidrug-resistant tuberculosis (MDR-TB) is commonly found in Thailand especially in the public health region 5, the Western region of Thailand. This study's aim was to characterize katG, inhA, rpoB and pncA genes in Mycobacterium tuberculosis. METHODOLOGY: One hundred strains of Mycobacterium tuberculosis (MTB) were isolated from sputum samples of MDR-TB risk patients in the laboratory of the Office of Disease Prevention and Control 5th Ratchaburi province, Thailand from January to December 2015. Drug susceptibility testing (DST) was performed using a BACTEC MGIT 960 system. Furthermore, the genes katG, inhA, rpoB and pncA were characterized by DNA sequencing. RESULTS: Of a total of 100 MTB samples which underwent drug susceptibility testing, 42% showed isoniazid (INH) and rifampicin (RIF) resistance, and a further 25% showed INH mono-resistance (25%). The most common gene mutations found using DNA sequencing were katG_Ser315Thr (70%), rpoB_Ser531leu (81%) and pncA_Ile31Thr (84%). The common mutation of pncA_Ile31Thr substitution was detected in 26 of 91 (29%) pyrazinamide (PZA) susceptible isolates. CONCLUSION: Using DNA sequencing to screen for gene mutations conferring drug resistance may be feasible and use less time than using DST to detect resistance patterns.

Prevalence of Providencia Strains among Patients with Diarrhea and in Retail Meats in Thailand.

In the present study, we examined the prevalence of Providencia spp. in retail meats and in stool samples from diarrheal patients in Thailand. Providencia-specific (Psp)-PCR amplification was positive for 58% (15/26) of the chicken, 64% (16/25) of the pork, and 68% (17/25) of the beef samples. Subsequently, Providencia strains were isolated from 65% (17/26) of the chicken, 68% (17/25) of the pork, and 72% (18/25) of the beef samples. Although P. alcalifaciens (40-58%) was the most prevalent Providencia strain, P. rettgeri, P. stuartii, and P. rustigianii were also isolated. Of the patient stool samples tested, 7.5% (16/214) had Psp-PCR products, of which 4 were P. alcalifaciens (1.9%), 4 were P. rettgeri (1.9%), and 1 was P. stuartii (0.5%). In conclusion, retail meats were substantially contaminated with Providencia, especially P. alcalifaciens. Therefore, retail meats are possibly the source of Providencia infections in humans.

Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea

Thirty one out of 153 strains of Shigella sonnei isolated from Thai patients with diarrhoea showed antibacterial activity against S. sonnei by agar well diffusion method. All of them harbor plasmids with the genetic determination of colicin type 7 (Js) gene but without colicin E and colicin U gene. The PCR product obtained from strain 35/44 was shown to be the gene for colicin type 7 lytic protein (cja). The partially purified bacteriocin (PPB) containing colicin type 7 of strain 35/44 was prepared and used for characterization. The antibacterial activity of PPB against a total of 17 selected Gram-positive and Gram-negative bacteria was tested. It was found that PPB of strain 35/44 was active against E. coli O157, S. sonnei and S. boydii. The sensitivity of PPB from this strain to proteinase K, trypsin and α-chymotrypsin suggests the proteinaceous nature of these antimicrobial substances. Therefore, this isolated bacterium can be regarded as bacteriocin producing bacteria. The bacteriocin produced by this isolated S. sonnei was heat stable as evidenced by its ability to maintain the activity at 80 °C for 60 min. In addition, it was stable within a wide range of pH (3-9). The molecular weight of colicin type 7 from isolated S. sonnei strain 35/44 analyzed by SDS-PAGE was 54.4 kDa composing of at least five subunits. It is to our knowledge; the first report of Thai patients with diarrhoea that S. sonnei isolated from them contained colicin type 7.

Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea.

Thirty one out of 153 strains of Shigella sonnei isolated from Thai patients with diarrhoea showed antibacterial activity against S. sonnei by agar well diffusion method. All of them harbor plasmids with the genetic determination of colicin type 7 (Js) gene but without colicin E and colicin U gene. The PCR product obtained from strain 35/44 was shown to be the gene for colicin type 7 lytic protein (cja). The partially purified bacteriocin (PPB) containing colicin type 7 of strain 35/44 was prepared and used for characterization. The antibacterial activity of PPB against a total of 17 selected Gram-positive and Gram-negative bacteria was tested. It was found that PPB of strain 35/44 was active against E. coli O157, S. sonnei and S. boydii. The sensitivity of PPB from this strain to proteinase K, trypsin and α-chymotrypsin suggests the proteinaceous nature of these antimicrobial substances. Therefore, this isolated bacterium can be regarded as bacteriocin producing bacteria. The bacteriocin produced by this isolated S. sonnei was heat stable as evidenced by its ability to maintain the activity at 80 °C for 60 min. In addition, it was stable within a wide range of pH (3-9). The molecular weight of colicin type 7 from isolated S. sonnei strain 35/44 analyzed by SDS-PAGE was 54.4 kDa composing of at least five subunits. It is to our knowledge; the first report of Thai patients with diarrhoea that S. sonnei isolated from them contained colicin type 7.

A cost function analysis of shigellosis in Thailand.

OBJECTIVE: The purpose of this study was to develop a cost function model to estimate the public treatment cost of shigellosis patients in Thailand. METHODS: This study is an incidence-based cost-of-illness analysis from a provider's perspective. The sample cases in this study were shigellosis patients residing in Kaengkhoi District, Saraburi Province, Thailand. All diarrhea patients who came to the health-care centers in Kaengkhoi District, Kaengkhoi District Hospital and Saraburi Regional Hospital during the period covering May 2002 to April 2003 were tested for Shigella spp. The sample for our study included all patients with culture that confirmed the presence of shigellosis. Public treatment cost was defined as the costs incurred by the health-care service facilities arising from individual cases. The cost was calculated based on the number of services that were utilized (clinic visits, hospitalization, pharmaceuticals, and laboratory investigations), as well as the unit cost of the services (material, labor and capital costs). The data were summarized using descriptive statistics. Furthermore, the stepwise multiple regressions were employed to create a cost function, and the uncertainty was tested by a one-way sensitivity analysis of varying discount rate, cost category, and drug prices. RESULTS: Cost estimates were based from 137 episodes of 130 patients. Ninety-four percent of them received treatment as outpatients. One-fifth of the episodes were children aged less than 5 years old. The average public treatment cost was US$8.65 per episode based on 2006 prices (95% CI, 4.79, and 12.51) (approximately US$1 = 38.084 Thai baht). The majority of the treatment cost (59.3%) was consumed by the hospitalized patients, though they only accounted for 5.8% of all episodes. The sensitivity analysis on the component of costs and drug prices showed a variation in the public treatment cost ranging from US$8.29 to US$9.38 (-4.20% and 8.43% of the base-case, respectively). The public treatment cost model has an adjusted R(2) of 0.788. The positive predictor variables were types of services (inpatient and outpatient), types of health-care facilities (health center, district hospital, regional hospital), and insurance schemes (civil servants medical benefit scheme, social security scheme and universal health coverage scheme). Treatment cost was estimated for various scenarios based on the fitted cost model. CONCLUSION: The average public treatment cost of shigellosis in Thailand was estimated in this study. Service types, health-care facilities, and insurance schemes were the predictors used to predict nearly 80% of the cost. The estimated cost based on the fitted model can be employed for hospital management and health-care planning.

Estimating the burden of shigellosis in Thailand: 36-month population-based surveillance study.

OBJECTIVE: To estimate incidence of shigellosis in the Kaengkhoi district, Saraburi Province, Thailand. METHODS: Population-based surveillance of shigellosis based in treatment centres. The detected rates of treated shigellosis were corrected for the number of cases missed due to the low sensitivity of microbiological culture methods and participants' use of health-care providers not participating in the study. FINDINGS: The overall uncorrected incidence of shigellosis was 0.6/1000 population per year (95% confidence interval (CI) = 0.5-0.8). The unadjusted incidence of treated shigellosis was highest among children less than 5 years old (4/1000 children per year; 95% CI = 3-6) and significantly lower among people aged > 5 years (0.3/1000 population per year; 95% CI = 0.2-0.5; P < 0.001). Adjusting for cases likely to be missed as a result of culture and surveillance methods increased estimates approximately five times. The majority of Shigella isolates (122/146; 84%) were S. sonnei; the rest were S. flexneri. Of the 22 S. flexneri isolates, the three most frequently encountered serotypes were 2a (36%), 1b (23%) and 3b (28%). A total of 90-95% of S. sonnei and S. flexneri isolates were resistant to tetracycline and co-trimoxazole. In contrast to S. sonnei isolates, more than 90% of the S. flexneri isolates were also resistant to ampicillin and chloramphenicol (P < 0.0001). CONCLUSION: Estimates of incidence of Shigella infection in the community are 10-fold to 100-fold greater than those found from routine government surveillance. The high prevalence of Shigella strains resistant to multiple antibiotics adds urgency to the development of a vaccine to protect against shigellosis in this region of Thailand.

Preferences for treatment of diarrhoea and dysentery in Kaengkhoi district, Saraburi province, Thailand.

To estimate the proportion of cases missed in a passive surveillance study of diarrhoea and dysentery at health centres and hospitals in Kaengkhoi district, Saraburi province, Thailand, a community-based cluster survey of treatment-seeking behaviours was conducted during 21-23 June 2002. Interviews were conducted at 224 households among a study population of 78,744. The respondents reported where they sought care for diarrhoea and dysentery in children aged less than five years and adults aged over 15 years. Health centres or hospitals were the first treatment choice for 78% of children with dysentery (95% confidence interval [CI] 63-94%), 64% of children with diarrhoea (95% CI 54-74%), 61% of adults with dysentery (95% CI 40-82%), and 35% of adults with diarrhoea (95% CI 17-54%). A high degree of heterogeneity in responses resulted in a relatively large design effect (D=3.9) and poor intra-cluster correlation (rho=0.3). The community survey suggests that passive surveillance estimates of disease incidence will need to be interpreted with caution, since this method will miss nearly a quarter of dysentery cases in children and nearly two-thirds of diarrhoea cases in adults.