Evaluation of synthetic DNA probes for confirmation of Clostridium perfringens enterotoxin gene PCR products

A new diagnostic reagent was developed that is capable of detecting the presence of Clostridium perfringens rapidly and accurately compared to the conventional methods. C. perfringens enterotoxin (cpe) gene is the gene of interest since it encodes the enterotoxin responsible for food poisoning. Two new cpe-specific labeled DNA probes were evaluated using Southern and dot blot hybridization. Bacterial DNA was amplified by a duplex PCR procedure. The results showed that 40 enterotoxin producing C. perfringens strains generated two bands of amplicons with sizes of 420 and 280 bp, whereas 40 non-enterotoxin producing strains produced a single band of 280 bp on agarose gel-electrophoresis. No bands were observed from 32 strains of Clostridium spp and other bacteria. Southern blot analysis using either cpe-specific DNA or oligonucleotide probe showed hybridization specifically to the 420 bp band in enterotoxin-positive C. perfringens. On the dot blot membrane, both cpe-specific DNA and oligonucleotide probes were able to hybridize specifically with the corresponding DNA templates but with different efficacy (100% vs 91.1%).     

Anticoagulation control of pharmacist-managed collaborative care versus usual care in Thailand

Objective There has been a lack of evidence of the effects of pharmacist-managed warfarin therapy (PMWT) in developing countries (e.g. Southeast Asian countries) where the patients?? characteristics, genetic make-up, clinical practice and healthcare system are different from the Western world. This study aimed to compare the anticoagulation control and clinical outcomes associated with warfarin therapy provided by PMWT to usual care (UC) in the Thai population. Setting A 1,000-bed tertiary-care hospital in Nakornratchasima province of Thailand. Method A quasi-experimental study comparing PMWT and UC in patients receiving long-term warfarin therapy. For PMWT group, clinical pharmacists optimised the warfarin therapy and suggested recommendations (e.g. dose adjustment, safer alternative drugs, and follow-up time) to physicians. The UC group received the standard care. Main outcome measure Time in therapeutic range (TTR), both actual- and expanded-TTR, bleeding and thromboembolic complications, and physician?? acceptance of pharmacist suggestions. Results Of 433 patients enrolled, 220 and 213 were in the PMWT and UC groups respectively. At baseline, patient??s characteristics of both groups were comparable. At the end of follow-up period, patients in the PMWT group had significantly higher actual-TTR (48.3% vs. 40.1%; P?<?0.001) and expanded-TTR (62.7% vs. 53.9%; P?<?0.001) compared to those in the UC group. Rates of major bleeding were 4.4 vs 4.5 events per 100 person-years for the PMWT and UC groups, respectively. Pharmacists performed 284 interventions with an acceptance rate of 80.3% from physicians. Conclusion Pharmacist-managed warfarin therapy resulted in a significantly better anticoagulation control. This study showed that a collaborative approach in anticoagulation management can be successfully implemented in a developing country. Implementation of such care model in other developing countries should be considered.     

Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water

Multiplex PCR amplification of lacZ, uidA and plc genes was developed for the simultaneous detection of total coliform bacteria for Escherichia coli and Clostridium perfringens, in drinking water. Detection by agarose gel electrophoresis yielded a band of 876 bp for the lacZ gene of all coliform bacteria; a band of 147 bp for the uidA gene and a band of 876 bp for the lacZ gene of all strains of E. coli; a band of 280 bp for the p/c gene for all strains of C. perfringens; and a negative result for all three genes when tested with other bacteria. The detection limit was 100 pg for E. coli and C. perfringens, and 1 ng for coliform bacteria when measured with purified DNA. This assay was applied to the detection of these bacteria in spiked water samples. Spiked water samples with 0-1,000 CFU/ml of coliform bacteria and/or E. coli and/or C. perfringens were detected by this multiplex PCR after a pre-enrichment step to increase the sensitivity and to ensure that the detection was based on the presence of cultivable bacteria. The result of bacterial detection from the multiplex PCR was comparable with that of a standard plate count on selective medium (p=0.62). When using standard plate counts as a gold standard, the sensitivity for this test was 99.1% (95% CI 95.33, 99.98) and the specificity was 90.9 % (95% CI 75.67, 98.08). Multiplex PCR amplification with a pre-enrichment step was shown to be an effective, sensitive and rapid method for the simultaneous detection of these three microbiological parameters in drinking water.     

Circulating and unique recombinant forms of HIV type 1 containing subsubtype A2

HIV-1 strains containing subsubtype A2 are relatively rare in the pandemic but have been repeatedly identified in Kenya, where candidate vaccines based in part on subtype A, but not A2 strains, may be evaluated. Among the most recent is CRF16_A2D, a circulating recombinant form (CRF) whose prototypes are complete or partial HIV-1 sequences from Kenya, Korea, and Argentina. Using samples from blood bank discards in Kenya and complete genome sequencing, this report further documents CRF16_A2D and related recombinants and identifies a second CRF, CRF21_A2D. The two A2-containing CRFs, and two recombinants related to CRF16_A2D, share common structural elements but appear to have been independently derived. Concerted selection may have influenced the emergence and spread of certain A2-containing strains in Kenya. The second complete subtype C sequence from Kenya is also reported here. Monitoring of A2-containing recombinants and subtype C strains, both relatively rare in Kenya, may be informative in the course of cohort development and evaluation of candidate vaccines.     

Antibiotic resistance of enterococci isolated from frozen foods and environmental water

We evaluated 239 isolates of enterococci (113 from frozen foods and 126 from environmental water) for their resistance to 8 antibiotics by agar disk diffusion method. Most isolates from both sources were resistant to tetracycline (64.1% food strains; 46.8% water strains) and ciprofloxacin (53.4% food strains; 48.4% water strains). A relatively high prevalence of chloramphenicol, trimethoprim-sulfamethoxazole and vancomycin resistance was present, ranging from 9.7 to 27.2% for food strains and 10.3 to 15.9% for water strains; while other drug resistance (ampicillin, gentamicin and teicoplanin) was minimal (< or = 0.9% for food strains; < or = 1.6% for water strains). No significant differences in resistant rates between the two sources were found for any of the drugs (p>0.05) except tetracycline (p<0.05). The majority of isolates from both sources were multi-resistant strains (50% for food strains and 42% for water strains). Most of them showed resistance to two drugs. There was no significant difference in the non-resistance patterns and the multidrug resistance patterns (p>0.05) between the frozen food and environmental water strains, but a significant difference was seen in the single drug resistance pattern (p<0.05). Vancomycin resistant enterococci (VRE) were isolated from nearly all sources studied, 9.7% food isolates and 10.3% water isolates, with no significant difference between the two sources (p>0.05). This study shows a high prevalence of multidrug resistance among enterococci isolated from foods of animal origin and environmental water. This may serve as a potential transfer route of antibiotic-resistant bacteria and resistant genes into the human food-chain and environment which could potentially pose a health threat to humans in the future. The use of antibiotics for purposes other than human health, ie in animal feeds and in the treatment of infection in animals, should be reduced and eventually eliminated. Improved hygiene practices and controlled use of antibiotics in agriculture, animal husbandry, and fisheries are desirable for environmental management and public health protection.     

Comparison of microplate hybridization with gel electrophoresis and dot blot hybridization for the rapid detection of Mycobacterium tuberculosis PCR products

A microplate ELISA hybridization assay has been developed for the detection of the IS6110 PCR products of M. tuberculosis from sputum specimens. In this study, its efficacy was evaluated by comparison with agarose gel electrophoresis (AGE) and dot blot hybridization (DBH), with culture results as the 'gold standard'. The assay was used with 190 sputum samples: the PCR results detected by ELISA and AGE showed close agreement, with sensitivity, specificity and accuracy of 90%, 100% and 96% respectively. The same values for DBH were 92%, 98% and 96% respectively. The validities of these methods were not statistically significantly different (p>0.05). The agreement rates of PCR product detection by AGE comparing with DBH and ELISA were 0.964 and 0.964 respectively, while that of DBH and ELISA was 1.0 by Kappa analysis. The overall agreement was not statistically significantly different (p>0.05). Use of DBH or ELISA hybridization increased the sensitivity of detection by AGE 10-fold from 10 pg to 1 pg of purified DNA per reaction; ie from about 30 to about 3 organisms. The amount of PCR product detected by ELISA was only one half of that detected by the other methods; the total assay time of ELISA following the PCR was 4 hours. In conclusion, the microplate hybridization assay may replace AGE and DBH for the detection of the PCR products of M. tuberculosis because of its sensitivity, specificity and accuracy. Additional advantages of the microplate assay over AGE and DBH include rapidity, ease of use, greater safety, cost effectiveness and greater objectivity in the reading of results; the technique is suitable for use in epidemiological studies for the analysis of a large number of samples.     

Cost-utility and budget impact analyses of gefitinib in second-line treatment for advanced non-small cell lung cancer from Thai payer perspective

Aim: To evaluate the cost utility and budget impact of second‐line gefitinib for non‐small cell lung cancer from a Thai payer perspective. Methods: A Markov model with three health states (pre‐progression, post‐progression and death) was constructed to estimate direct medical costs and outcomes comparing four treatment options, i.e., gefitinib, erlotinib, pemetrexed and docetaxel. The model followed patients for 2 years with discount rate of 3% annually. Clinical inputs and patients' characteristics were based on a randomized phase III trial (INTEREST). Costs were based on reference prices published by the Ministry of Public Health, Thailand, and other information related to treatment from expert opinion and presented in 2010. Deterministic and probabilistic sensitivity analyses were performed to determine the impact of model parameters on results. Results: In the base case model, gefitinib and erlotinib yielded equal quality‐adjusted life years (QALY) but 0.0140 and 0.0110 more QALY compared with docetaxel and pemetrexed, respectively. Total costs were 188 848 Baht (US$6237) for gefitinib, 196 313 Baht (US$6483) for docetaxel, 249 177 Baht (US$8229) for erlotinib and 275 303 Baht (US$9092) for pemetrexed. Drug acquisition contributed the greatest component. A series of sensitivity analyses demonstrated the robustness to various parameter variations except for docetaxel cost and duration of treatment. The budget impact analyses demonstrate the greater the percentage of substitution of gefitinib for docetaxel (ranging from 10–60%) the greater the cost saving. Conclusion: Gefitinib is a dominant cost saving strategy compared with docetaxel for the second‐line treatment of advanced NSCLC from the Thai payer perspective.     

Antimicrobial resistance among Clostridium perfringens isolated from various sources in Thailand

Antimicrobial resistance among Clostnridium perfringens isolated from feces of humans and pigs, food and other environmental sources was examined by testing of 201 PCR-confirmed strains for resistance to 7 antimicrobial agents. The minimal inhibitory concentrations (MICs) were determined by the agar dilution method. Overall, C. perfringens showed the highest resistance to tetracycline (56.2%), followed by imipenem (24.9%), metronidazole (9.5%), penicillin G (9%), vancomycin (4.5%), chloramphenicol (3%) and ceftriaxone (1%). The majority of the isolated strains from pig feces (77.8%), environment (72.7%), human feces (44.9%) and food (28%) showed resistance to tetracycline. Strains isolated from human feces only showed low resistance to ceftriaxone (2.5%) and vancomycin (10.1%). Penicillin G had high activity, with overall MIC50 and MIC90 of 0.06 and 1.0 microg/ml, respectively, and low rate of resistance (10-12% for strains isolated from humans, animals and food). Among 62.7% of antimicrobial resistant strains, 39.3% were resistant to a single drug and 23.4% were multiple-drug resistant (MDR). Of overall 47 MDR strains, 63.8% were derived from human feces and were resistant to two to six drugs.     

Cost-effectiveness analysis of pharmacogenetic-guided warfarin dosing in Thailand

Introduction

Pharmacogenetic (PGx) test is a useful tool for guiding physician on an initiation of an optimal warfarin dose. To implement of such strategy, the evidence on the economic value is needed. This study aimed to determine the cost-effectiveness of PGx-guided warfarin dosing compared with usual care (UC).

Methods

A decision analytic model was used to compare projected lifetime costs and quality-adjusted life years (QALYs) accrued to warfarin users through PGx or UC for a hypothetical cohort of 1,000 patients. The model was populated with relevant information from systematic review, and electronic hospital-database. Incremental cost-effectiveness ratios (ICERs) were calculated based on healthcare system and societal perspectives. All costs were presented at year 2013. A series of sensitivity analyses were performed to determine the robustness of the findings.

Results

From healthcare system perspective, PGx increases QALY by 0.002 and cost by 2,959 THB (99 USD) compared with UC. Thus, the ICER is 1,477,042 THB (49,234 USD) per QALY gained. From societal perspective, PGx results in 0.002 QALY gained, and increases costs by 2,953 THB (98 USD) compared with UC (ICER 1,473,852 THB [49,128 USD] per QALY gained). Results are sensitive to the risk ratio (RR) of major bleeding in VKORC1 variant, the efficacy of PGx-guided dosing, and the cost of PGx test.

Conclusion

Our finding suggests that PGx-guided warfarin dosing is unlikely to be a cost-effective intervention in Thailand. This evidence assists policy makers and clinicians in efficiently allocating scarce resources.     

High glucose‐ROS conditions enhance the progression in cholangiocarcinoma via upregulation of MAN2A2 and CHD8

Diabetes is a major risk factor in the development and progression of several cancers including cholangiocarcinoma (CCA). However, the molecular mechanism by which hyperglycemia potentiates progression of CCA is not clearly understood. Here, we showed that a high glucose condition significantly increased reactive oxygen species (ROS) production and promoted aggressive phenotypes of CCA cells, including proliferation and migration activities. Mannosidase alpha class 2a member 2 (MAN2A2), was upregulated at both mRNA and protein levels in a high glucose‐ and ROS‐dependent manner. In addition, cell proliferation and migration were significantly reduced by MAN2A2 knockdown. Based on our proteome and in silico analyses, we further found that chromodomain helicase DNA‐binding protein 8 (CHD8) was induced by ROS signaling and regulated MAN2A2 expression. Overexpression of CHD8 increased MAN2A2 expression, while CHD8 knockdown dramatically reduced proliferation and migration as well as MAN2A2 expression in CCA cells. Moreover, both MAN2A2 and CHD8 were highly expressed with positive correlation in CCA tumor tissues. Collectively, these data suggested that high glucose conditions promote CCA progression through ROS‐mediated upregulation of MAN2A2 and CHD8. Thus, glucose metabolism is a promising therapeutic target to control tumor progression in patients with CCA and diabetes.