Duplex PCR-hybridization based detection of pathogenic Leptospira in environmental water samples obtained from endemic areas in northeast region of Thailand.

Leptospirosis, a major health problem worldwide, is known to be endemic in the northeastern part of Thailand with the risk of infection by exposure to pathogenic Leptospira in contaminated aquatic environment. A method based on PCR-hybridization detection of pathogenic Leptospira in water was established. The method included filtration of water sample through membrane filters of two pore sizes, DNA extraction from filters using a guanidine thiocyanate extraction method, a duplex-PCR assay with two primer pairs, and hybridization with a synthetic LipL32 DNA probe. The duplex-PCR allowed detection of two products of 279 bp for LipL32 gene and 430 bp for 16S rRNA gene. In water samples artificially seeded with serovar bratislava, at least 10(3) cells could be analyzed by PCR-agarose gel electrophoresis and 1-10 cells by PCR-Southern blot hybridization. The protocol was applied to the detection of pathogenic Leptospira in environmental waters collected from endemic areas in the northeast region of Thailand. Of 100 water samples analyzed, 23 samples were positive for pathogenic Leptospira with PCR performed with Southern blot hybridization only, but none was detected by PCR-agarose gel-electrophoresis. However, PCR performed with the chemiluminescent LipL32 probe using the Fluorescein ULS labeling facilitated the detection of low numbers of pathogenic Leptospira in water. This method should prove useful for monitoring of pathogenic Leptospira pollution in environmental waters, and has the potential to become a valuable tool to the surveillance of leptospirosis in endemic areas, thus leading to enhanced public health protection.

Development of a duplex-polymerase chain reaction for rapid detection of pathogenic Leptospira.

A duplex-polymerase chain reaction (PCR) for the rapid detection of pathogenic leptospires was developed by using two sets of newly designed primers which amplified in the same reaction two different DNA fragments simultaneously: 279-bp of LipL32 and 430-bp of 16S rRNA. For DNA extraction from bacterial cultures, the silica-based spin column method was found to be more suitable and was selected for the extraction of DNAs from all 92 bacterial strains including 56 strains of pathogenic Leptospira, 15 strains of non-pathogenic Leptospira and 21 other strains of bacteria. The PCR products were analyzed by agarose gel-electrophoresis with confirmation by Southern and dot hybridization using synthetic DNA probe prepared from LipL32 gene of a pathogenic reference strain, L. interrogans serovar pyrogenes. The duplex-PCR allowed detection of two products of 279 bp and 430 bp in all pathogenic Leptospira. Non-pathogenic Leptospira generated a single product of 430 bp. Other bacterial strains failed to reveal any amplification products. As little as 1 pg of pure DNA corresponding to 100 cells could be detected by agarose gel-electrophoresis, and 1-10 fg of pure DNA by hybridization.

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.

An enrichment broth culture-duplex PCR combination assay for the rapid detection of enterotoxigenic Clostridium perfringens in fecal specimens.

An enrichment broth culture-duplex PCR combination assay was devised to identify Clostridium perfringens directly from fecal samples. The method consists of a combination of short enrichment of samples in selective media, DNA isolation, and performing duplex PCR using two pairs of primers which identify C. perfringens strains that harbor the virulence enterotoxin gene. Comparison of two selective enrichment media and two incubation temperatures showed that the reinforced clostridial medium with neomycin was better than the fluid thioglycollate medium with neomycin (p<0.001); and incubation at 37 degrees C vs 45 degrees C showed no statistically significant difference (p=0.238). The optimal short time for pre-enrichment culture was 4 hours. The developed assay was applied to detect phospholipase C (plc) and enterotoxin (cpe) genes for C. perfringens in feces inoculated artificially with enterotoxigenic C. perfringens. The method could detect both gene products in samples inoculated with a minimum of 10(4) CFU per ml. When the method was applied to detect enterotoxigenic C. perfringens in 198 diarrhea patients, C. perfringens was found in 121 samples; 7 out of 121 samples were positive for both plc and cpe (prevalence of 5.8%). These results indicate that the developed assay was a suitable method for the rapid and specific detection of enterotoxigenic C. perfringens directly in fecal specimens of diarrhea patients, which will assist epidemiological investigations of food poisoning outbreaks and quality control of food products.

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.

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%).

Two simple immunoassays using endemic leptospiral antigens for serodiagnosis of human leptospirosis.

Two simple enzyme immunoassays, a conventional microplate and dot-ELISA, were developed to detect specific IgM antibodies using pool sonicated antigen prepared from three of the most reactive serovars of Leptospira associated with disease in Thailand. Both assays were evaluated and compared with the standard microscopic agglutination test (MAT) performed with 343 serum samples. A battery of 16 pathogenic serovars of L. interrogans were used as antigens in the MAT assay. The result of MAT at serum titers > or = 1:400 showed three pathogenic serovars of leptospira, Bratislava (71.88%), Sejroe (63.54%) and Pyrogenes (36.46%), were among the most commonly reacted serovars and they were selected for preparation of pool sonicated antigen for both IgM ELISA tests. The microplate IgM-ELISA, performed with sera at 1:80 dilution using the cutoff OD of 0.60, demonstrated sensitivity, specificity and efficiency of 87.50, 97.57, and 94.75%, respectively. The same values for IgM dot-ELISA performed with sera at 1:160 dilution were 98.96, 93.93, and 95.33%, respectively. Both ELISA methods showed results with statistically significant differences from MAT (p < 0.05). The agreement rate of IgM dot-ELISA compared with microplate IgM-ELISA was 0.85 by Kappa analysis. Both assays offered relatively high negative predictive values (95.26-99.57%), thus making the assays ideally suited for rapid screening. Future applications of the IgM dot-ELISA as a test kit would be suitable for use at the peripheral level as a rapid screening test for human leptospirosis.

A test strip IgM dot-ELISA assay using leptospiral antigen of endemic strains for serodiagnosis of acute leptospirosis.

A test strip IgM dot-ELISA assay for the detection of leptospire-specific IgM antibodies in human sera was developed. Antigen dotted on a nitrocellulose paper strip was the pool sonicated antigen prepared from three predominant reactive Leptospira serovars currently in endemic area, i.e., Bratislava, Sejroe and Pyrogenes. The ability of the test to diagnose acute leptospiral infection was assessed by testing 343 single serum samples from 96 laboratory-confirmed leptospirosis case patients with positive result in the standard microscopic agglutination test (MAT), 55 serum samples from patients with various diseases other than leptospirosis, and 192 serum samples from healthy individuals. Using the results of the MAT as a gold standard, the sensitivity and specificity of the test strip IgM dot-ELISA assay were 98.96 and 93.93 per cent, respectively. The assay offered relatively high negative predictive values (99.57%) thus making the assay ideally suited for rapid screening. The stability of the test strip was assessed with a panel of five positive and five negative control sera after storage at 4 degrees C and -20 degrees C at different times. The results showed a good performance of the test strip at both storage temperatures for up to one year. In conclusion, the test strip IgM dot-ELISA assay was sufficiently sensitive for use as a screening test for serodiagnosis of acute leptospirosis. The assay was simple, inexpensive, and easy to perform for both a single test format and a large number of specimens. However, further studies are still needed to improve the stability of the test strip and assay reagents at ambient temperature, and to make the assay more rapidly and more user friendly.

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.

Direct identification of Mycobacterium tuberculosis from sputum on Ziehl-Neelsen acid fast stained slides by use of silica-based filter combined with polymerase chain reaction assay.

This paper describes a method for isolation of deoxyribonucleic acid (DNA) from Ziehl-Neelsen stained sputum smears on glass slides; and isolated DNA was used for the IS6110 polymerase chain reaction (PCR)-based identification of M. tuberculosis. A total of 221 samples from newly diagnosed suspected tuberculosis cases were first examined by microscopic examination. For DNA extraction by silica-based filter, a home-made modified spin column gave the efficacy as did the nucleospin tissue reagent kit and therefore was selected for PCR template preparation. The extracted DNA was amplified by the IS6110 PCR using a primer pair that amplifies a 377-bp target, and the product was analyzed by agarose gel electrophoresis with confirmation by Southern blot hybridization. In comparison with culture, PCR with template prepared by the silica based filter showed overall sensitivity and specificity of 91.7 and 100 per cent, respectively. This study used the over one year and less than one year slides samples to study the effect of storage time. In the more than one year storage group, PCR assay gave a sensitivity and specificity of 83.3 and 100 per cent, respectively. In conclusion, the applicability of the PCR directly to DNA extracted from Ziehl-Neelsen stained smears could become a valuable alternative approach for rapid identification of M. tuberculosis, and could be used to evaluate quality of the control of local laboratories in tuberculosis (TB) screening and solve the problem of specimen transportation. In addition, the method could be used in retrospective studies involving a wide range of PCR-based analyses, such as detection of rifampicin resistant gene in multidrug-resistant tuberculosis (MDR-TB) study.

Drug-resistant tuberculosis among prisoners of three prisons in Bangkok and the vicinity.

The purpose of this study was to determine the prevalence of drug-resistant tuberculosis and some factors associated with drug resistance among prisoners of three prisons in Bangkok and the vicinity. Susceptibility testing to four first-line antituberculous drugs was performed on 165 M. tuberculosis strains isolated from prisoners of three prisons including Klongprem Central (KC) prison, Bangkwang Central (BC) prison and the Correctional Institution (CI) for Male Drug Addicts. Of 165 smear positive tuberculosis (TB) cases with drugs susceptibility results, resistance to one or more drugs was 49.7 per cent. Resistance to one, two, three, and four drugs was 20.0, 13.3, 4.2 and 12.1 per cent, respectively. Multidrug resistant tuberculosis (MDR-TB) was 18.8 per cent. Patients classified as primary and acquired drug resistant were 6.7 and 50.0 per cent. The primary drug resistance to one or more drugs among prisoners at KC, BC and CI were 42.5, 36.4 and 53.9 per cent, respectively and MDR-TB were 8.2, 3.0, and 7.7 per cent, respectively. Of several factors analyzed in the present study, only a history of previous TB treatment was significantly associated with drug resistance (p < 0.05). In conclusion, the results indicate the high prevalence of drug-resistant tuberculosis and the seriousness of the TB problem in prisons. The public health sector and prison authorities should work in close collaboration and co-ordination to continue improving TB case detection. Directly Observed Treatment Short course (DOTS) is highly recommended. Moreover, discharged prisoners with tuberculosis should be appropriately referred to hospitals or TB control centers.

PCR detection and prevalence of enterotoxin (cpe) gene in Clostridium perfringens isolated from diarrhea patients.

Clostridium perfringens isolated from patients with diarrhea (n=233) were analysed by a duplex PCR assay, in order to determine the prevalence of enterotoxin (cpe) gene and various factors involved in patients with cpe-positive isolates. This duplex PCR uses two sets of primers which amplify in the same reaction two different gene fragments: the phospholipase C (plc, alpha-toxin) and the enterotoxin (cpe) genes in C. perfringens. PCR analysis of 477 colonies of fecal spore isolates, from 159 patients who had a spore count > or = 10(3) cfu/g, gave positive plc gene detection in 436 colonies. The results were consistent with those obtained by using the standard method of C. perfringens species identification. 21 of 436 colonies gave positive results for both plc and cpe genes, indicating a prevalence of 4.8 per cent of C. perfringens that carried the cpe gene in cases of diarrhea. The majority of cases with cpe-positive isolates were women over 50 years of age (71.4%). These patients had diarrhea more than 6 times per day (71.4%) with a duration of 1-3 days (100%). Furthermore, 85.7 per cent of cases developed diarrhea after food consumption, 28.6 per cent had high spore counts of more than 106/g in their feces, and 71.4 per cent were co-infected with other enteric pathogens. The spore count should be interpreted with caution because not all isolates of C. perfringens from diarrhea patients with high fecal spore count carried the cpe gene, which encodes a sporulation-associated enterotoxin. CONCLUSION: The duplex PCR assay can thus become a tool for C. perfringens species identification together with the detection of enterotoxin gene. This PCR assay is faster, less expensive and more suitable for large-scale use in epidemiological studies than conventional procedures. The authors recommend this assay to screen for enterotoxigenic C. perfringens isolates from primary fecal spore isolation cultures, particularly in elderly patients with food-borne diarrhea and non-food related diarrhea.

Evaluation of sputum staining by modified cold method and comparison with Ziehl-Neelsen and fluorochrome methods for the primary diagnosis of tuberculosis.

An improved acid-fast staining technique for sputum examination for the primary diagnosis of tuberculosis is described. The technique was modified and simplified by the elimination of heating and by combining the stages of counterstaining: making the technique easier and safer, with less risk of phenol aerosols. The efficiency of this method was evaluated by comparison with two conventional methods, Ziehl-Neelsen (ZN) staining and fluorochrome staining; culture was deemed the gold standard for tuberculosis diagnosis. Of the 392 sputum samples examined, 22.7%, 19.4% and 22.9% were positive by the ZN, fluorochrome and modified cold (MC) staining methods respectively. In comparison with culture results, the sensitivities of ZN, fluorochrome and MC methods were 68.9%, 59.7% and 70.6% respectively; the specificities were 97.4%, 98.2% and 97.8% respectively and the efficiencies were 88.8%, 86.5% and 89.5% respectively. The fluorochrome method was statistically less sensitive than the ZN and MC (p < 0.05), but no significant differences between the ZN and MC were found (p > 0.05). The results of the MC and ZN methods were in close agreement (97.2%); the slides stained by these techniques could be stored for a long time and the staining reagents were stable for several weeks. In conclusion, the MC method proved to be a valuable alternative to ZN staining for the primary diagnosis of tuberculosis.

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.

A comparison of sputum examination for acid fast bacilli By modified cold stain and ziehl-neelsen stain for screening of pulmonary tuberculosis.

A comparative study of the conventional Ziehl-Neelsen (ZN) stain and a new modified cold (MC) stain was carried out to evaluate the efficiency of this staining method in sputum examination for acid fast bacilli (AFB). The MC technique was simplified by avoiding the need for heat and combining the stage of counterstaining to overcome the problem aerosolized phenol and the more laborious heating method. Of the 392 sputum samples examined, 84 were positive and 297 were negative on both staining techniques, with an agreement of 97.2%. In comparison with culture as the gold standard for the diagnosis of tuberculosis, the ZN stain exhibited a sensitivity, specificity, positive and negative predictive values and efficiency of 68.9, 97.4, 92.1, 87.8 and 88.8%, respectively. The same values for the MC stain were 70.6, 97.8, 93.3, 88.4 and 89.5%, respectively with no statistically significant differences (P > 0.05) between the 2 methods. The MC stain was also as reliable as the ZN stain in retaining the color of the stained slide after prolonged storage; an agreement with the first reading was 90% after 4 weeks storage and 80% after 16 weeks storage. The staining reagents had a long shelf life; with agreement between both staining methods of 100% at every time of re-stocking aliquots. To apply this new MC stain for future use at the peripheral level of the health care system, we made a survey by using questionnaires sent to 200 hospitals. Most of the respondants also accepted that the MC stain was easier to perform, more comfortable and much less expensive than the ZN stain. Together, these factors make the MC stain suitable for use as a practical and rapid sputum staining test for screening for patients with pulmonary tuberculosis and for assessment of their treatment.

Rapid detection of haemophilus influenzae type B by PCR.

Rapid and reliable diagnosis of meningitis caused by Haemophilus influenzae type b (Hib) is essential for early treatment to reduce the mortality rate and neurological damage among survivors. In this study, a PCR assay for Hib was developed as a reliable method in the clinical laboratory. Two methods for DNA extraction from H. influenzae isolates were compared. The extraction by a commercial “DNAzol reagent” was more rapid and convenient than conventional phenol-chloroform extraction. DNA yield from both methods was not significantly different. DNA from standard strains was used for optimizing the PCR reaction with our new designed primers based on the genes coding for capsule type-specific Hib. The sensitivity, specificity and agreement rate of the primers were tested by comparison with one pair of the published primers. There was a perfect agreement between the newly designed and the published primers with K = 1; however, the new primers had higher sensitivity and could detect as low as 1 pg of DNA. When the blind colonies of 187 bacterial meningitis isolates were used in the PCR assay, the sensitivities, specificities and efficiencies of the PCR with both primer sets, comparing the results of the culture and slide agglutination with Hib specific antiserum as the “gold” standard, were 100, 99.32 and 99.47%, respectively. The one capsule-deficient type b mutant strain could be detected by PCR while the serological result with Hib antiserum was negative. The PCR developed in this study shows rapidity, high sensitivity and specificity and may be a useful adjunct to conventional methods for early diagnosis of Hib meningitis in the clinical microbiology laboratory.