Evaluation of culture and PCR-based assay for detection of Mycobacterium tuberculosis from sputum collected and stored on filter paper.

We evaluated the use of culture and PCR-based assay for the direct detection of Mycobacterium tuberculosis (MTB) from sputum collected and stored on filter paper at room temperature for 5 days; the results were compared with those of staining and conventional culture of fresh sputum before storage (the 'gold standard'). Out of 231 sputum specimens examined, MTB was recovered from 124 samples by culture before storage. The culture positivity rate was significantly decreased to 70% after 5 days storage. For PCR assay, a fragment of 377 bp of the IS6110 sequence was amplified and detected using three methods: first PCR product combined with agarose gel electrophoresis (AGE); first PCR product with dot blot hybridization (DBH); nested PCR with AGE. Compared with culture, the sensitivity, specificity, and efficiency for first PCR with AGE were 71.8, 100 and 84.9% respectively; PCR with DBH gave results of 89.5, 96.3 and 92.6% respectively; the same values for nested PCR were 96.0, 97.2, and 96.5% respectively. Of these three methods, nested PCR gave excellent sensitivity and specificity with no significant difference (p = 0.727) from conventional culture. The storage of sputum on filter paper and storage at room temperature for 5 days had no apparent effect on the performance of nested PCR. We propose that this collection and storage method be considered for transporting sputum specimens from peripheral health centers or from the field; specimens may be sent by post to a central point for both culture and PCR analysis by trained technicians supervised in accordance with a well-established quality control system.

Simple microplate hybridization assay for detection of amplified products of Mycobacterium tuberculosis.

We describe a simple microplate hybridization assay for the rapid detection of the IS6110 PCR products of Mycobacterium tuberculosis from clinical cultures and from sputum specimens. The assay is based on the specific detection with a fluorescein-labeled detection probe of biotinylated PCR products which are captured on avidin coated microplate. Hybridized products with fluorescein were identified by using anti-fluorescein antibody, horseradish peroxidase conjugate and colorimetric peroxidase substrate. The specificity of the assay was assessed by analysis of 56 bacterial strains: the assay discriminated perfectly between the positive and negative groups when an OD490 of 0.18 was used as the cut-off point. The assay was sensitive enough to detect as little as 1 pg of M. tuberculosis H37Rv DNA, which is equivalent to approximately three bacilli. To evaluate the assay performance clinically, 190 sputum samples from newly diagnosed TB patients were tested; 79 were classified as TB positive, and 111 were classified as TB negative by culture and acid-fast staining as the 'gold standard'. The sensitivity, specificity and accuracy of the PCR-microplate hybridization assay were 90, 100 and 96%, respectively. The total assay time of hybridization following the PCR was 4 hours. The PCR-microplate hybridization assay is fast, simple and accurate and is suitable for use in the microbiology laboratory or for the analysis of large numbers of samples.

Comparison of the resistance ratio and proportion methods for antimicrobial susceptibility testing of Mycobacterium tuberculosis.

This study compared two in vitro antimicrobial susceptibility methods for determining drug susceptibilities of Mycobacterium tuberculosis isolated from newly diagnosed pulmonary tuberculosis patients to four front-line drugs. Of 250 strains of M. tuberculosis tested, 74.4 per cent were susceptible by the resistance ratio method, with 72.0 per cent by the proportion method. The results showed high agreement for both methods (P<0.0001) and agreement rates to streptomycin, isoniazid, rifampicin and ethambutol were 96.8, 98.0, 94.8 and 96.8 per cent, respectively. For drug resistance patterns, both methods showed the highest resistance to one drug, followed by two, three, and four drugs, respectively. Of the single drug resistance, both methods gave the highest resistance to streptomycin, followed by resistance to isoniazid, rifampicin and ethambutol, respectively. The correlation between both methods for determining susceptibility of M. tuberculosis to four drugs was not statistically significantly different by Mc Nemar chi2 (p>0.05). Thus, the resistance ratio method may be substituted. However, WHO recommended the use of the proportion method to be used for determining drug susceptibility of M. tuberculosis. The susceptibility testing result can be used as the guidance for proper treatment and is valuable for confirmation of drug resistance in patients showing unsatisfactory response to treatment, useful for identifying primary and acquired drug resistance trends in a community and for minimizing the spread of drug-resistant strains.

Detection of Mycobacterium tuberculosis from sputum collected on filter paper and stored at room temperature for 5 days by PCR assay and culture.

The efficacy of PCR assay and culture for direct detection of M. tuberculosis (MTB) from sputum specimens collected on filter paper and stored at room temperature for 5 days was evaluated in comparison with conventional culture of fresh sputum specimen. A total of 231 sputum specimens were examined. MTB was recovered from 124 samples by culture from fresh sputum samples before storage. The culture positivity rate was significantly decreased to 70 per cent after 5 day's storage on filter paper. For PCR assay, a fragment of 377-bp of the IS6110 sequence was amplified and detected using nested PCR. Compared with culture results performed on fresh sputum samples, the sensitivity, specificity, and efficiency for the nested PCR were 96.0, 97.2 and 96.5 per cent, respectively. The nested PCR showed sensitivity and specificity with no significant difference (p>0.05) from culture of fresh sputum specimens. CONCLUSION: The collection and storage of sputum on filter paper at room temperature for 5 days had no apparent effect on the performance of nested PCR. Sputum samples collected by this method could be sent by post in a minimum of space and inexpensive way and will enable a large number of samples collected in the field or from peripheral health centers to be sent to central laboratories for analysis by trained technicians and under a well-equipped and well-established quality control system. The rapid and reliable detection by PCR-based assay will be helpful for optimal patient management of therapy and effective control of tuberculosis.

Development of duplex PCR assay for rapid detection of enterotoxigenic isolates of Clostridium perfringens.

A duplex PCR assay was developed for the rapid and specific amplification of the alpha-toxin (phospholipase C, plc) gene and the enterotoxin (cpe) gene from Clostridium perfringens. Two pairs of primers were newly designed for the species identification and also for the differentiation between enterotoxin-positive and enterotoxin-negative C. perfringens strains in a single reaction. The detection by agarose gel electrophoresis yielded 2 bands of 280-bp of plc and 420-bp of cpe for all four enterotoxin-positive reference strains tested without the need for further hybridization, and one band of 280-bp of plc for all seven enterotoxin-negative reference strains. While 50 strains of other Clostridium species and other bacteria tested by PCR were negative for both genes. The detection limit, as measured with purified DNA was 10 fg or as few as 4 organisms could be detected. This assay was used to identify primary fecal spore isolates from 244 fecal specimens of patients with diarrhea. Of total 432 colonies from 144 positive growth cultures determined, 21 revealed both plc and cpe genes and 411 were positive for plc gene only. This suggested a prevalent of 5% of all C. perfringens strains that carry the enterotoxin gene. The results indicate the duplex PCR as a simple, sensitive, specific, cost-effective and time saving assay for detection of potentially enterotoxigenic isolates of C. perfringens, and has potential application for epidemiological investigations of food poisoning outbreaks and quality control of food products for humans and animal feeds.

Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR.

This study describes the rapid detection of polioviruses in environmental waters by a simple reverse transcriptase-polymerase chain reaction (RT-PCR) using two primer pairs for differentiation of poliovirus from non-polio enteroviruses in a single reaction by a one-step method, combining RT and PCR in a single tube. The detection by agarose gel electrophoresis yielded 2 bands of 153-bp and 293-bp for poliovirus tested without the need for further hybridization. The detection sensitivity of this one-step duplex RT-PCR, as measured with RNA extracted by heat treatment from supernatant of infected cell extracts, was 10(-1) 50% tissue culture effective doses (TCID50). This assay was used to evaluate the ability of sample concentration by membrane filter-based adsorption and elution, and purification by a simple RNA isolation based on guanidine isothiocyanate-phenol-chloroform extraction; the system yielded a detection limit of 5 x 10(-1) TCID50 seeded in 5 liters of tap water. This protocol was applied to the poliovirus detection in environmental water collected from 2 communities in Bangkok, Thailand during February and May 1998. Of 100 samples tested, 2 water samples collected from the same open sewage pipeline at one location were positive for polioviruses and one sample collected from another sewage pipeline was positive for non-polio enterovirus while a further 97 water samples were negative for both polioviruses and non-polio enteroviruses. With poliovirus detection by cell culture technique, none of the 100 samples tested was positive for poliovirus type 1, 2 or 3. RT-PCR was more sensitive, rapid, simple and cost-effective than the cell culture technique since the two water samples which were positive for polioviruses by RT-PCR failed to be detected by cell culture. Sequence data of 293-bp amplicons from positive samples were compared with those of reference poliovirus strains in the Genbank and the EMBL databases and identity to the sequence of type 1 strain Sabin was found to be 99%.

Crude side tomato lectins for identification of group B beta-hemolytic streptococci.

Crude extracte from the side tomato fruit have been shown to contain lectins which specifically bind to group B Beta-hemolytic streptococci. Some additional properties of crude lectins were investigated. The lectins could agglutinate human erythrocytes of type A,B and O. Agglutination was enhanced by trypsin treatment of erythrocytes. The carbohtydrate-binding specificity indicated a specificity of tri-acetyl-glucosamine. They were stable at a high temperature and had a long half-life. The crude lectins agglutinated 149 to 160 strains of group B Beta-hemolytic streptococci with 93.1 percent sensitivity, and did not react with any other of 150 streptococcal sergroups (100% specificity). This finding indicates that the test using crude Sida tomato lectins offers some profound sensitive agglutination technique, they may provide a suitable alternative to other conventional tests for a rapid, simple, cost-effective and specific differentiation of group B from other of Beta-hemolytic streptococci on primary isolation.