PCR hot start using primers with the structure of molecular beacons (hairpin-like structure).

A new technique of PCR hot start using oligonucleotide primers with a stem-loop structure is developed here. The molecular beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3'-end sequence of the primers was complementary to the target and five or six nucleotides complementary to the 3'-end were added to the 5'-end. During preparation of the reaction mixture and initial heating, the oligonucleotide has a stem-loop structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension can begin.

Treatment monitoring and prevalence of drug resistance in tuberculosis patients in Tehran.

SETTING: Health care clinics and private practitioners in Tehran. OBJECTIVE: To analyse rates of drug resistance and response to treatment in tuberculosis patients. DESIGN: A prospective study of 257 patients undergoing treatment for whom data were collected on drug susceptibility testing and outcome as well as age, sex and history of treatment. RESULTS: Of 774 initially diagnosed patients, 380 were female and 394 were male; 520 (67%) of the cases had pulmonary disease. The overall rate of primary drug resistance among Mycobacterium tuberculosis isolates resistant to at least one drug was 87/563 (15.5%). Twenty-three patients were multidrug-resistant. Among 215 patients with drug-susceptible cultures recruited for follow-up, rapid response to short-course chemotherapy was observed in 190 (88%) who were successively both smear and culture negative after 2 and 4 months of treatment. After 6 months of treatment, 12 of the 25 patients with slow response to treatment had positive cultures; one was smear-positive. Of the 42 patients with drug-resistant isolates, satisfactory bacteriological response was observed after 6 months of treatment in 30 (71%). CONCLUSIONS: These observations support regional recommendations for short-course treatment regimens. Culture rather than smear result could be a key parameter for individually guiding the duration of treatment in patients with poor response to treatment.

Non-tuberculous mycobacteria: patterns of isolation. A multi-country retrospective survey.

OBJECTIVE: To collect data on non-tuberculous mycobacteria (NTM) isolated from clinical laboratories in different countries to establish: 1) whether the isolation of NTM was increasing, 2) which species were increasing, and 3) whether there was any pattern of geographical distribution. DESIGN: In 1996, the Working Group of the Bacteriology and Immunology Section of the International Union Against Tuberculosis and Lung Disease contacted 50 laboratories in different countries for the necessary information. RESULTS: The number of patients reported with NTM was 36099 from 14 countries. Mycobacterium avium complex, M. gordonae, M. xenopi, M. kansasii and M. fortuitum were the five species most frequently isolated. There was a significant upward trend for M. avium complex and M. xenopi. Pigmented mycobacteria predominated in Belgium, the Czech Republic and the Mediterranean coast of Spain. Non-chromogenic mycobacteria were found to be predominant in the area of the Atlantic coast of Brazil and in Turkey, the United Kingdom, Finland and Denmark. CONCLUSIONS: There was an increase in the number of NTM isolated from clinical samples of patients. Isolation of the most frequent species is constantly changing in most of the geographical areas, and newer species are emerging due to better diagnostic techniques to detect and identify NTM.

HSP65-PRA identification of non-tuberculosis mycobacteria from 4892 samples suspicious for mycobacterial infections.

Various molecular methods have been used for the rapid identification of mycobacterial species. In this survey, evaluation of antibiotic resistance and PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was carried out for identification of non-tuberculosis mycobacteria (NTM) isolates from different clinical specimens. Forty-eight different mycobacterial isolates were selected and followed by the conventional and PRA of hsp65 for species identification. The antibiotic susceptibility test was carried out according to standard methods. A 439 bp PCR product of hsp65 in all selected isolates was amplified and digested with the BstEII and HaeIII restriction enzymes. The restriction fragment length polymorphism (RFLP) patterns were analyzed for species identification. Using PRA for 48 mycobacterial selected isolates, including 15 M. tuberculosis, one M. bovis and all 32 isolates of NTM, revealed 11 different species among the NTM isolates. The most frequent NTM isolates were M. kansasii, M. gordonae III, M. marinum, M. chelonae, M. scrofluaceum and M. gastri. In most cases, the PRA results were perfectly in accordance with the classical biochemical method. Combination of resistance to rifampin and isoniazid was present among M. kansasi, M. gordoniae III, M. scrofluaceum, M. chelonae, M. marinum, M. gastri, M. gordoniae II and M. trivale isolates. A high incidence of co-resistance to six, five, four and three anti-TB drugs was observed in 18.5%, 9.1%, 6.6% and 11.7% of all NTM isolates, respectively. Our results showed that PRA, in comparison with classical methods, is rapid and accurate enough for the identification of mycobacterial species from LJ medium. Additionally, we found that in Iran we have a highly diverse population of NTM isolates among patients suspected of having TB.

Antimicrobial resistance pattern and plasmid profile of Salmonella typhi isolated from an outbreak in Tehran province.

The antimicrobial resistance patterns and plasmid profiles of Salmonella typhi isolates from sporadic cases (n = 33) and an outbreak (n = 48) were compared. Of 28 sporadic drug-resistant isolates, 24 (85.7%) were multiply resistant. The predominant antibiotic resistance pattern was TeCmSmSxTAp, which was also the most common pattern of the outbreak isolates. 13 drug-resistant strains isolated before the outbreak (46.4%) were able to transfer the whole resistance pattern or part of it to Escherichia coli K 12 by conjugation. Although 20 of the sporadic strains contained plasmid DNA, transferable R plasmids were only detected in 13 (65%) of them. Among the outbreak strains, the rate of R plasmid transfer was 92.3%, with only the TeCmSmSxTAp pattern transferred. Plasmid profiling and Hind III endonuclease digestion of plasmid DNA identified a 91.2 megadaltons (Mda) plasmid that was recovered from most of the outbreak isolates and from 4 strains collected before the outbreak. This plasmid coded for TeCmSmSxtAp and transferred the pattern of resistance in toto. The results indicate multidrug-resistant S. typhi as a potential cause of infection in the region.

Use of polymerase chain reaction for primary diagnosis of pulmonary tuberculosis in the clinical laboratory.

A nested polymerase chain reaction (PCR) has been used for the rapid detection of tubercule bacilli in respiratory specimens from 287 patients suspected of tuberculosis. The results of PCR testing were compared with isolation methods (conventional culture and Bactec system) in 110 smear-positive and 177 smear-negative patients. There were only 4 false negative results by PCR in the 171 specimens that were M. tuberculosis complex culture-positive. Of 92 PCR-positive samples prepared from the smear-positive specimens 90 (97.8%) were confirmed by culture. However, a poor correlation was obtained between initial 122 PCR-positive results and combined 81 culture recovered organisms in smear-negative patients. After verification of the efficacy of isolation method, retesting PCR-positive culture-negative samples, and studies of patients' clinical histories, only 18 of the cases were found to be associated with the disease. The other 29 results out of the original 47 discrepants were considered PCR false positives, possibly due to contamination. In conclusion, the PCR assay described is suitable for implementation in daily routine work with respiratory specimens, however it should be validated with culture, especially for the smear-negative patients.

Use of restriction enzyme analysis of amplified DNA coding for the hsp65 gene and polymerase chain reaction with universal primer for rapid differentiation of mycobacterium species in the clinical laboratory.

Two rapid procedures, restriction enzyme analysis of the amplified segment of the gene encoding for the 65000 mol. wt heat shock protein and a polymerase chain reaction with single universal primer (UP-PCR), were used for the identification of Mycobacterium tuberculosis complex (n = 47) and proving the species identity of non-tuberculous mycobacteria (NTM, n=36) cultured from clinical samples by comparing the resulting DNA banding pattern with patterns derived from mycobacterial type strains (n = 24). UP-PCR assay provided a rather wide limit of tolerance for variations in procedure. Although mycobacterial strains were found to generate species-specific banding patterns in both assays, M. tuberculosis and M. bovis strains and isolates produced nearly the same DNA patterns, which were very distinctive from that of all NTM tested. Investigation of the majority of M. fortuitum (n = 14) and M. kansasii (n = 7), mycobacteria most frequently causing mycobacterioses in the region, as well as other NTM isolates, showed reproducible patterns characteristic of corresponding type strains. Both methods combine the advantages of ordinary PCR and PCR 'fingerprinting', namely, the species-specific DNA pattern and primers applicable to different species. They may be applied as rapid tests for proving the identity of Mycobacterium species in a clinical laboratory.

[New solid culture media for growing Borrelia persica and Borrelia microti]. / Nuevo medio sólido para el crecimiento de Borrelia persica y Borrelia microti.

A new solid means for the fast detection of Borrelia persica and Borrelia microti is described. Generally, culture and isolation of Borrelia takes about 21 days. The serological test, which is carried out more often, takes less time but it is associated with false positive reactions relatively high. However, our new solid means reduces the culture time to 72 hours, allowing to have a fast diagnosis of the disease caused by Borrelia persica and Borrelia microti, and to start the early treatment of these patients.

[Report of drug resistance in strains of Mycobacterium tuberculosis isolated from patients in Iran]. / Reporte de la resistencia a las drogas en cepas de Mycobacterium tuberculosis aisladas de pacientes en Irán.

6,472 clinical samples of patients with tuberculosis suspicion between March, 1993 and March, 1994, were studied. Positive results were obtained in 443 patients; 238 females (53.7%) and 205 males (46.3%). The predominant age group was that between 30 and 39 years (31.5%). The cutaneous test of sensitivity to the purified protein derivate (PPD) was positive in 178 patients with a range of 10-14 mm. Abnormal radiological images were found in 222 patients (50.1%). Higher resistance frequency was detected in Mycobacterium tuberculosis strains among cases suffering from pulmonary tuberculosis. 42 (9.5%) strains were resistant to isoniazid and 31 (7.0%) to streptomycin. Resistance to one drug was observed in 25 isolations (5.4%). A few strains (1.3%) were resistant to 3 drugs, and 1 of them to 5 drugs. Clinical and epidemiological data suggest that resistance to drugs in tuberculosis is becoming an important problem in the region. The fast diagnosis of this infection and the use of antibiotics with a reduced spectrum may enable the control of this form of tuberculosis.

Chemical cleavage of mismatches in heteroduplexes of the rpoB gene for detection of mutations associated with resistance of Mycobacterium tuberculosis to rifampin.

In order to detect mutations in the core region of the RNA polymerase B (rpoB) subunit gene of Mycobacterium tuberculosis that are known to be associated with resistance to rifampin, we applied rapid chemical cleavage of mismatches (CCM) to heteroduplexes formed between the DNA of M. tuberculosis H37Rv and strains resistant to rifampin. DNA fragments amplified from normal and mutant rpoB genes by polymerase chain reaction were mixed, denatured and re-annealed to create heteroduplexes containing mispaired bases reactive to modification by hydroxylamine (cytosine mismatches) or osmium tetroxide (thymine mismatches) and cleavage of DNA by piperidine at the position of modified base. The cleaved products and the heteroduplexes were separated by polyacrylamide-urea gel electrophoresis and detected by autoradiography. The position of mutations was confirmed by DNA sequencing of the amplified DNA fragments. The results suggest further applicability of the CCM method as a means to screen M. tuberculosis isolates for mutations associated with drug resistance.

Detection and identification of non-tuberculous mycobacterial infections in 6,472 tuberculosis suspected patients.

Between March 1993 and March 1994, 82 patients with infection by non-tuberculous mycobacteria (NTM) and 443 patients with tuberculosis (TB) were registered among 6,472 patients with suspected tuberculosis. Skin-test reactivity to purified protein derivative (PPD) in patients demonstrated indurations of 10-14 mm or more for the majority of patients in both groups. Most patients with NTM infection had abnormal chest roentgenograms showing sporadic infiltrations, nodular abscesses, and cavities resembling TB radiological evidence. The similarity in age range, PPD skin reaction, and radiological evidence in patients infected with NTM or Mycobacterium tuberculosis (MTB) can mislead the physician. Some NTM species were recovered more often than others. Mycobacterium fortuitum from 22 clinical specimens (26.8%); Mycobacterium gastric 19 (23.1%); and Mycobacterium terrae complex 15 (18.3%). The antimicrobial drug susceptibility tests of the isolated organisms showed that 42 (9.5%) isolates of MTB were resistant to isoniazid and 31 (7.0%) to streptomycin. a few strains (1.3%) were identified as being resistant to a combination of 3 primary drugs. These findings suggest that drug-resistant mycobacterial infections are becoming an important problem in the region.

Polymerase chain reaction of bacterial genomes with single universal primer: application to distinguishing mycobacteria species.

The polymerase chain reaction with a single universal primer (UP-PCR) was applied to bacterial strains and mycobacteria isolates alongside conventional methods. A universal protocol of preparation of PCR samples from cultures representing Escherichia coli, Enterobacter aerogenes, Serratia marcescens, Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Mycobacterium tuberculosis, Mycobacterium bovis, and several non-tuberculous mycobacteria was found to be reproducible and efficient with these organisms. The bands of UP-PCR products observed in an agarose gel after electrophoresis were species-specific and provided an efficient means of distinguishing bacterial species. The applicability of this approach to mycobacteria identification was assessed by comparing the DNA bands obtained for different strains. Three reference strains and 22 clinical isolates of M. tuberculosis and M. bovis produced very similar DNA banding patterns. They comprised a triplet of prominent and several minor fragments within the 200-500 base pair (bp) size range and were the easiest to interpret. The DNA profiles of unrelated mycobacteria clearly differed from each other when subjected to electrophoretic analysis and correlated well with results of culture method. The method provides a real promise of its application in clinical studies as a simple assay for distinguishing between tubercle bacilli.

Reporte de la resistencia a las drogas en cepas de Mycobacterium tuberculosis aisladas de pacientes en Iran / Report of resistance to drugs in Mycobacterium tuberculosis isolated from patients in Iran

Se estudiaron 6 472 muestras clinicas de pacientes con sospecha de tuberculosis entre marzo de 1993 a marzo de 1994. Se obtuvieron resultados positivos en 443 pacientes; 238 correspondieron al sexo femenino (53,7 por ciento) y 205 (46,3 por ciento) al masculino, predomino el grupo de edad entre 30 y 39 anos (31,5 por ciento). La prueba cutanea de sensibilidad al derivado proteico purificado (PPD) fue positiva en 178 pacientes con un rango de 10-14 mm. Se encontraron imagenes radiologicas anormales en 222 pacientes (50,1 por ciento). Se detecto mayor frecuencia de resistencia en las cepas de Mycobacterium tuberculosis en casos con tuberculosis pulmonar. Cuarenta y dos cepas (9,5 por ciento) fueron resistentes a la isoniacida y 31 (7,0 por ciento) a la estreptomicina. Se registro resistencia a 1 droga en 25 aislamientos (5,4 por ciento). Pocas cepas (1,3 por ciento) resultaron resistentes a 3 drogas y 1 de ellas a 5 drogas. Los datos clinicos y epidemiologicos sugieren que la resistencia a las drogas en la tuberculosis comienza a ser un problema importante en la region. El diagnostico rapido de esta infeccion y el uso de antibioticos con un espectro reducido puede facilitar el control de esta forma de tuberculosis

Nuevo medio solido para el crecimiento de Borrelia persica y Borrelia microti / New solid means for the growing of Borrelia persica and Borrelia microti

Se describe un nuevo medio solido para la rapida deteccion de Borrelia persica y Borrelia microti. Corrientemente el cultivo y aislamiento de Borrelia demora alrededor de 21 dias. El examen serologico mas frecuentemente realizado demora menos tiempo pero esta asociado con resultados falsos positivos relativamente altos. Sin embargo, nuestro nuevo medio solido reduce el tiempo de cultivo a 72 horas, lo que permite un rapido diagnostico de la enfermedad causada por Borrelia persica y Borrelia microti y el inicio temprano del tratamiento en estos pacientes