Application Of Mycobacterium Tuberculosis Genotyping Method In Molecular Epidemiological Surveillance Study

ObjectiveTuberculosis is increasing in the Shandong provinces of China, but it is not clear how much of the disease is caused by reactivated latent infection and how much is attributed to interpersonal transmission. The discovery of the transposable DNA insertion sequence, IS6110, provided the desired polymorphism among different strains to track routes of transmission, study the degree of inter-person transmission versus reactivation, to detect laboratory contamination and disease outbreaks. Alternative methods include spoligotyping and the Mycobacterial Interspersed Repetitive Units (MIRU). Sustained studies performed on 15 counties in the Shandong Province of China have found person-to-person transmission of tuberculosis to be high in these populations. In addition, resistance determinants to key antituberculosis drugs have remained unknown among tuberculosis causative organisms circulating. Thus, extensive genotyping method of Mycobacterium tuberculosis studies is needed to address these problems. Methods.An area in the 15 counties suitable for long-term surveillance studies was defined using available information from the governmental database, the Shandong Centre for tuberculosis Control case loads and transfer data obtained from the National Tuberculosis Database. Clinical patients' information was provided by the clinic managers and doctors. Sputum samples were collected and Mycobacterium tuberculosis isolated from tuberculosis positive patients during the cases intake period from July 1, 2005 to June 30, 2006.Drug susceptibility test was determined by the proportion method on Lowenstein - Jensen egg - based slopes containing critical concentrations of INH, SM, EMB and RFP (0. 2 μg/ml, 1 μg/ml, 5 μg/ml, and 40 μg/ml, respectively).All the isolates were genotyped by MIRU method. Polymerase chain reaction was carried out for in vitro amplification of 12 known loci containing MIRU using the HotStart Taq DNA polymerase kit. Incubation cycles in the thermocycler were as follows: 95℃ for 15 min (denaturing) followed by 35 cycles at 95℃for 1 min, annealing at 65℃for 1 min, extension at 72℃ for 1 min 30 sec, and a final extension step at 72℃ for 7 min. The resultant PCR product was fractionated electrophoretically in 2.5% agarose gels prepared in 1×TBE buffers. Electrophoresis was performed at 120 V for 5 h using a 100 bp ladder as the size marker. The gel was observed under ultraviolet light and photographed were also included in the study. The cause of tuberculosis recurrence was determined by comparing the MIRU pattern of Mycobacterium tuberculosis isolates responsible for different tuberculosis episodes from patients. Results.598 M. tuberculosis isolates and 19 Mycobacterium Other Than Tuberculosis (MOTT) isolates were isolated from the 1471 sputum specimens collected from the 15 counties. 108 M. tuberculosis isolates were tested resistant to any of INH, SM, EMB, or RFP, and the resistant rate was 18. 1 percent. 12 loci of MIRU were detected in 528 isolates, and a total of 140 MIRU patterns were confirmed. 75 isolates had distinct patterns. The remaining 453 (85.8%) strains were in clusters, and the largest cluster, pattern as 223325173533, included 164 strains. The number of MIRU patterns varied from two to 164. A cluster of eight isolates collected from eight patients was confirmed as one strain by MIRU typing while the patients were confirmed as the students of two different schools by follow-up.Discussion and conclusions.The aim of this study was to investigate the molecular epidemiology and the spread of resistance determinants of tuberculosis, including multidrug-resistant tuberculosis in 15 counties of Shandong provinces using conventional and genetic methods. Traditional laboratory methods used in the control of tuberculosis have inherent limitations, but molecular techniques can provide useful hidden data.As for the 15 counties area, the high intracommunity movement observed might have resulted in the loss of TB patients from the clinics participating in the study. This might explain the high strain diversity observed, contrasting reports from high TB incidence areas. In addition, the more cluster strains (85.8% similarity index) observed suggest that recent transmission of strains able to survive within the TB control programme may have established themselves in the provinces. The use of PCR-based methods (MIRU typing) was of value in differentiating strains with difficult drug resistant patterns by the genotyping. Since TB was not cultured from a large proportion of sputa collected f, either drug susceptibility test or MIRU typing can investigate transmission of TB in this area better. Since less exposure to live tuberculosis is needed with these methods, risk of infection of overworked clinic staff is reduced and this may eliminate the possible apathy towards research.