Martha Inírida Guerrero

IS6110 fingerprinting of sensitive and resistant strains (1991-1992) of Mycobacterium tuberculosis in Colombia

The standardized method to study the polymorphism of IS 6110 was used to characterize 53 isolates of Mycobacterium tuberculosis obtained during 1991-1992 from 14 regions in Colombia. In Valle region cluster rate was 25% (4/16). The mean number of IS6110 band was 10 +/- 3. Similarity between strains was of 60% in 81% of strains and this tended to be correlated with geographic origin. For the first time M. tuberculosis without IS6110 bands in restriction fragment length polymorphism analysis was found in Colombia. Additional studies are necessaries in order to best characterize the situation in relation to human immunodeficiency virus epidemic and recent changes in tuberculosis control program.

Developing and using a PCR test to detect subclinical Mycobacterium leprae infection

OBJECTIVEnWhile the prevalence of leprosy has declined around the world, there has not been a corresponding decrease in its incidence, thus indicating that it has not been possible to prevent transmission of the disease. Despite the small number of patients with lepromatous leprosy, the majority of the inhabitants of endemic areas show signs of exposure to Mycobacterium leprae, which could be explained by the presence of subclinical bacilliferous infections in the community. The objective of this study was to investigate the use of a polymerase chain reaction (PCR) test to detect M. leprae in samples of nasal mucus from asymptomatic household contacts of patients with leprosy.nnnMETHODSnWe standardized and optimized a PCR technique to amplify a 321 base pair DNA fragment, using a pair of primers complementary to a segment of an LSR/A15 gene that codes for the 15 kDa M. leprae antigen. We investigated the optimal concentrations of all the test components. We used dimethyl sulfoxide (DMSO) to achieve a more specific amplification. We applied the PCR test to 70 healthy household contacts of leprosy patients from eight municipalities in Colombia where there was a high prevalence of the disease.nnnRESULTSnThe tests detection limit was 100 fg of DNA. With the optimized technique, bacillus was detected in the nasal mucus samples of 9 (12.8%) of the 70 household contacts. The 3 PCR-positive household contacts of paucibacillary cases were from municipalities with very high prevalence levels. In comparison to contacts who were PCR-negative, the contacts who were PCR-positive had spent significantly less time, as a proportion of their age, living with a patient (P = 0.028). This finding demonstrates the tests capacity for early detection.nnnCONCLUSIONSnThe PCR test that we developed is useful as a tool for detection and early follow-up of possible leprosy cases. It can be used to monitor high-risk populations and also to maintain the achievements of leprosy elimination programs in countries where the diseases prevalence has been significantly reduced.

The epidemiological evidence for an association between extrapulmonary tuberculosis and AIDS in Colombia

Mycobacteria may act as a cofactor in the development of AIDS since the human immunodeficiency virus (HIV) needs to reproduce within an activated macrophage. Mycobacterium tuberculosis is most commonly involved in this stage of cellular activation and tuberculosis (TBC) is often one of the first manifestations of AIDS. For this reason the number of cases of extrapulmonary TBC in a community can be used as an epidemiological indicator of the progression of the AIDS epidemic. Extrapulmonary TBC is usually precipitated by a decrease in cellular immunity. In Colombia an AIDS epidemiological surveillance system based on the confirmation of cases by Western Blot was set up in 1986. In this country only 30% of all cases of extrapulmonary TBC are confirmed by bacteriological culture. Between 1971 and 1985 only about 2% of all cases of TBC represented extrapulmonary forms of the disease. However this proportion has increased to 8% of all cases of TBC since 1986 coinciding with the beginning of the AIDS epidemic in this country. An ecological analysis was carried out based on linear regression analysis of the association between extrapulmonary TBC and the number of AIDS cases in 35 regional health services in Colombia from 1986 to 1991. The strongest linear association was observed in 1988 (p < 0.05). Between 1987 and 1989 the association decreased (p < 0.05). The weakest association was observed in 1986 (p < 0.05) although there was considerable underreporting of cases. These data demonstrate a strong linear relation between the number of cases of AIDS and extrapulmonary TBC in regional health services. In two other studies carried out in Colombia mycobacteria were isolated from about 30% of HIV-positive patients. These results show that extrapulmonary TBC is an important opportunistic infection in AIDS patients in Colombia. HIV testing should be routinely offered to all patients with extrapulmonary TBC.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.