Epidemiology and Drug Resistance Pattern of Mycobacterial Isolates among HIV positive and HIV negative TB patients using Conventional and Molecular Methods in South east Ethiopia
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Date
2007-05
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Addis Ababa University
Abstract
Background:- Tuberculosis is the leading cause of death in hospitalized patients. It has not
yet been investigated whether MDR-TB contributes to the reported high mortality. There
has also been no study on the molecular epidemiology of TB and M. tuberculosis strain
types have not yet been characterized in the area. Nationwide, there have been no previous
reports on drug resistance in individuals treated with 4 fixed dose combination (FDC) anti-
TB drugs. Shashemene is one of the known locations for high prevalence of TB and HIV in
Ethiopia. The TB-HIV co-infection rate in the age group of 15-49 years was estimated to
be 47% in the area (MoH, 2004).
Objectives:-To determine the prevalence of resistance to the four first line anti-TB drugs
with and without HIV co-infected TB patients and to investigate association of TB bacteria
strain types with drug resistance.
Methods: - A cross-sectional survey on anti-TB drug resistance was conducted in four
health centres and one Hospital in and around Shashemene in Ethiopia. Sputum and blood
samples were collected from all consenting smear positive pulmonary TB patients visiting
the sites between May 2006 and December 2006. Sputum was digested and decontaminated
using Petroff s method with 4% NaOH and cultured on Lowenstein Jensen media. Drug
sensitivity tests were performed on isolates using the proportion method on Middlebrook
7H10 media and 10% OADC enrichment. Pateints were screened for HIV with rapid assays
(DetermineĀ®, CapillusĀ® and UnigoldĀ®) according to national guidelines. Species were
identified with biochemical (Thiophene-2-Carboxylic acid Hydrazide TCH test) and
DNA based methods (species specific PCR amplification with RD4 primer). Drug resitance
was further characterized using PCR based mutation analysis as a rapid and simple
technique for diagnosis of drug resistance in tuberculosis. We described a simple multiplex
allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene
codon 315, including AGC ACC and ACA (Ser Thr) substitutions that confer resistance
to isoniazid (INH) in M. tuberculosis clinical isolates. We used PCR-RFLP assay for
rpsL43 mutation analysis, in the absence of mutation in AAG AGG position in MboII
digest rpsL43 and the fragment was a 210-bp and 60-bp. If a mutation in codon 43 resulted
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in a 270-bp fragment.
Results and Discussion: - A total of 292 (264 new and 28 previously treated) patients were
included in this study. Out of these, 82.5% (241/264) were culture positive. Sensitivity test
results were available for 217 isolates from new cases and 24 isolates from previously
treated patients. Among the isolates, 80% (174/217) of those from new patients and 54.2%
(13/24) of those from previously treated patients were sensitive to all drugs tested.
Prevalence of MDR-TB among new cases was 0.9% (2 isolates). Resistance to INH, RMP,
STM and EMB was 12.9% (28/217), 1.8% (4/217), 16.6% (36/217) and 4.1% (9/217)
respectively. In previously treated patients INH, RMP, STM and EMB resistance was
37.5% (9/24), 8.3% (2/24), 41.7% (10/24) and 12.5% (3/24) respectively. There was also
no association observed between drug resistance among new cases and HIV. The high
prevalence of the katG315 ACC mutant allele among INH resistant and rpsL43 AGG
mutant allele among STM resistant M. tuberculosis clinical isolates in the Shashemene
area, Southeast Ethiopia, 83.8% (31/37) and 55.56% (20/36) for all isolates was observed.
Conclusion: Prevalence of Multi-drug resistant TB in the Shashemene area is comparable
with the national report. M. bovis was not isolated from pulmonary TB in the area.
Key words: Proportion methods; Multi-drug resistant tuberculosis; Tuberculosis; Human
Immunodeficiency Virus; Analysis of TB drug resistant mutants;
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Keywords
Proportion methods, Multi-drug resistant tuberculosis, Tuberculosis, Human Immunodeficiency Virus;, Analysis of TB drug resistant mutants