Susceptibility to Reactive Nitrogen Intermediates and Anti Tuberculosis Drugs Resistance in Northwest, Ethiopia.
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Date
2012-06
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Addis Ababa University
Abstract
Background: Tuberculosis, caused by M. tuberculosis, is the deadliest disease causing 3 million annual deaths globally. M. tuberculosis is an intracellular pathogen that survives and replicates within cells, primarily macrophages. An important part of the host defense against tuberculosis is nitric oxide and other reactive intermediates produced by activated macrophages. tuberculosis differ in their strain types, susceptibility to the conventional anti Tb drugs and to the effect of nitric oxide which in turn may influence clinical outcome of active tuberculosis patients.
Objectives: The aim of this project, therefore, was to investigate strains differences, the effect of the potent RNI, NO (nitric oxide), and the convectional anti-Tb drugs on the clinical isolate of M. tuberculosis, in vitro, from patients receiving arginine (Nitric oxide) in the form of peanuts in an ongoing clinical trial study and to correlate the findings with clinical outcome of those subjects.
Methods: Both, prospective and retrospective study had been conducted to investigate the molecular epidemiology, NO (nitric oxide) susceptibility and anti-mycobacterial sensitivity patterns of the clinical isolates MTB in order to asses correlation of results to clinical outcome of active tuberculosis patients. Smear-positive sputums from 180 study participants were cultured to obtain pure clinical isolates. Standard PCR method and spoligo typing were done to identify the strains of M. tuberculosis. The isolates were also tested for their drug susceptibility pattern using proportion method. The in vitro effects of RNI and susceptibility profile of those isolates were determined after series of optimizations experiments using DETA/NO (potent nitric oxide donor chemical).
Results: Of 176 smear-positive TB patients enrolled, 93 (52.8%) were female, 68 (38.6%) were HIV sero-positive and the mean age was 27.5 (SD = 9.55; R = 15 – 59). HIV seroprevalence was high among age groups between 25 – 45 yrs of age (P=0.000). Of 176 smear-positive sputa 67.6% were culture positives. Conventional PCR of isolates revealed that all strains belong to M. tuberculosis. Spoligotyping was done for 108 strains, of which 83 (76.8%) were clustered in to 4 major M. tuberculosis families being CAS families the dominant ones (43%). The remaining 25 (23.2%) were unique and no non-tuberculos mycobcteria was detected. The overall proportion of drug resistant strains was 14.3% (14/98), poly-resistant 1% and no MDR-strains were identified. The clinical strains generally showed variability in percent survival to NO exposure with median survival rate of 14.1% (IQR, 3.0 – 30.3%), and exposure of strains to 1mM of DETA (NO donor) for 24hrs significantly reduced (P=0.000) the bacterial colony forming units (cfu). In logistic regression analysis, nitric oxide survival rate of clinical isolates was significantly associated with some of the clinical conditions of the patients like BMI (kg/m 2) (P= 0.04), smear conversion (P= 0.03) and cure rate (P= 0.04).
Conclusions: Mycobacterium tuberculosis was the only mycobacterial strain found among consecutive smear positive isolates, the rate of anti-microbial resistance was high. Patients carry drug resistant strains significantly exhibited decreased cure rate and smear conversion rate at 2 months. Clinical isolates of M. tuberculosis exhibiting nitric oxide resistance showed significantly more anti mycobacterial drug resistance compared to nitric oxide susceptible strains. Further research is needed on the importance and use of nitric oxide to prevent and control of the spread of tuberculosis.
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Keywords
Tuberculosis, nitric oxide, drug resistance, spoligotypes, clinical outcome