Antimycobacterial activity of persea americana and conventional drugs susceptibility against molecular isolates of mycobacteria from new cases of pulmonary tuberculosis in kisumu county, kenya

Abstract

ABSTRACT Pulmonary tuberculosis (PTB) remains a major cause of morbidity and mortality worldwide. Challenges in the diagnosis and identification of the causative agent among members of mycobacteria in TB patients due to low sensitivity of smear microscopy leading to misdiagnosis and subsequently treated blindly is of great concern. Emergence and spread of multi-drug resistant Mycobacterium tuberculosis complex (MTBC) strains and non tuberculous mycobacteria (NTM) whose treatment is not directly analogous to that of M. tuberculosis poses challenges to disease control. Continued surveillance of drug susceptibility patterns help select effective or modify treatment regimen for better management of patients and timely control of the spread of MDR-TB. Tuberculosis drugs used for a long duration are toxic leading to adverse effects which has necessitated patients to look for alternative therapy which is medicinal plants such as Persea americana. The aim of this study was to investigate the molecular types and antimycobacterial activity of P. americana pod extracts and conventional first line anti-tuberculosis susceptibility pattern against mycobacteria isolates from new cases of pulmonary TB patients. Specific objectives were to determine the sensitivity of smear microscopy, geneXpert MTB/RIF and culture tests in sputum samples, identify the molecular types of mycobacteria isolates, determine drug susceptibility patterns of first-line anti-TB drugs against MTBC isolates and to determine the antimycobacterial activity of P. americana chloroform, n-hexane and ethanol pod extracts. A cross-sectional descriptive study was conducted between February 2016 and August 2017 that engaged 343 new cases of PTB patients at Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH) and Kisumu County Hospital who gave sputum samples. Study participants were recruited consecutively from consenting individuals with smear and/or GeneXpert positive. Sputum samples were collected to saturation. Age and gender of the participants were recorded into standard data capture forms. Sensitivity of smear microscopy and GeneXpert MTB/RIF was done using culture as the reference standard. Mycobacteria subspecies were identified from positive MGIT tubes using GenoType® Mycobacterium common mycobacteria/ additional species and MTBC assays. Drug susceptibility test was done using BACTEC MGIT 960 SIRE (streptomycin, isoniazid, rifampicin and ethambutol) and PZA (pyrazinamide) systems. Antimycobacterial activity of P. americana pod extracts was performed on twenty selected MTBC (16) and NTM (4) isolates. Descriptive statistics were used to find the sensitivity of smear microscopy culture and GeneXpert test results. Fisher’s exact test was used to assess the associations between patient characteristics and MTBC and NTM species identified. All statistical analyses were performed in STATA version 13.0 with significant threshold set to P < 0.05 at 95% confidence interval (CI). Culture results indicated that 290 (75.8%) were smear positive and 53 (24.2%) negative; while 317 (92.4%) and 26 (7.6%) were GeneXpert positive and negative, respectively, while 316/343 (92.1% were culture positive and 27/343 (7.9%) culture negative. Smear microscopy and GeneXpert had a sensitivity of 83.5% and 91.8% respectively compared to culture test. 290 (91.8%) of the 316 were MTBC and 26 (8.2%) were NTM. Subspecies among the MTBC isolates were M. tuberculosis 283/290 (97.6%), M .africanum 5/290 (1.7%) and M. bovis 2/290 (0.7%); whereas the NTM were: M .intracellulare 16/26 (61.5%), M .fortuitum 2/26 (7.7%), M. kansasii 3/26 (11.5%) and M. abscessus 5/26 (19.2%). Out of 283 M. tuberculosis isolates, mono resistance to specific drugs was as follows: highest in INH and PZA 17 (6%), EMB 8 (2.8%), STR 3 (1.1%) and the least 1 (0.4%) RIF. Double resistance was: 4 (1.4%) INH + PZA and 2 (0.7%) INH + EMB. Triple resistance was: 1 (0.4%) in both STR + INH + PZA and INH + EMB + PZA. Quadrupal resistance was 1 (0.4%) STR + INH + EMB + PZA. Pentadrupal resistance was 4 (1.4%). Chloroform pod extract inhibited the growth of all isolates, while ethanol extract was active against 19 isolates with MIC range between 18.75 – 75mg/ml. N-hexane extract was less potent, active against 17 isolates with MIC range between 150-375mg/ml. Culture and GeneXpert tests were more sensitive in the detection of mycobacteria. About 98% of PTB due to MTBC was caused by M. tuberculosis and about 62% due to NTM was caused by M. intracellulare. The highest mono resistance was in INH and PZA and low MDR rate was revealed. Chloroform extract had the highest antimycobacterial activity, followed by ethanol, and n-hexane extract had the least. Accessibility to GeneXpert and culture tests in health facilities should be increased. There is need for molecular characterization of strains underlying MTBC resistance. Treatment of PTB should be guided and reviewed based on regional DST results. Chloroform should be used as an extraction solvent and further separation of the bioactive compounds in P. americana that can be used in the development of therapeutic agents for the treatment of pulmonary TB.