Importance of Ethnicity, CYP2B6 and ABCB1 Genotype for Efavirenz Pharmacokinetics and Treatment Outcomes: A Parallel-group Prospective Cohort Study in two sub-Saharan Africa Populations.

We evaluated the importance of ethnicity and pharmacogenetic variations in determining efavirenz pharmacokinetics, auto-induction and immunological outcomes in two African populations. ART naïve HIV patients from Ethiopia (n = 285) and Tanzania (n = 209) were prospectively enrolled in parallel to start efavirenz based HAART. CD4+ cell counts were determined at baseline, 12, 24 and 48 weeks. Plasma and intracellular efavirenz and 8-hydroxyefvairenz concentrations were determined at week 4 and 16. Genotyping for common functional CYP2B6, CYP3A5, ABCB1, UGT2B7 and SLCO1B1 variant alleles were done. Patient country, CYP2B6*6 and ABCB1 c.4036A>G (rs3842A>G) genotype were significant predictors of plasma and intracellular efavirenz concentration. CYP2B6*6 and ABCB1 c.4036A>G (rs3842) genotype were significantly associated with higher plasma efavirenz concentration and their allele frequencies were significantly higher in Tanzanians than Ethiopians. Tanzanians displayed significantly higher efavirenz plasma concentration at week 4 (p<0.0002) and week 16 (p = 0.006) compared to Ethiopians. Efavirenz plasma concentrations remained significantly higher in Tanzanians even after controlling for the effect of CYP2B6*6 and ABCB1 c.4036A>G genotype. Within country analyses indicated a significant decrease in the mean plasma efavirenz concentration by week 16 compared to week 4 in Tanzanians (p = 0.006), whereas no significant differences in plasma concentration over time was observed in Ethiopians (p = 0.84). Intracellular efavirenz concentration and patient country were significant predictors of CD4 gain during HAART. We report substantial differences in efavirenz pharmacokinetics, extent of auto-induction and immunologic recovery between Ethiopian and Tanzanian HIV patients, partly but not solely, due to pharmacogenetic variations. The observed inter-ethnic variations in efavirenz plasma exposure may possibly result in varying clinical treatment outcome or adverse event profiles between populations

Anti-Tuberculosis Therapy-Induced Hepatotoxicity among Ethiopian HIV-Positive and Negative Patients

Background: To assess and compare the prevalence, severity and prognosis of anti-TB drug induced hepatotoxicity (DIH) in HIV positive and HIV negative tuberculosis (TB) patients in Ethiopia. Methodology/Principal Findings: In this study, 103 HIV positive and 94 HIV negative TB patients were enrolled. All patients were evaluated for different risk factors and monitored biochemically and clinically for development of DIH. Sub-clinical hepatotoxicity was observed in 17.3 % of the patients and 8 out of the 197 (4.1%) developed clinical hepatotoxicity. Seven of the 8 were HIV positive and 2 were positive for HBsAg. Conclusions/Significance: Sub-clinical hepatotoxicity was significantly associated with HIV co-infection (p = 0.002), concomitant drug intake (p = 0.008), and decrease in CD4 count (p = 0.001). Stepwise restarting of anti TB treatment was also successful in almost all the patients who developed clinical DIH. We therefore conclude that anti-TB DIH is a major problem in HIV-associated TB with a decline in immune status and that there is a need for a regular biochemical and clinical follow up for those patients who are at risk

Pharmacogenetic & Pharmacokinetic Biomarker for Efavirenz Based ARV and Rifampicin Based Anti-TB Drug Induced Liver Injury in TB-HIV Infected Patients

BACKGROUND: Implication of pharmacogenetic variations and efavirenz pharmacokinetics in concomitant efavirenz based antiviral therapy and anti-tubercular drug induced liver injury (DILI) has not been yet studied. We performed a prospective case-control association study to identify the incidence, pharmacogenetic, pharmacokinetic and biochemical predictors for anti-tubercular and antiretroviral drugs induced liver injury (DILI) in HIV and tuberculosis (TB) co-infected patients. METHODS AND FINDINGS: Newly diagnosed treatment naïve TB-HIV co-infected patients (n = 353) were enrolled to receive efavirenz based ART and rifampicin based anti-TB therapy, and assessed clinically and biochemically for DILI up to 56 weeks. Quantification of plasma efavirenz and 8-hydroxyefaviernz levels and genotyping for NAT2, CYP2B6, CYP3A5, ABCB1, UGT2B7 and SLCO1B1 genes were done. The incidence of DILI and identification of predictors was evaluated using survival analysis and the Cox Proportional Hazards Model. The incidence of DILI was 30.0%, or 14.5 per 1000 person-week, and that of severe was 18.4%, or 7.49 per 1000 person-week. A statistically significant association of DILI with being of the female sex (p = 0.001), higher plasma efavirenz level (p = 0.009), efavirenz/8-hydroxyefavirenz ratio (p = 0.036), baseline AST (p = 0.022), ALT (p = 0.014), lower hemoglobin (p = 0.008), and serum albumin (p = 0.007), NAT2 slow-acetylator genotype (p = 0.039) and ABCB1 3435TT genotype (p = 0.001). CONCLUSION: We report high incidence of anti-tubercular and antiretroviral DILI in Ethiopian patients. Between patient variability in systemic efavirenz exposure and pharmacogenetic variations in NAT2, CYP2B6 and ABCB1 genes determines susceptibility to DILI in TB-HIV co-infected patients. Close monitoring of plasma efavirenz level and liver enzymes during early therapy and/or genotyping practice in HIV clinics is recommended for early identification of patients at risk of DILI

Pulmonary tuberculosis and pneumocystis jiroveci pneumonia in HIV-infected patients in Ethiopia

The objective of this study initially was to determine the prevalence of culture-verified pulmonary tuberculosis in TB suspects and investigate the impact of human immunodeficiency virus (HIV) infection on the prevalence, clinical and radiological presentation and diagnosis of tuberculosis. During the study, it soon became clear that the HIV sero-prevalence in tuberculosis (TB) suspects who could not be verified to be culture-positive was too high to deserve an explanation. The second objective, therefore, was to prospectively look for possible causes of other pulmonary opportunistic infections including pneumocystis pneumonia (PCP) to explain the excess HIV. In paper I, among 509 consecutive PTB suspects attending the outpatient department of a university hospital in Addis Ababa, 33% could be culture-verified as having PTB. PTB patients, non-TB patients (culture-negative PTB suspects) and controls were HIV-1 positive in 57.1%, 38.5% and 8.3% of cases respectively. Independent predictors of culture-verified TB were age <25yrs, male gender and the presence of HIV and fever whereas profound weight loss indicated HIV infection. Diagnosis of PTB based on clinical symptoms, direct sputum microscopy and chest radiography (CXR) was significantly less sensitive and specific in HIV+ patients. In paper II, a look at the relationship between disease pattern and disease burden by chest x-ray, mycobacterial load and HIV infection also demonstrated that: (1) atypical chest x-ray with interstitial infiltrates, pleural effusion, miliary mottling, normal CXR and absent cavitations occurred more frequently in HIV-infected than non-HIV-infected patients (2) Mycobacterial load as assessed by the number of colonies of Mycobacterium tuberculosis (MTB) culture was significantly less in HIV-infected patients than non-HIV-infected, (3) the occurrence of high number of culture-verified PTB cases with normal CXR was identified as one of the challenges in the diagnosis of PTB in patients with HIV infection. In paper III, amongst 119 culture-negative, HIV+ TB suspects, P. jiroveci was detected in 10.9% by single polymerase chain reaction (PCR) and immunofluorescence (IF) and 30.3% by nested PCR in expectorated sputum sample. In HIV- negative TB and Non-TB patients, the prevalence of P.carini was significantly lower. Besides, in the IF-positive and nPCR-positive HIV+ non-TB patients,more than 40% were interpreted as PTB by CXR whereas only one patient was diagnosed with clinical PCP. This observation prompted us to design a follow up prospective study to find out the relative importance of P. jiroveci and other pulmonary opportunistic diseases in smear-negative patients presenting with atypical chest x-rays. In paper IV, 131 consecutive HIV-1 infected patients presenting with respiratory symptoms and atypical chest x-rays who were sputum smear-negative for AFB and sero-reactive for HIV were enrolled into the study. They underwent clinical evaluation and investigation for P. jiroveci (using TBO and IF stain) and M. tuberculosis from expectorated sputum and BAL samples and fungal and bacterial culture from BAL alone. The results of this study showed that the prevalence of PCP was 29.8%, that of bacterial infection 33.6% and tuberculosis 23.7%. Pulmonary Kaposi sarcoma (PKS) and interstitial pneumonitis (NIP) occurred in 4 patients each. Double infection occurred in 18(13.7%) patients. Cryptococcal pneumonitis was conspicuously absent in this study population. In paper V, we evaluated the usefulness of a simple diagnostic method, Toluidine Blue O stain for the diagnosis of P. jiroveci in expectorated sputum sample and bronchoalveolar lavage (BAL) and compared it to immunofluorescence and PCR. Comparison of these diagnostic tests showed that the sensitivity of TBO in sputum and BAL samples was 71.4 % and 68% compared to immunofluorescence respectively. The overall sensitivity for the diagnosis of PJ was 42.7 % by PCR, 29.8% by IF and 20.6% by TBO. PCR was the most sensitive test and detected additional 18 patients than immunofluorescence. Considering cost, simplicity and efficacy, we recommend TBO as the most practical diagnostic test and expectorated sputum (ES) as the most practical biologic specimen for use in resource-constrained, high HIV-settings such as Ethiopia

Comparison of acid-fast stain and culture for Mycobacterium tuberculosis in pre- and post-bronchoscopy sputum and bronchoalveolar lavage in HIV-infected patients with atypical chest X-ray in Ethiopia

<b>Background :</b> Smear-negative tuberculosis occurs more frequently in human immunodeficiency virus (HIV)-infected patients than in non-HIV-infected patients. Besides, there are substantial numbers of patients who cannot produce sputum, making the diagnosis of pulmonary tuberculosis (PTB) difficult. <b> Aims :</b> To evaluate the relative yield of pre- and post-bronchoscopy sputum and bronchoalveolar lavage (BAL) in &#x2032;sputum smear&#x2032;-negative, HIV-positive patients. <b> Settings :</b> A tertiary care referral hospital in Addis Ababa. <b> Materials and Methods :</b> Acid-fast stain (AFS) using the concentration technique was done on 85 pre-bronchoscopy sputum and 120 BAL samples. Direct AFS was done on all BAL and 117 post-bronchoscopy sputum samples. Culture for <i> Mycobacterium tuberculosis</i> (MTB) was done for all sputa and BAL. <b> Results :</b> MTB was isolated from 26 (21.7&#x0025;), 23 (19.7&#x0025;) and 13 (15.3&#x0025;) of BAL, post- and pre-bronchoscopy sputum cultures respectively. AFS on pre-bronchoscopy sputum using concentration technique and direct AFS on BAL together detected 11 (41&#x0025;) of the 27 culture-positive cases. In patients who could produce sputum, the sensitivity of pre-bronchoscopy sputum culture (13/85, 15.3&#x0025;) was comparable to BAL (12/85, 14&#x0025;) and post-bronchoscopy sputum (12/85, 14&#x0025;). In patients who could not produce sputum, however, both BAL (12/35, 40&#x0025;) and post-bronchoscopy sputum (12/32, 31.4&#x0025;) detected significantly more patients than those who could produce sputum (<i> P</i> =0.002, <i> P</i> =0.028 respectively). <b> Conclusion</b>: In HIV-infected patients, AFS by concentration method on pre-bronchoscopy sputum and direct AFS on BAL in patients who cannot produce sputum are the preferred methods of making a rapid diagnosis. BAL culture seems to add little value in patients who can produce sputum; therefore, bronchoscopy should be deferred under such circumstances

Molecular Epidemiology and Drug Resistance of Mycobacterium tuberculosis Isolates from Ethiopian Pulmonary Tuberculosis Patients with and without Human Immunodeficiency Virus Infection

We have analyzed the molecular epidemiology and drug resistance of 121 Mycobacterium tuberculosis isolates from consecutive patients with culture-positive pulmonary tuberculosis attending a university hospital outpatient department in Addis Ababa, Ethiopia. Restriction fragment length polymorphism analysis and spoligotyping were used to analyze the DNA fingerprinting patterns. Fifty-one (41.2%) of the isolates were found in 13 clusters with two or more identical DNA patterns. Two such clusters contained 49.0% of all clustered isolates. In a multivariate logistic regression model, human immunodeficiency virus (HIV)-positive serostatus was significantly associated with clustering of isolates for patients of both sexes (odds ratio [OR], 2.55; 95% confidence interval [CI], 1.17 to 5.80). There was a trend toward increased clustering of isolates from tuberculous women residing in Addis Ababa (OR, 2.10; 95% CI, 0.85 to 5.25). In total, 17 of 121 isolates (14.0%) were resistant to one or more of the antituberculosis drugs isoniazid (8.3%), streptomycin (7.4%), rifampin (2.5%), and ethambutol (1.7%). The high rate of drug-resistant isolates (29.6%) coincided with the peak prevalence of HIV infection (77.8%) in patients 35 to 44 years old. The majority (62.5%) of resistant isolates in this group were found within clusters. The simultaneous accumulation of certain bacterial clones in a patient population likely reflects recent transmission. Hence, we conclude that tuberculosis is commonly caused by recent infection with M. tuberculosis in HIV-positive Ethiopian patients. Furthermore, with the rapidly increasing prevalence of HIV infection in Ethiopia, the burden of tuberculosis, including drug-resistant tuberculosis, is likely to increase. Strengthening of classical tuberculosis control measures by promoting active case finding among HIV-positive adults with tuberculosis is warranted to reduce rates of transmission

Daily Nutritional Supplementation with Vitamin D3 and Phenylbutyrate to Treatment-Naïve HIV Patients Tested in a Randomized Placebo-Controlled Trial

Poor nutritional status is common among human immunodeficiency virus (HIV)-infected patients including vitamin D (vitD3) deficiency. We conducted a double-blinded, randomized, and placebo-controlled trial in Addis Ababa, Ethiopia, to investigate if daily nutritional supplementation with vitD3 (5000 IU) and phenylbutyrate (PBA, 2 &times; 500 mg) could mediate beneficial effects in treatment-na&iuml;ve HIV patients. Primary endpoint: the change in plasma HIV-1 comparing week 0 to 16 using modified intention-to-treat (mITT, n = 197) and per-protocol (n = 173) analyses. Secondary endpoints: longitudinal HIV viral load, T cell counts, body mass index (BMI), middle-upper-arm circumference (MUAC), and 25(OH)D3 levels in plasma. Baseline characteristics were detectable viral loads (median 7897 copies/mL), low CD4+ (median 410 cells/&micro;L), and elevated CD8+ (median 930 cells/&micro;L) T cell counts. Most subjects were vitD3 deficient at enrolment, but a gradual and significant improvement of vitD3 status was demonstrated in the vitD3 + PBA group compared with placebo (p &lt; 0.0001) from week 0 to 16 (median 37.5 versus 115.5 nmol/L). No significant changes in HIV viral load, CD4+ or CD8+ T cell counts, BMI or MUAC could be detected. Clinical adverse events were similar in both groups. Daily vitD3 + PBA for 16 weeks was well-tolerated and effectively improved vitD3 status but did not reduce viral load, restore peripheral T cell counts or improve BMI or MUAC in HIV patients with slow progressive disease. Clinicaltrials.gov NCT01702974

Vitamin D3 Status and the Association with Human Cathelicidin Expression in Patients with Different Clinical Forms of Active Tuberculosis

Low vitamin D (vitD3) is one of the most common nutritional deficiencies in the world known to be associated with numerous medical conditions including infections such as tuberculosis (TB). In this study, vitD3 status and its association with the antimicrobial peptide, human cathelicidin (LL-37), was investigated in Ethiopian patients with different clinical forms of TB. Patients with active TB (n = 77) and non-TB controls (n = 78) were enrolled in Ethiopia, while another group of non-TB controls (n = 62) was from Sweden. Active TB included pulmonary TB (n = 32), pleural TB (n = 20), and lymph node TB (n = 25). Concentrations of 25-hydroxyvitamin D3 (25(OH)D3) were assessed in plasma, while LL-37 mRNA was measured in peripheral blood and in samples obtained from the site of infection. Median 25(OH)D3 plasma levels in active TB patients were similar to Ethiopian non-TB controls (38.5 versus 35.0 nmol/L) and vitD3 deficiency (&lt;50 nmol/L) was common in both groups (73%). Ethiopians (low latitude) had significantly lower 25(OH)D3 levels compared with Swedish non-TB controls (51.0 nmol/L, high latitude), but vitD3 status was not affected by tuberculin-positivity or HIV infection. Patients with local lymph node TB had significantly higher 25(OH)D3 levels compared with pulmonary TB patients (48.0 versus 29.0 nmol/L). Moreover, plasma 25(OH)D3 levels correlated with local LL-37 expression in granulomatous lesions in TB infected lymph nodes. Instead, systemic LL-37 mRNA expression in blood cells was elevated compared with the site of infection in pulmonary and pleural TB. Low vitD3 status may be associated with an enhanced peripheral expression of LL-37 in patients with intrathoracic TB that could result from chronic inflammation

DataSheet_2_Inflammatory immune profiles associated with disease severity in pulmonary tuberculosis patients with moderate to severe clinical TB or anemia.xlsx

BackgroundImmune control of Mycobacterium tuberculosis (Mtb) infection is largely influenced by the extensive disease heterogeneity that is typical for tuberculosis (TB). In this study, the peripheral inflammatory immune profile of different sub-groups of pulmonary TB patients was explored based on clinical disease severity, anemia of chronic disease, or the radiological extent of lung disease.MethodsPlasma samples were obtained from n=107 patients with active pulmonary TB at the time of diagnosis and after start of standard chemotherapy. A composite clinical TB symptoms score, blood hemoglobin status and chest X-ray imaging were used to sub-group TB patients into 1.) mild and moderate-severe clinical TB, 2.) anemic and non-anemic TB, or 3.) limited and extensive lung involvement. Plasma levels of biomarkers associated with inflammation pathways were assessed using a Bio-Plex Magpix 37-multiplex assay. In parallel, Th1/Th2 cytokines were quantified with a 27-multiplex in matched plasma and cell culture supernatants from whole blood stimulated with M. tuberculosis-antigens using the QuantiFERON-TB Gold assay.ResultsClinical TB disease severity correlated with low blood hemoglobin levels and anemia but not with radiological findings in this study cohort. Multiplex protein analyses revealed that distinct clusters of inflammation markers and cytokines separated the different TB disease sub-groups with variable efficacy. Several top-ranked markers overlapped, while other markers were unique with regards to their importance to differentiate the TB disease severity groups. A distinct immune response profile defined by elevated levels of BAFF, LIGHT, sTNF-R1 and 2, IP-10, osteopontin, chitinase-3-like protein 1, and IFNα2 and IL-8, were most effective in separating TB patients with different clinical disease severity and were also promising candidates for treatment monitoring. TB patients with mild disease displayed immune polarization towards mixed Th1/Th2 responses, while pro-inflammatory and B cell stimulating cytokines as well as immunomodulatory mediators predominated in moderate-severe TB disease and anemia of TB.ConclusionsOur data demonstrated that clinical disease severity in TB is associated with anemia and distinct inflammatory immune profiles. These results contribute to the understanding of immunopathology in pulmonary TB and define top-ranked inflammatory mediators as biomarkers of disease severity and treatment prognosis.</p

DataSheet_1_Inflammatory immune profiles associated with disease severity in pulmonary tuberculosis patients with moderate to severe clinical TB or anemia.docx

BackgroundImmune control of Mycobacterium tuberculosis (Mtb) infection is largely influenced by the extensive disease heterogeneity that is typical for tuberculosis (TB). In this study, the peripheral inflammatory immune profile of different sub-groups of pulmonary TB patients was explored based on clinical disease severity, anemia of chronic disease, or the radiological extent of lung disease.MethodsPlasma samples were obtained from n=107 patients with active pulmonary TB at the time of diagnosis and after start of standard chemotherapy. A composite clinical TB symptoms score, blood hemoglobin status and chest X-ray imaging were used to sub-group TB patients into 1.) mild and moderate-severe clinical TB, 2.) anemic and non-anemic TB, or 3.) limited and extensive lung involvement. Plasma levels of biomarkers associated with inflammation pathways were assessed using a Bio-Plex Magpix 37-multiplex assay. In parallel, Th1/Th2 cytokines were quantified with a 27-multiplex in matched plasma and cell culture supernatants from whole blood stimulated with M. tuberculosis-antigens using the QuantiFERON-TB Gold assay.ResultsClinical TB disease severity correlated with low blood hemoglobin levels and anemia but not with radiological findings in this study cohort. Multiplex protein analyses revealed that distinct clusters of inflammation markers and cytokines separated the different TB disease sub-groups with variable efficacy. Several top-ranked markers overlapped, while other markers were unique with regards to their importance to differentiate the TB disease severity groups. A distinct immune response profile defined by elevated levels of BAFF, LIGHT, sTNF-R1 and 2, IP-10, osteopontin, chitinase-3-like protein 1, and IFNα2 and IL-8, were most effective in separating TB patients with different clinical disease severity and were also promising candidates for treatment monitoring. TB patients with mild disease displayed immune polarization towards mixed Th1/Th2 responses, while pro-inflammatory and B cell stimulating cytokines as well as immunomodulatory mediators predominated in moderate-severe TB disease and anemia of TB.ConclusionsOur data demonstrated that clinical disease severity in TB is associated with anemia and distinct inflammatory immune profiles. These results contribute to the understanding of immunopathology in pulmonary TB and define top-ranked inflammatory mediators as biomarkers of disease severity and treatment prognosis.</p