Genomic transmission clusters and circulating lineages of Mycobacterium tuberculosis among refugees residing in refugee camps in Ethiopia.

BACKGROUND: Understanding the transmission dynamics of Mycobacterium tuberculosis (Mtb) could benefit the design of tuberculosis (TB) prevention and control strategies for refugee populations. Whole Genome Sequencing (WGS) has not yet been used to document the Mtb transmission dynamics among refugees in Ethiopia. We applied WGS to accurately identify transmission clusters and Mtb lineages among TB cases in refugee camps in Ethiopia. METHOD AND DESIGN: We conducted a cross-sectional study of 610 refugees in refugee camps in Ethiopia presenting with symptoms of TB. WGS data of 67 isolates was analyzed using the Maximum Accessible Genome for Mtb Analysis (MAGMA) pipeline; iTol and FigTree were used to visualize phylogenetic trees, lineages, and the presence of transmission clusters. RESULTS: Mtb culture-positive refugees originated from South Sudan (52/67, 77.6%), Somalia (9/67, 13.4%). Eritrea (4/67, 6%), and Sudan (2/67, 3%). The majority (52, 77.6%) of the isolates belonged to Mtb lineage (L) 3, and one L9 was identified from a Somalian refugee. The vast majority (82%) of the isolates were pan-susceptible Mtb, and none were multi-drug-resistant (MDR)-TB. Based on the 5-single nucleotide polymorphisms cutoff, we identified eight potential transmission clusters containing 23.9% of the isolates. Contact investigation confirmed epidemiological links with either family or social interaction within the refugee camps or with neighboring refugee camps. CONCLUSION: Four lineages (L1, L3, L4, and L9) were identified, with the majority of strains being L3, reflecting the Mtb L3 dominance in South Sudan, where the majority of refugees originated from. Recent transmission among refugees was relatively low (24%), likely due to the short study period. The improved understanding of the Mtb transmission dynamics using WGS in refugee camps could assist in designing effective TB control programs for refugees.

Performances of four Nucleic Acid Amplification Tests for the identification of SARS-CoV-2 in Ethiopia.

Since Coronavirus Disease-2019 (COVID-19) outbreak was reported, many commercial Nucleic Acid Amplification Tests (NAAT) have been developed all over the world, and it has been the standard method. Even though several assays were rapidly developed and applied to laboratory diagnostic testing, the performance of these assays was not evaluated in different contexts. Thus, this study aimed to assess the performance of Abbott SARS-CoV-2, Daan Gene, BGI and Sansure Biotech assays by using Composite Reference Standard (CRS). The study was conducted at the Ethiopian Public Health Institute (EPHI) from December 1 to 30/2020. Of the 164 nasopharyngeal samples were extracted by using a QIAamp RNA mini kit and Abbott DNA sample preparation system. Out of 164 samples, 59.1% were positive and 40.9% were negative by CRS. Sansure Biotech positivity was significantly low compared to CRS (p < 0.05). The overall agreement of the four assays compared to CRS was 96.3-100%. The performance of the four assays had almost comparable diagnostic performance, except for a low positive rate of Sansure Biotech assay. Hence, Sansure Biotech assay [Research Use Only (RUO)] needs further verification on its use in Ethiopia. Finally an additional study should be considered for evaluating assays with respective manufacturer claims.

Reference Intervals for Absolute and Percentage CD4+ T Lymphocytes among an Apparently Healthy Population in Addis Ababa, Ethiopia.

Background: Reference intervals for clinical laboratory parameters differ based on several factors, including age, sex, genetic variation, and geographic location. This variation influences clinical decisions and treatment monitoring. Currently, Ethiopia has used adopted reference intervals from manufacturer values derived from non-Africans. Therefore, the aim this study was to determine reference intervals for absolute and percentage CD4+ T cells for an apparently healthy population in Addis Ababa, Ethiopia. Methods: A community-based cross-sectional study was conducted on 361 apparently healthy people in four subcities in Addis Ababa from January to June 2019. Sociodemographic and clinical data were collected using a structured questionnaire after informed consent had been obtained. Blood samples were collected and CD4+ T-lymphocyte enumeration performed using a BD FACSPresto near-patient CD4 counter. Data were entered and analyzed using SPSS 20. Reference intervals were determined by a nonparametric test estimating percentiles 2.5 (lower limit) and 97.5 (upper limit) with 95% CIs. P<0.05 was considered statistically significant. Results: A total of 337 (183 female and 154 male) healthy participants of median age 28 (IQR 17-35) years were included in the final analysis. Medians of absolute and percentage CD4+ T-cell counts (932.0 and 42.9, respectively) of female participants were significantly higher than male participants (802.5 and 38.7, respectively; P<0.05). Reference intervals for absolute CD4+ T-cell count and percentages in males were 483.8-1,310 cells/µL and 18.1-57.3 and in females 447.8-1,479.8 cells/µL and 25.6-58.9, respectively. Conclusion: The CD4+ T-count reference intervals established in this study showed some inconsistency from the manufacturer's provided values and other studies and also revealed sex differences, necessitating sex-specific locally established reference intervals.

Low transcriptomic of PTPRCv1 and CD3E is an independent predictor of mortality in HIV and tuberculosis co-infected patient.

A comprehensive assessment of immunological profiles during HIV-TB co-infection is essential to predict mortality, and facilitate the development of effective diagnostic assays, therapeutic agents, and vaccines. Expression levels of 105 immune-related genes were measured at enrolment and 6th month follow-up from 9 deceased HIV and TB coinfected patients who died between 3 and 7th months follow-up and at enrolment, 6th and 18th month from 18 survived matched controls groups for 2 years. Focused gene expression profiling was assessed from peripheral whole blood using a dual-color Reverse-Transcription Multiplex Ligation-dependent Probe Amplification assay. Eleven of the 105 selected genes were differentially expressed between deceased individuals and survivor-matched controls at baseline. At baseline, IL4δ2 was significantly more highly expressed in the deceased group than survivor matched controls, whereas CD3E, IL7R, PTPRCv1, CCL4, GNLY, BCL2, CCL5, NOD1, TLR3, and NLRP13 had significantly lower expression levels in the deceased group compared to survivor matched controls. At baseline, a non-parametric receiver operator characteristic curve was conducted to determine the prediction of mortality of single genes identified CCL5, PTPRCv1, CD3E, and IL7R with Area under the Curve of 0.86, 0.86, 0.86, and 0.85 respectively. The expression of these genes in the survived control was increased at the end of TB treatment from that at baseline, while decreased in the deceased group. The expression of PTPRCv1, CD3E, CCL5, and IL7R host genes in peripheral blood of patients with TB-HIV coinfected can potentially be used as a predictor of mortality in the Ethiopian setting. Anti-TB treatment might be less likely to restore gene expression in the level expression of the deceased group. Therefore, other new therapeutics that can restore these genes (PTPRCv1, CD3E, IL7R, and CCL5) in the deceased groups at baseline might be needed to save lives.

Incidence of Hepatotoxicity and Factors Associated During Highly Active Antiretroviral Therapy in People Living with HIV in Ethiopia: A Prospective Cohort Study.

INTRODUCTION: Hepatotoxicity is one of the risk factors associated with treatment non-adherence, which is the main risk factor for drug resistance. Therefore, this study aimed to determine the incidence and risk factors of hepatotoxicity during highly active antiretroviral therapy (HAART) among people living with HIV in Ethiopia. METHODS: A prospective cohort study was conducted between April 2007 and January 2011 at Saint Peter Specialized Hospital, Akaki and Kality Health Centers, Addis Ababa, Ethiopia. A total of 316 HIV-infected adult individuals (70 participants were HIV and TB co-infected and 246 were infected with HIV alone) were included in this study. The study participants were followed for a total of 18 months with or without HAART. Socio-demographic data were collected using a structured questionnaire, and venous blood samples were collected for laboratory tests. Logistic regression and Poisson regression were used to determine the independent effect of each variable on hepatotoxicity at baseline and end of follow-up. RESULTS: Of 316 HIV-infected people, 72 (22.8%) participants had an elevated ALT/AST which was 100% mild-to moderate hepatotoxicity at baseline. Baseline CD4 T-cell count (p = 0.027) and HIV co-infection with TB (p < 0.001) were independently associated with hepatotoxicity at baseline. The overall incidence rate of hepatotoxicity in participants on HAART (21.8 per 100 person-years) was lower than participants who were HAART naïve (33.3 per 100 person-years) (p = 0.009). CONCLUSION: High incidence of mild-to-moderate hepatotoxicity and low severe hepatotoxicity were observed in HIV-infected individuals who were on HAART or were HAART naïve. HAART may minimize the occurrence of hepatotoxicity. Although HAART could minimize hepatotoxicity among HIV-infected people, to manage mild and moderate hepatotoxicity liver function test monitoring is required.

Gene expression profiles classifying clinical stages of tuberculosis and monitoring treatment responses in Ethiopian HIV-negative and HIV-positive cohorts.

BACKGROUND: Validation of previously identified candidate biomarkers and identification of additional candidate gene expression profiles to facilitate diagnosis of tuberculosis (TB) disease and monitoring treatment responses in the Ethiopian context is vital for improving TB control in the future. METHODS: Expression levels of 105 immune-related genes were determined in the blood of 80 HIV-negative study participants composed of 40 active TB cases, 20 latent TB infected individuals with positive tuberculin skin test (TST+), and 20 healthy controls with no Mycobacterium tuberculosis (Mtb) infection (TST-), using focused gene expression profiling by dual-color Reverse-Transcription Multiplex Ligation-dependent Probe Amplification assay. Gene expression levels were also measured six months after anti-TB treatment (ATT) and follow-up in 38 TB patients. RESULTS: The expression of 15 host genes in TB patients could accurately discriminate between TB cases versus both TST+ and TST- controls at baseline and thus holds promise as biomarker signature to classify active TB disease versus latent TB infection in an Ethiopian setting. Interestingly, the expression levels of most genes that markedly discriminated between TB cases versus TST+ or TST- controls did not normalize following completion of ATT therapy at 6 months (except for PTPRCv1, FCGR1A, GZMB, CASP8 and GNLY) but had only fully normalized at the 18 months follow-up time point. Of note, network analysis comparing TB-associated host genes identified in the current HIV-negative TB cohort to TB-associated genes identified in our previously published Ethiopian HIV-positive TB cohort, revealed an over-representation of pattern recognition receptors including TLR2 and TLR4 in the HIV-positive cohort which was not seen in the HIV-negative cohort. Moreover, using ROC cutoff ≥ 0.80, FCGR1A was the only marker with classifying potential between TB infection and TB disease regardless of HIV status. CONCLUSIONS: Our data indicate that complex gene expression signatures are required to measure blood transcriptomic responses during and after successful ATT to fully diagnose TB disease and characterise drug-induced relapse-free cure, combining genes which resolve completely during the 6-months treatment phase of therapy with genes that only fully return to normal levels during the post-treatment resolution phase.

The serum concentration of vitamin B12 as a biomarker of therapeutic response in tuberculosis patients with and without human immunodeficiency virus (HIV) infection.

BACKGROUND: Prior to clinical trials of new tuberculosis (TB) drugs or therapeutic vaccines, it is necessary to develop monitoring tools to predict treatment outcomes in TB patients. METHODS: Micronutrients concentration level was determined from a total of 262 study participants with five clinical groups: 57 TB patients coinfected with HIV (HIV+TB+), 87 active TB Patients (TB cases), 71 HIV infected without active and latent TB infection (HIV+TST-), 22 latent TB infection (TST+) and 25 healthy controls (TST-). Vitamin A concentration was measured using high-performance liquid chromatography (HPLC), whereas iron and vitamin B12 concentrations were measured using Cobas® 6000 analyzer. RESULT: The serum concentration levels of iron, vitamin A and vitamin B12 had a significant difference between active TB and latent (LTBI) or healthy controls. Six months after treatment, the serum concentration levels of vitamin A, vitamin B12 and iron in tuberculosis became indistinguishable from the levels of LTBIs and healthy control individuals. The concentration levels of iron and vitamin B12 in HIV+TB+patients at the end of TB treatment were normalized to the levels observed in healthy controls (TST-) regardless of HAART treatment. However, the concentration level of vitamin A in HIV+TB+patients HAART untreated at the end of TB treatment was not normalized to the levels observed in healthy controls (TST-) or HAART untreated HIV+TST-. CONCLUSION: Detecting serum concentration levels of vitamin B12 and vitamin A might be used as a biomarker of the diagnostic method of active TB regardless of HIV-infected individuals. Moreover, detecting serum concentration of vitamin B12 might also be used for TB treatment responses monitoring biomarker in TB-HIV-co-infected individuals regardless of HAART (in)eligibility and therapy.

Host Gene Expression Kinetics During Treatment of Tuberculosis in HIV-Coinfected Individuals Is Independent of Highly Active Antiretroviral Therapy.

Background: Limitations in diagnostic tools to discriminate between active tuberculosis and latent Mycobacterium tuberculosis infection and for monitoring antituberculosis treatment responses are major challenges in tuberculosis control, especially in human immunodeficiency virus (HIV)-coinfected individuals. Methods: Expression levels of 105 immune-related genes were determined in 131 HIV-infected patients with active tuberculosis (n = 48), patients with latent M. tuberculosis infection (LTBI; n = 37), and controls with no M. tuberculosis infection (n = 46) in Addis Ababa, Ethiopia, using focused gene expression profiling with a dual-color reverse-transcription multiplex ligation-dependent probe amplification assay. Results: Within the cohort of HIV-positive subjects, the expression profiles of 7 genes at baseline (FCGR1A, RAB24, TLR1, TLR4, MMP9, NLRC4, and IL1B) could accurately discriminate between active tuberculosis and both latent and no M. tuberculosis infection, largely independently of (in)eligibility for highly active antiretroviral therapy (HAART). Six months after antituberculosis treatment, biomarker profiles of patients with tuberculosis became indistinguishable from those of patients with LTBI and controls. Importantly, host gene expression kinetics during antituberculosis treatment in HIV-coinfected individuals was found to be independent of HAART use. Conclusions: Blood transcriptomic profiles can potentially be used as biomarkers to discriminate the different clinical stages of tuberculosis in HIV-coinfected individuals and to monitor tuberculosis treatment responses in both HAART recipients and untreated individuals.

Lipid Profile in Tuberculosis Patients with and without Human Immunodeficiency Virus Infection.

BACKGROUND: Understanding whether the preceding low lipid profile leads to active tuberculosis (TB) or active TB leads to low lipid profile is crucial. METHODS: Lipid profile concentrations were determined from 159 study participants composed of 93 active TB patients [44 HIV coinfected (HIV+TB+) and 49 HIV negative (HIV-TB+)], 41 tuberculin skin test (TST) positive cases [17 HIV coinfected (HIV+TST+) and 24 HIV negative (HIV-TST+)], and 25 healthy controls (HIV-TST-). Cobas Integra 400 Plus was used to determine lipid profiles concentration level. RESULTS: The concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in HIV-TB+ patients were significantly lower compared to HIV-TST+ and to HIV-TST- individuals. Similarly, the concentrations of the TC, LDL-C, and HDL-C in HIV+TB+ were significantly lower compared to HIV-TB+ patients. After the 6 months of anti-TB treatment (ATT), the concentration levels of TC, LDL-C, and HDL-C in HIV-TB+ patients were higher compared to the baseline concentration levels, while they were not significantly different compared to that of HIV-TST+ concentration. CONCLUSION: The low concentration of lipid profiles in TB patients may be a consequence of the disease and significantly increased in TB patients after treatment.

The performance of BD FACSPresto™ for CD4 T-cell count, CD4% and hemoglobin concentration test in Ethiopia.

INTRODUCTION: In Ethiopia, CD4+ T-cell counting is still required for all patients at baseline before antiretroviral therapy (ART) and to determine eligibility and follow-up of opportunistic infection prophylaxis. However, access to CD4+ T cell count in rural health facilities remains a major challenge in Ethiopia like other resource-limited settings. METHODOLOGY: Both capillary and venous blood was drawn from each of 325 study participant recruited in Addis Ababa and surroundings. The CD4+ T-cell count, CD4%, and hemoglobin (Hgb) were tested at one of the four study health facilities using capillary blood and BD FACSPresto™ device. These tests were also done at the national HIV reference laboratory, using venous blood with BD FACSCalibur™, Sysmex XT-1800i™, and BD FACSPresto™. RESULTS: BD FACSPresto™ had an absolute mean bias of -13.3 cells/ul (-2.99%) and 28.3 cells/µl (6.4%) using venous and capillary blood, respectively, compared with BD FACSCalibur™. The absolute CD4 assay on the BD FACSPresto™ had a regression coefficient (R2) of 0.87 and 0.92 using capillary blood and venous blood samples, respectively, compared with BD FACSCalibur™. The percentage similarity of the BD FACSPresto™ using capillary and venous blood was 105.2% and 99.3%, respectively. The sensitivity of the FACSPresto™ using threshold of 500 cells/µl for ART eligibility using capillary and venous blood was 87.9 and 94.3%, while the specificity was 91.4 and 83.8%, respectively. Furthermore, the BD FACSPresto™ had an absolute mean bias of -0.2 dl/µl (0.0%) (95% LOA: -1.7, 1.3) and -0.59 dl/µl (0.1%) (95% LOA: -1.49, 0.31) for Hgb using capillary and venous blood compared with the Sysmex XT-1800i™, respectively. CONCLUSION: Our results showed acceptable agreement between the BD FACSPresto™ and BD FACSCalibur™ for CD4+ T-cell counting and CD4%; and between the BD FACSPresto™ and Sysmex XT-1800i™for measuring Hgb concentration.