Phylogenetic lineages of tuberculosis isolates and their association with patient demographics in Tanzania.

BACKGROUND: Mycobacterium tuberculosis presents several lineages each with distinct characteristics of evolutionary status, transmissibility, drug resistance, host interaction, latency, and vaccine efficacy. Whole genome sequencing (WGS) has emerged as a new diagnostic tool to reliably inform the occurrence of phylogenetic lineages of Mycobacterium tuberculosis and examine their relationship with patient demographic characteristics and multidrug-resistance development. METHODS: 191 Mycobacterium tuberculosis isolates obtained from a 2017/2018 Tanzanian drug resistance survey were sequenced on the Illumina Miseq platform at Supranational Tuberculosis Reference Laboratory in Uganda. Obtained fast-q files were imported into tools for resistance profiling and lineage inference (Kvarq v0.12.2, Mykrobe v0.8.1 and TBprofiler v3.0.5). Additionally for phylogenetic tree construction, RaxML-NG v1.0.3(25) was used to generate a maximum likelihood phylogeny with 800 bootstrap replicates. The resulting trees were plotted, annotated and visualized using ggtree v2.0.4 RESULTS: Most [172(90.0%)] of the isolates were from newly treated Pulmonary TB patients. Coinfection with HIV was observed in 33(17.3%) TB patients. Of the 191 isolates, 22(11.5%) were resistant to one or more commonly used first line anti-TB drugs (FLD), 9(4.7%) isolates were MDR-TB while 3(1.6%) were resistant to all the drugs. Of the 24 isolates with any resistance conferring mutations, 13(54.2%) and 10(41.6%) had mutations in genes associated with resistance to INH and RIF respectively. The findings also show four major lineages i.e. Lineage 3[81 (42.4%)], followed by Lineage 4 [74 (38.7%)], the Lineage 1 [23 (12.0%)] and Lineages 2 [13 (6.8%)] circulaing in Tanzania. CONCLUSION: The findings in this study show that Lineage 3 is the most prevalent lineage in Tanzania whereas drug resistant mutations were more frequent among isolates that belonged to Lineage 4.

Disproportionate Distribution of HBV Genotypes A and D and the Recombinant Genotype D/E in the High and Low HBV Endemic Regions of Uganda: A Wake-Up Call for Regional Specific HBV Management.

BACKGROUND: Hepatitis B virus (HBV) is the leading cause of liver-related diseases. In Uganda, there is a regional disparity in the HBV burden. Our study was aimed at establishing the circulating genotypes in a low and a high endemic region to give plausible explanations for the differences in regional burden and guide the future management of the disease. METHODS: A total of 200 HBsAg-seropositive subjects were recruited into the study by convenience sampling. The HBsAg Rapid Test Strip (Healgen Scientific Limited Liability Company, Houston, TX77047- USA) was used to screen for HBsAg while the Roche machine (Roche, Basel Switzerland/Abbot Technologies (USA)) was used to determine the viral load. The Chemistry Analyzer B120 (Mindray, China) was used for chemistry analysis. For HBV genotyping, total DNA was extracted from whole blood using the QIAamp® DNA extraction kit. Nested PCR amplification was performed using Platinum Taq DNA Polymerase (Invitrogen Corporation, USA) to amplify the 400 bp HBV polymerase gene. Purification of nested PCR products was performed using Purelink PCR product purification kit (Life Technologies, USA). Automated DNA sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit on 3130 Genetic Analyzer (Applied Biosystems, USA). The NCBI HBV genotyping tool (https://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) was used for determination of genotype for each HBV sequence. Pearson's chi-square, multinomial logistic regression, and Mann-Whitney U tests were used for the analysis. All the analyses were done using SPSS version 26.0 and MedCalc software version 19.1.3 at 95% CI. A p < 0.05 was considered statistically significant. RESULTS: Majority of our study subjects were female (64.5%), youth (51.0%), and married (62.0%). Overall, genotype A was the most prevalent (46%). Genotype D and the recombinant genotype D/E were proportionately more distributed in the high endemic (38.2%) and low endemic (36.5%) regions, respectively. Genotype D was significantly more prevalent in the high endemic region and among the elderly (p < 0.05). Genotype D was significantly associated with elevated viral load and direct bilirubin (p < 0.05). The recombinant genotype D/E was significantly associated with elevated viral load (p < 0.05). Similarly, genotype A was significantly associated with elevated AST and GGT, lowered viral load, and normal direct bilirubin levels (p < 0.05). CONCLUSION: There is disproportionate distribution of genotypes A and D and the recombinant genotype D/E in the low and high endemic regions of Uganda. This probably could explain the differences in endemicity of HBV in our country signifying the need for regional specific HBV management and control strategies.

First report of whole-genome analysis of an extensively drug-resistant Mycobacterium tuberculosis clinical isolate with bedaquiline, linezolid and clofazimine resistance from Uganda.

BACKGROUND: Uganda remains one of the countries with the highest burden of TB/HIV. Drug-resistant TB remains a substantial challenge to TB control globally and requires new strategic effective control approaches. Drug resistance usually develops due to inadequate management of TB patients including improper treatment regimens and failure to complete the treatment course which may be due to an unstable supply or a lack of access to treatment, as well as patient noncompliance. METHODS: Two sputa samples were collected from Xpert MTB/RIF® assay-diagnosed multi-drug resistant tuberculosis (MDR-TB) patient at Lira regional referral hospital in northern Uganda between 2020 and 2021 for comprehensive routine mycobacterial species identification and drug susceptibility testing using culture-based methods. Detection of drug resistance-conferring genes was subsequently performed using whole-genome sequencing with Illumina MiSeq platform at the TB Supranational Reference Laboratory in Uganda. RESULTS: In both isolates, extensively drug-resistant TB (XDR-TB) was identified including resistance to Isoniazid (katG p.Ser315Thr), Rifampicin (rpoB p.Ser450Leu), Moxifloxacin (gyrA p.Asp94Gly), Bedaquiline (Rv0678 Glu49fs), Clofazimine (Rv0678 Glu49fs), Linezolid (rplC Cys154Arg), and Ethionamide (ethA c.477del). Further analysis of these two high quality genomes revealed that this 32 years-old patient was infected with the Latin American Mediterranean TB strain (LAM). CONCLUSIONS: This is the first identification of extensively drug-resistant Mycobacterium tuberculosis clinical isolates with bedaquiline, linezolid and clofazimine resistance from Uganda. These acquired resistances were because of non-adherence as seen in the patient's clinical history. Our study also strongly highlights the importance of combating DR-TB in Africa through implementing next generation sequencing that can test resistance to all drugs while providing a faster turnaround time. This can facilitate timely clinical decisions in managing MDR-TB patients with non-adherence or lost to follow-up.