Advancing proficiency testing for ultra in resource-limited settings using dried tube specimen: A study by SRL-Uganda.

BACKGROUND: Proficiency testing (PT) has been hard to set up due to cost limitations and technical capacity. Conventional Xpert MTB/RIF PT programs use liquid and culture spots which require stringent storage and transportation conditions with cross-contamination chances prevalent. These setbacks prompted the use of dried tube specimens (DTS) for Ultra assay PT. For continuity of PT provision, stability of DTS and compatibility with testing protocols when kept for a long period needs to be established. METHODS: DTS were prepared from known isolates inactivated using a hot air oven at 85°C. 100µl of bacterial suspensions were aliquoted and dried inside a Biosafety cabinet. Panel validation was done to establish the baseline Deoxyribonucleic acid (DNA) concentration in terms of cycle threshold (Ct) value. DTS aliquots were shipped to participants to test and report within six weeks. The remaining DTS were kept at 2-8°C and room temperature for one year with testing at six months. Twenty (20) DTS samples per set remaining at one year were heated at 55°C for two weeks before testing. The means of the different samples were compared to validation data using paired t-tests. Boxplots were designed to visualize the differences in the medians of the DTS. RESULTS: Overall mean Ct value increased by 4.4 from the validation to testing after one year at the different storage conditions. Samples heated at 55°C showed a 6.4 Ct difference from validation data. Testing done at six months on 2-8°C stored items showed no statistical difference. At all the remaining testing times and conditions, P-values were less than 0.008 although the absolute mean Ct when compared showed slight increments and accommodated differences for the detection of MTB and rifampicin resistance. Median values for samples stored at 2-8°C were lower compared to those at room temperature. CONCLUSION: DTS stored at 2-8°C remain more stable for one year compared to higher temperatures and can be consistently used as PT materials in more than one PT round for biannual PT providers.

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.

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.

Molecular characterisation of second-line drug resistance among drug resistant tuberculosis patients tested in Uganda: a two and a half-year's review.

BACKGROUND: Second-line drug resistance (SLD) among tuberculosis (TB) patients is a serious emerging challenge towards global control of the disease. We characterized SLD-resistance conferring-mutations among TB patients with rifampicin and/or isoniazid (RIF and/or INH) drug-resistance tested at the Uganda National TB Reference Laboratory (NTRL) between June 2017 and December 2019. METHODS: This was a descriptive cross-sectional secondary data analysis of 20,508 M. tuberculosis isolates of new and previously treated patients' resistant to RIF and/or INH. DNA strips with valid results to characterise the SLD resistance using the commercial Line Probe Assay Genotype MTBDRsl Version 2.0 Assay (Hain Life Science, Nehren, Germany) were reviewed. Data were analysed with STATAv15 using cross-tabulation for frequency and proportions of known resistance-conferring mutations to injectable agents (IA) and fluoroquinolones (FQ). RESULTS: Among the eligible participants, 12,993/20,508 (63.4%) were male and median (IQR) age 32 (24-43). A total of 576/20,508 (2.8%) of the M. tuberculosis isolates from participants had resistance to RIF and/or INH. These included; 102/576 (17.7%) single drug-resistant and 474/576 (82.3%) multidrug-resistant (MDR) strains. Only 102 patients had test results for FQ of whom 70/102 (68.6%) and 01/102 (0.98%) had resistance-conferring mutations in the gyrA locus and gyrB locus respectively. Among patients with FQ resistance, gyrAD94G 42.6% (30.0-55.9) and gyrA A90V 41.1% (28.6-54.3) mutations were most observed. Only one mutation, E540D was detected in the gyrB locus. A total of 26 patients had resistance-conferring mutations to IA in whom, 20/26 77.0% (56.4-91.0) had A1401G mutation in the rrs gene locus. CONCLUSIONS: Our study reveals a high proportion of mutations known to confer high-level fluoroquinolone drug-resistance among patients with rifampicin and/or isoniazid drug resistance. Utilizing routinely generated laboratory data from existing molecular diagnostic methods may aid real-time surveillance of emerging tuberculosis drug-resistance in resource-limited settings.

Rifampicin susceptibility discordance between Xpert MTB/RIF G4 and Xpert Ultra before MDRT-TB treatment initiation: A case report from Uganda.

Tuberculosis (TB) resistance to rifampicin, the most powerful drug leads to increase in mortality. Globally, half a million new patients develop such resistant TB each year, coupled with both inappropriate diagnosis and treatment initiation. We report a case of rifampicin resistant Mycobacterium tuberculosis whose rifampicin resistance was missed by Xpert MTB/RIF Assay G4 but detected by the Xpert MTB/RIF Ultra assay at different time points leading to increased delays for MDR-TB treatment initiation at Mulago Hospital, Kampala, Uganda. Our case report compels greater urgency in accelerating the transition to the newer assay, Ultra, to benefit from higher sensitivity of rifampicin resistance detection.

Quality of Sputum Specimen Samples Submitted for Culture and Drug Susceptibility Testing at the National Tuberculosis Reference Laboratory-Uganda, July-October 2013.

Setting: The Uganda National Tuberculosis Reference Laboratory (NTRL) in Kampala. Objective: The proportion of poor quality specimens received for drug susceptibility testing (DST) at the NTRL and factors contributing to poor specimen quality were assessed. Design: A cross-sectional study was conducted of sputum samples received at the NTRL from patients at high risk for multidrug-resistant tuberculosis (MDR TB) during July-October 2013. Demographic, clinical, and bacteriological data were abstracted from laboratory records. A poor quality sample failed to meet any one of four criteria: ≥3 milliliter (ml) volume, delivered within 72 hours, triple packaged, and non-salivary appearance. Results: Overall, 365 (64%) of 556 samples were of poor quality; 89 (16%) were not triple packaged, 44 (8%) were <3 mls, 164 (30%) were not delivered on time, and 215 (39%) were salivary in appearance. Poor quality specimens were more likely to be collected during the eighth month of TB treatment (OR = 2.5, CI = 1.2 - 5.1), from the East or Northeast zones (OR = 2.2, CI = 1.1 - 4.8), and from patients who previously defaulted from treatment (OR = 1.9, CI = 1.1 - 3.2). Conclusion: The majority of sputum samples had poor quality. Additional efforts are needed to improve quality of samples collected at the end of treatment, from East and Northeast zones, and from patients who had previously defaulted.