Supranational Reference Laboratory Research Articles

Evaluating the effectiveness of Uganda’s Supranational TB Reference Laboratory quality management system training program

BackgroundAchieving the targeted organizational goals through effective training can increase employee satisfaction. Since 2015, the Supranational Reference Laboratory Uganda (SRL Uganda) has trained National Tuberculosis Reference Laboratories (NTRLs) from 21 countries in a variety of areas that cover both technical and programmatic aspects pertinent to TB laboratories. The Laboratory Quality Management System (LQMS) under SRL coordinates actions intended to ensure sustained quality of the laboratory services offered by the National TB Reference Laboratories. In order for laboratory results to be helpful in a clinical or public health setting, they must be accurate, reliable, and timely. The LQMS course aims to provide learners with knowledge on how to attain and maintain this quality. Prior to this study, there was hardly any data available on the effectiveness of LQMS trainings provided by SRL Uganda; using Kirkpatrick model, which is popular among researchers for evaluating the efficacy of the training program, this paper seeks to establish the effectiveness of the LQMS training offered by the SRL Uganda.MethodWe evaluated the effectiveness of LQMS training within the Uganda’s SRL network for courses offered during the period 2017 and 2021 for participants from the Southern and East African sub-Saharan region.ResultsIn 2017 and 2021, respectively, test results from 10/17 and 9/17 showed overall post-test scores above 80%. Of the 18 labs evaluated, 14 showed improvement; of these, 7 labs were from the Eastern region and the other 7 were from Southern Africa; one facility in this region also maintained its accreditation. In the post-evaluation assessment, attendees of the LQMS course gave feedback of strongly agree and agree variety.ConclusionMore SRL Uganda network laboratories in the regions achieved a 5-star SLIPTA level rating and among these, 5 NTRLs got ISO 15189:2012 accredited by the end of 2021, while one maintained its accreditation status. This proves that the Laboratory Quality Management System training program was an effective tool in improving the quality of laboratory services, work practices, and processes.

Face mask sampling (FMS) for tuberculosis shows lower diagnostic sensitivity than sputum sampling in Guinea

BackgroundPulmonary tuberculosis (PTB) diagnosis relies on sputum examination, a challenge in sputum-scarce patients. Alternative non-invasive sampling methods such as face mask sampling (FMS) have been proposed.ObjectiveTo evaluate the value of FMS for PTB diagnosis by assessing its agreement with sputum samples processed by GeneXpert MTB/RIF (Ultra)(Xpert) testing, and describe FMS sensitivity and specificity.MethodsThis was a prospective study conducted at the Carrière TB clinic in Guinea. Presumptive TB patients willing to participate were asked to wear a surgical mask containing a polyvinyl alcohol (PVA) strip for thirty minutes. Subsequently, two spot sputum samples were collected, of which one was processed by microscopy on site and the other by Xpert in Guinea’s National Reference Laboratory of Mycobacteriology (LNRM). The first 30 FMS were processed at the Supranational Reference Laboratory in Antwerp, Belgium, and the following 118 FMS in the LNRM.ResultsOne hundred fifty patients participated, of whom 148 had valid results for both mask and sputum. Sputum smear microscopy was positive for 47 (31.8%) patients while sputum-Xpert detected MTB in 54 (36.5%) patients. Among the 54 patients testing sputum-Xpert positive, 26 (48.1%) yielded a positive FMS-Xpert result, while four sputum-Xpert negative patients tested positive for FMS and 90 patients were Xpert-negative for both sputum and mask samples, suggesting a moderate level of agreement (k-value of 0.47). The overall mask sensitivity was 48.1%, with 95.7% specificity.ConclusionIn our setting, Xpert testing on FMS did not yield a high level of agreement to sputum sample.

Resistance Profiles to Second-Line Anti-Tuberculosis Drugs and Their Treatment Outcomes: A Three-Year Retrospective Analysis from South India.

Background: Patients with first-line drug resistance (DR) to rifampicin (RIF) or isoniazid (INH) as a first-line (FL) line probe assay (LPA) were subjected to genotypic DST using second-line (SL) LPA to identify SL-DR (including pre-XDR) under the National TB Elimination Program (NTEP), India. SL-DR patients were initiated on different DR-TB treatment regimens and monitored for their outcomes. The objective of this retrospective analysis was to understand the mutation profile and treatment outcomes of SL-DR patients. Materials and Methods: A retrospective analysis of mutation profile, treatment regimen, and treatment outcome was performed for SL-DR patients who were tested at ICMR-NIRT, Supra-National Reference Laboratory, Chennai between the years 2018 and 2020. All information, including patient demographics and treatment outcomes, was extracted from the NTEP Ni-kshay database. Results: Between 2018 and 2020, 217 patients out of 2557 samples tested were identified with SL-DR by SL-LPA. Among them, 158/217 were FQ-resistant, 34/217 were SLID-resistant, and 25/217 were resistant to both. D94G (Mut3C) of gyrA and a1401g of rrs were the most predominant mutations in the FQ and SLID resistance types, respectively. Favorable (cured and treatment complete) and unfavorable outcomes (died, lost to follow up, treatment failed, and treatment regimen changed) were recorded in a total of 82/217 and 68/217 patients in the NTEP Ni-kshay database. Conclusions: As per the testing algorithm, SL- LPA is used for genotypic DST following identification of first-line resistance, for early detection of SL-DR in India. The fluoroquinolone resistance pattern seen in this study population corelates with the global trend. Early detection of fluoroquinolone resistance and monitoring of treatment outcome can help achieve better patient management.

A comparison of phenotypic and WGS drug susceptibility testing in Mycobacterium tuberculosis isolates from the Republic of Korea.

WGS has significant potential to help tackle the major public health problem of TB. The Republic of Korea has the third highest rates of TB of all Organisation for Economic Cooperation and Development countries but there has been very limited use of WGS in TB to date. A retrospective comparison of Mycobacterium tuberculosis (MTB) clinical isolates from 2015 to 2017 from two centres in the Republic of Korea using WGS to compare phenotypic drug susceptibility testing (pDST) and WGS drug susceptibility predictions (WGS-DSP). Fifty-seven MTB isolates had DNA extracted and were sequenced using the Illumina HiSeq platform. The WGS analysis was performed using bwa mem, bcftools and IQ-Tree; resistance markers were identified using TB profiler. Phenotypic susceptibilities were carried out at the Supranational TB reference laboratory (Korean Institute of Tuberculosis). For first-line antituberculous drugs concordance for rifampicin, isoniazid, pyrazinamide and ethambutol was 98.25%, 92.98%, 87.72% and 85.96%, respectively. The sensitivity of WGS-DSP compared with pDST for rifampicin, isoniazid, pyrazinamide and ethambutol was 97.30%, 92.11%, 78.95% and 95.65%, respectively. The specificity for these first-line antituberculous drugs was 100%, 94.74%, 92.11% and 79.41%, respectively. The sensitivity and specificity for second-line drugs ranged from 66.67% to 100%, and from 82.98% to 100%, respectively. This study confirms the potential role for WGS in drug susceptibility prediction, which would reduce turnaround times. However, further larger studies are needed to ensure current databases of drug resistance mutations are reflective of the TB present in the Republic of Korea.

Primary drug resistance of M. tuberculosis according to the Center of Excellence of the WHO Network of Supranational Reference Laboratories of the CTRI in 2016–2021

ntroduction. Culture research and testing of Mycobacterium tuberculosis (MBT) for drug susceptibility in the Russian Federation is carried out with uneven qual-ity, which distorts the idea of primary drug resistance (DR). Aim. To study the primary DR of MBT to the main combinations of anti-TB drugs for 2016–2021 according at the Center of Excellence of the WHO Supranational Reference Laboratories Network at the Central Tuber-culosis Research Institute. Methods. Data on drug sus-ceptability and frequency of resistance to the main com-binations of anti-TB drugs (in accordance with updated definitions) of MBT isolated before treatment were stud-ied in 966 new TB cases from 69 regions of the Russian Federation examined at the Center of Excellence of the WHO Supranational Reference Laboratories Network at the Central Tuberculosis Research Institute from 2016 to 2021. Results. The rate of any primary drug resistance to rifampicin was 46.2%; 95% CI [43.0–49.3], MDR — 45.8%; 95% CI [42.6–48.9], pre-XDR — 24.2%; 95% CI [21.5–27.0], XDR — 5.4%; 95% CI [3.3–8.0]. Over time, there was a decrease in primary MDR MBT from 48.7% in 2016 and 54.9% in 2017 to 36.8% in 2021. The frequency of pre-XDR did not change during the observation peri-od. There was a statistically significant increase in the incidence of primary XDR from 2.1%; 95% CI [0.4–11.1] in 2016 and 0.0% in 2017 to 11.0%; 95% CI [6.1–19.1] in 2021 (p=0.03). Conclusion. The rate of primary MBT drug resistance to the main anti-TB combinations, ac-cording to our study, significantly exceeds the overall figures for Russian Federation. This suggests that the situation with MDR, pre-XDR, and XDR tuberculosis in the Russian Federation is much more alarming than we see from the regular reports, while the quality of local microbiological diagnostics on is insufficient

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

BackgroundMycobacterium 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.Methods191 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 v2.0.4ResultsMost [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.ConclusionThe 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.

Spot specimen testing with GeneXpert MTB/RIF results compared to morning specimen in a programmatic setting in Cotonou, Benin

BackgroundThe diagnosis of tuberculosis (TB) using smear microscopy has been based on testing two specimens: one spot and one early morning sputa. Recently, the World Health Organization (WHO) has recommended to replace, whenever possible, microscopy with GeneXpert® MTB/RIF performed on a single specimen. However, as the bacterial load is higher in early morning specimens than in spot specimens, one could expect lower sensitivity of GeneXpert® MTB/RIF performed only on spot specimens. In this study, we compared results of GeneXpert® MTB/RIF on spot specimens versus early morning specimens, under programmatic conditions in Cotonou, Benin.MethodsFrom June to September 2018, all sputa received from presumptive TB patients at the Supranational Reference Laboratory for Tuberculosis of Cotonou were included in the study. From each patient, two specimens were collected (one spot and one early morning) and GeneXpert® MTB/RIF was performed on both specimens.ResultsIn total, 886 participants were included in the study, of whom 737 provided both sputa and 149 (16.8%) gave only the spot specimen. For the 737 participants who provided both sputa, GeneXpert® MTB/RIF was positive for both specimens in 152 participants; for three participants GeneXpert® MTB/RIF was positive on spot specimen but negative on morning specimen while for another three, the test was positive on morning specimen but negative on spot specimen. The overall percentage of agreement was excellent (99.2%) with a positive and negative percent agreement greater than 98%.ConclusionFor TB diagnosis under programmatic conditions in Cotonou, GeneXpert® MTB/RIF in spot specimens gave similar results with the test in morning specimens. Performing GeneXpert® MTB/RIF in both specimens did not significantly increase the number of cases detected. To avoid losing patients from the diagnostic cascade, it is preferable to test sputa produced at the time of the first visit at the health center.

Tuberculosis Drug Susceptibility, Treatment, and Outcomes for Belarusian HIV-Positive Patients with Tuberculosis: Results from a National and International Laboratory.

Background To cure drug-resistant (DR) tuberculosis (TB), the antituberculous treatment should be guided by Mycobacterium tuberculosis drug-susceptibility testing (DST). In this study, we compared conventional DST performed in Minsk, Belarus, a TB DR high-burden country, with extensive geno- and phenotypic analyses performed at the WHO TB Supranational Reference Laboratory in Copenhagen, Denmark, for TB/HIV coinfected patients. Subsequently, DST results were related to treatment regimen and outcome. Methods Thirty TB/HIV coinfected patients from Minsk were included and descriptive statistics applied. Results Based on results from Minsk, 10 (33%) TB/HIV patients had drug-sensitive TB. Two (7%) had isoniazid monoresistant TB, 8 (27%) had multidrug-resistant (MDR) TB, 5 (17%) preextensive drug-resistant (preXDR) TB, and 5 (17%) had extensive drug-resistant (XDR) TB. For the first-line drugs rifampicin and isoniazid, there was DST agreement between Minsk and Copenhagen for 90% patients. For the second-line anti-TB drugs, discrepancies were more pronounced. For 14 (47%) patients, there were disagreements for at least one drug, and 4 (13%) patients were classified as having MDR-TB in Minsk but were classified as having preXDR-TB based on DST results in Copenhagen. Initially, all patients received standard anti-TB treatment with rifampicin, isoniazid, pyrazinamide, and ethambutol. However, this was only suitable for 40% of the patients based on DST. On average, DR-TB patients were changed to 4 (IQR 3-5) active drugs after 1.5 months (IQR 1-2). After treatment adjustment, the treatment duration was 8 months (IQR 2-11). Four (22%) patients with DR-TB received treatment for >18 months. In total, sixteen (53%) patients died during 24 months of follow-up. Conclusions We found high concordance for rifampicin and isoniazid DST between the Minsk and Copenhagen laboratories, whereas discrepancies for second-line drugs were more pronounced. For patients with DR-TB, treatment was often insufficient and relevant adjustments delayed. This example from Minsk, Belarus, underlines two crucial points in the management of DR-TB: the urgent need for implementation of rapid molecular DSTs and availability of second-line drugs in all DR-TB high-burden settings. Carefully designed individualized treatment regimens in accordance with DST patterns will likely improve patients' outcome and reduce transmission with drug-resistant Mycobacterium tuberculosis strains.

Acquisition of Cross-Resistance to Bedaquiline and Clofazimine following Treatment for Tuberculosis in Pakistan

Aims and objectives: In the present study, we report on the first seven cases of acquired bedaquiline and clofazimine resistance identified in Pakistan. The study was conducted within the framework of a retrospective surveillance project to monitor the acquisition of resistance to bedaquiline implemented by the National TB Reference Laboratory in Pakistan and TB Supranational Reference Laboratory. Methods: Seventy three sequential isolates from 31 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing in 7H11 medium, and for all isolates showing an increased MIC compared to the baseline isolate, further MIC testing for bedaquiline and clofazimine was conducted using the Bactec MGIT960 (BD, Franklin Lakes, NJ, USA). The H37Rv (ATCC 27294) strain was used as a susceptible control in MIC testing. Bedaquiline dry powder was supplied by Janssen-Pharmaceutica (Beerse, Belgium). WGS was carried out with the Illumina Nextera-XT DNA sample preparation kit to prepare paired-end libraries of 150-bp read length to sequence on an Illumina NextSeq platform. Data analysis and single nucleotide polymorphism (SNP) calling were performed using the MTBseq-Pipeline on low-frequency detection mode. Genes associated with resistance to bedaquiline and/or clofazimine (atpE, Rv0678, pepQ, and Rv1979c) were screened for mutations. Results: We studied 73 isolates from patients (n=31; 8 MDR, 16 pre-XDR, and 7 XDR) enrolled in bedaquiline-containing regimens. The sequential isolates tested included two strains from 22 patients, three from 7 patients, and four from 2 patients. All baseline strains included in the study were sensitive to bedaquiline. Seven patients developed an increase in bedaquiline MICs in 7H11 medium (range, 0.125 to >0.5mg·liter−1) during therapy, and six of them became resistant to bedaquiline according to the current critical concentration proposed for the drug. In all seven patients we detected genetic variants in Rv0678 that were not present at baseline isolates. Moreover in five out of seven patients the treatment was ultimately failed. Conclusions: We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.

Drug-resistant tuberculosis diagnosis since Xpert® MTB/RIF introduction in Papua New Guinea, 2012-2017.

Xpert® MTB/RIF was introduced in Papua New Guinea in 2012 for the diagnosis of tuberculosis (TB) and of rifampicin-resistant TB (RR-TB), a marker of multi-drug-resistant TB (MDR-TB). To assess the concordance of Xpert with phenotypic drug susceptibility testing (DST) performed at the supranational reference laboratory and to describe the patterns of drug-resistant TB observed. This was a retrospective descriptive study of laboratory data collected from April 2012 to December 2017. In 69 months, 1408 specimens with Xpert results were sent for mycobacterial culture and DST; Mycobacterium tuberculosis was cultured from 63% (884/1408) and DST was completed in 99.4%. The concordance between Xpert and culture for M. tuberculosis detection was 98.6%. Of 760 RR-TB cases, 98.7% were detected using Xpert; 98.5% of 620 MDR-TB cases were identified using phenotypic DST. Phenotypic resistance to second-line drugs was detected in 59.4% (522/879) of specimens tested, including 29 with fluoroquinolone resistance; the majority were from the National Capital District and Daru Island. The high concordance between phenotypic DST and Xpert in identifying RR-TB cases supports the scale-up of initial Xpert testing in settings with high rates of drug resistance. However, rapid DST in addition to the detection of RR-TB is required.

Towards tailored regimens in the treatment of drug-resistant tuberculosis: a retrospective study in two Italian reference Centres

BackgroundThe increased incidence of drug-resistant TB is a major challenge for effective TB control. Limited therapeutic options and poor treatment outcomes of DR-TB may increase drug-resistance rates. The objective of the study is to retrospectively compare MDR-TB and pre-XDR-TB treatment regimens and outcomes in two large TB reference centres in Italy from January 2000 to January 2015.MethodsA retrospective, multicentre study was conducted at the Regional TB Reference Centre Villa Marelli Institute (Milan) and at the Reference Center for MDR-TB and HIV-TB, Eugenio Morelli Hospital (Sondalo). The supra-national Reference Laboratory in Milan performed DST. Inclusion criteria were: age ≥ 18 and culture-confirmed diagnosis of MDR- or pre-XDR TB. Chi-square or Fisher exact test was used to detect differences in the comparison between treatment outcomes, therapeutic regimens, and drug-resistances. Computations were performed with STATA 15.ResultsA total of 134 patients were selected. Median (IQR) age at admission was 33 (26–41) years and 90 patients (67.2%) were male. Pulmonary TB was diagnosed in 124 (92.5%) patients. MDR- and pre-XDR-TB cases were 91 (67.9%) and 43 (32.1%), respectively. The WHO shorter MDR-TB regimen could have been prescribed in 16/84 (19.1%) patients. Treatment success was not statistically different between MDR- and pre-XDR-TB (81.3% VS. 81.4%; P = 0.99). Mortality in MDR-TB and pre-XDR-TB groups was 4.4 and 9.3%, respectively (P = 0.2). Median duration of treatment was 18 months and a total of 110 different regimens were administered. Exposure to linezolid, meropenem, and amikacin was associated with a better outcome in both groups (P = 0.001, P < 0.001, and P = 0.004, respectively).ConclusionsTailored treatment regimens based on DST results can achieve successful outcomes in patients with pre-XDR-TB.

PO 8372 CULTURE-FREE APPROACHES FOR THE DIAGNOSIS AND MANAGEMENT OF PATIENTS WITH RIFAMPICIN RESISTANT TUBERCULOSIS: THE DIAMA PROJECT

BackgroundRecent advances in molecular diagnostics, especially the Xpert MTB/Rif test, have reduced the time to diagnose rifampicin resistant tuberculosis (RR-TB). However, with this test only rifampicin resistance is diagnosed, leading to presumptive diagnosis of resistance to isoniazid and maybe other drugs. In addition, culture on monthly sputum samples is currently recommended by the World Health Organization (WHO) for follow-up of RR-TB patients under treatment. Unfortunately, culture is often not locally available, and samples need to be shipped from field to culture laboratories. The associated transport delays lead to high rates of contamination and false-negative culture, particularly in laboratories in low-resource settings. Many gaps for the diagnosis and management of RR-TB patients still need to be addressed and the DIAMA project (Diagnostics for multidrug-resistant tuberculosis in Africa) aims to address some of them.MethodsThe TB Supranational Reference Laboratory of Benin leads a consortium of 11 partners involved in multiple-drug resistant TB care in Africa. The DIAMA project will explore the feasibility and accuracy of: i) diagnosing TB resistance to first and second line drugs through novel molecular multiplex assays developed by the company Genoscreen; ii) setting-up alternative culture-free approaches for the monitoring of patients’ response to rifamipcin-resistant treatment; iii) piloting whether the implementation of software by Data2Care Technologies for real-time monitoring of molecular test results can reduce delays between diagnosis and treatment of RR-TB patients. This project is funded by EDCTP for a period of five years.ConclusionTogether, these advances could dramatically improve the currently dismal prognosis of multiple-drug resistant TB in health systems in resource-poor settings. Through this presentation, we will share the background information, the design of this project and its progress.

Proficiency of WHO Global Foodborne Infections Network External Quality Assurance System Participants in Identification and Susceptibility Testing of Thermotolerant Campylobacter spp. from 2003 to 2012.

Campylobacter spp. are foodborne and waterborne pathogens. While rather accurate estimates for these pathogens are available in industrialized countries, a lack of diagnostic capacity in developing countries limits accurate assessments of prevalence in many regions. Proficiency in the identification and susceptibility testing of these organisms is critical for surveillance and control efforts. The aim of the study was to assess performance for identification and susceptibility testing of thermotolerant Campylobacter spp. among laboratories participating in the World Health Organization (WHO) Global Foodborne Infections Network (GFN) External Quality Assurance System (EQAS) over a 9-year period. Participants (primarily national-level laboratories) were encouraged to self-evaluate their performance as part of continuous quality improvement. The ability to correctly identify Campylobacter spp. varied by year and ranged from 61.9% (2008) to 90.7% (2012), and the ability to correctly perform antimicrobial susceptibility testing (AST) for Campylobacter spp. appeared to steadily increase from 91.4% to 93.6% in the test period (2009 to 2012). The poorest performance (60.0% correct identification and 86.8% correct AST results) was observed in African laboratories. Overall, approximately 10% of laboratories reported either an incorrect identification or antibiogram. As most participants were supranational reference laboratories, these data raise significant concerns regarding capacity and proficiency at the local clinical level. Addressing these diagnostic challenges is critical for both patient-level management and broader surveillance and control efforts.

Multi-clonal evolution of multi-drug-resistant/extensively drug-resistant Mycobacterium tuberculosis in a high-prevalence setting of Papua New Guinea for over three decades.

An outbreak of multi-drug resistant (MDR) tuberculosis (TB) has been reported on Daru Island, Papua New Guinea. Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual of drug resistance mutations have not been described. Whole genome sequencing of 100 of 165 clinical isolates referred from Daru General Hospital to the Supranational reference laboratory, Brisbane, during 2012–2015 revealed that 95 belonged to a single modern Beijing sub-lineage strain. Molecular dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with potentially enhanced virulence mediated by an mycP1 mutation. The Beijing sub-lineage strain demonstrated a high degree of co-resistance between isoniazid and ethionamide (80/95; 84.2 %) attributed to an inhA promoter mutation combined with inhA and ndh coding mutations. Multi-drug resistance, observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together with a compensatory rpoC mutation in the 1980s. There was independent acquisition of fluoroquinolone and aminoglycoside resistance, and evidence of local transmission of extensively drug resistant (XDR) strains from 2009. These findings underline the importance of whole genome sequencing in informing an effective public health response to MDR/XDR TB.

Molecular characterization of Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Khartoum, Sudan.

The aim of this study was to characterize the drug resistance profile, and the specific lineages of Mycobacterium tuberculosis (MTB) strains isolated from patients with pulmonary TB in the state of Khartoum in Sudan. Consecutive sputum samples and clinical data were collected from 406 smear-positive TB patients with pulmonary TB in 2007-2009. The samples were cultured, and drug susceptibility testing (DST) was performed using the proportion method (PM) on solid Löwenstein-Jensen medium, and species were identified using biochemical methods at the National Reference Laboratory (NRL) in Khartoum. Extracted deoxyribonucleic acid from a total of 120, 60 suspected multidrug-resistant isolates (MDR), and 60 non-MDR isolates were subsequently sent to the WHO supranational reference laboratory (SRL) in Stockholm at the Public Health Agency of Sweden, for confirmation of the drug resistance profile, examinations by line probe assay (LPA), and molecular epidemiology analysis with Spoligotyping. LPA results correlated 100% for non-MDR and 62% for the suspected MDR strains when compared to the DST results obtained by PM at the NRL. Two strains were initially using the PM identified as MDR-TB but later shown by Hain GenoType Mycobacterium CM/AS to belong Mycobacterium avium complex (Mycobacterium intracelluare). These two strains were excluded from the study material for further analysis. The remaining 58 MDR strains were analyzed using LPA, and 36 strains were confirmed as MDR, 10 as rifampicin monoresistant, and eight as isoniazid-monoresistant. Spoligotyping for all the 118 MTB isolates revealed a total of 115 patterns in which four patterns represented major clusters with a total of 108 (91%) of the strains. The CAS1_Delhi/family was the predominant type and detected in 62 isolates (52%), of which 26 were MDR and 36 were susceptible. It was followed by H3/family with 19 (16%) strains, and 11 Latin American Mediterranean3/family, 16 T2/T1, and two strains each of the Beijing and S lineage. Comparison of DST results obtained using PM and LPA showed 100% agreement for the non-MDR strains but only 62% for the MDR strains. Taking in consideration the time, risk of contamination and the cost of labour to identify MDR TB, the LPA have clear advantages in early detection of MDRTB than the PM. Additionally in this study material Spoligotyping revealed the CAS1 Delhi as the most predominant family. We could not see no major difference in lineages between MDR and non-MDR strains.

Culture and Next-generation sequencing-based drug susceptibility testing unveil high levels of drug-resistant-TB in Djibouti: results from the first national survey

Djibouti is a small country in the Horn of Africa with a high TB incidence (378/100,000 in 2015). Multidrug-resistant TB (MDR-TB) and resistance to second-line agents have been previously identified in the country but the extent of the problem has yet to be quantified. A national survey was conducted to estimate the proportion of MDR-TB among a representative sample of TB patients. Sputum was tested using XpertMTB/RIF and samples positive for MTB and resistant to rifampicin underwent first line phenotypic susceptibility testing. The TB supranational reference laboratory in Milan, Italy, undertook external quality assurance, genotypic testing based on whole genome and targeted-deep sequencing and phylogenetic studies. 301 new and 66 previously treated TB cases were enrolled. MDR-TB was detected in 34 patients: 4.7% of new and 31% of previously treated cases. Resistance to pyrazinamide, aminoglycosides and capreomycin was detected in 68%, 18% and 29% of MDR-TB strains respectively, while resistance to fluoroquinolones was not detected. Cluster analysis identified transmission of MDR-TB as a critical factor fostering drug resistance in the country. Levels of MDR-TB in Djibouti are among the highest on the African continent. High prevalence of resistance to pyrazinamide and second-line injectable agents have important implications for treatment regimens.

Evaluation of Mycobacterium tuberculosis viability in OMNIgene-SPUTUM reagent upon multi-day transport at ambient temperature

BackgroundMaintaining the quality of clinical specimens for tuberculosis (TB) testing is a major challenge in many high TB burden-limited resources countries. Sample referral systems in low and middle income countries are often weak and the maintenance of the cold-chain challenging and very costly for TB programs. The development of transport media allowing the preservation of samples without refrigeration is critical for increasing access to TB diagnostic services and for reducing the costs related to testing.MethodsWe evaluated the performance of OMNIgene-SPUTUM (OM-S) reagent for the maintenance of Mycobacterium tuberculosis (MTB) viability in sputum samples in the absence of refrigeration and its capacity to stabilize nucleic acid for molecular testing. A total of 329 sputum specimens from presumptive TB cases collected at the National Reference Laboratory in Tirana, Albania, were either decontaminated by a conventional method or processed with OM-S reagent and stored at room temperature. Samples in OM-S were shipped to the Supranational Reference Laboratory in Milan, Italy, at various times and processed for liquid culture.ResultsOur data show that OM-S maintains MTB viability for at least three weeks in the absence of refrigeration and improves the quality of culture resulting in a contamination rate lower than 0.5%. However, a significant delay in the time to culture positivity was observed for samples stored for more than two weeks in OM-S.ConclusionsOverall, OM-S offers multiple benefits both at laboratory and TB national program level by increasing the availability to quality diagnostics, promoting access to health care services and strengthening TB patient care especially in hard to reach populations.

Drug resistance among extrapulmonary TB patients: Six years experience from a supranational reference laboratory.

Background & objectives:There is limited information available about the drug resistance patterns in extrapulmonary tuberculosis (EPTB), especially from high burden countries. This may be due to difficulty in obtaining extrapulmonary specimens and limited facilities for drug susceptibility testing. This study was undertaken to review and report the first and second-line anti-TB drug susceptibility patterns in extrapulmonary specimens received at the National Institute for Research in Tuberculosis (NIRT), Chennai, India, between 2005 and 2012.Methods:Extrapulmonary specimens received from referring hospitals were decontaminated and cultured using standard procedures. Drug susceptibility testing (DST) for Mycobacterium tuberculosis was done by absolute concentration or resistance ratio methods for the first and the second line anti-TB drugs.Results:Between 2005 and 2012, of the 1295 extrapulmonary specimens, 189 grew M. tuberculosis, 37 (19%) cases were multidrug resistant (MDR) while one was extensively drug resistant (XDR). Specimen-wise MDR prevalence was found to be: CSF-10 per cent, urine-6 per cent, fluids and aspirates-27 per cent, pus-23 per cent, lymph nodes-19 per cent. Resistance to isoniazid and ethionamide was found to be high (31 and 38%, respectively).Interpretation & conclusions:Drug resistance including MDR-TB was observed in a significant proportion of extrapulmonary specimens referred for DST. Access to culture and DST for extrapulmonary specimens should be expanded. Guidelines for MDR-TB management should have explicit sections on extra-pulmonary tuberculosis and training on laboratory techniques is urgently required.

Molecular characterization of multi drug-resistant tuberculosis isolated from Baghdad, Iraq

BackgroundTuberculosis (TB) remains a major cause of mortality and morbidity all over the world. Multi drug-resistant tuberculosis (MDR-TB) is an important cause for mortality among TB patients, and management of MDR-TB remains a major challenge for national TB programs. Early detection of MDR-TB provides better treatment outcomes and reduces the transmission of MDR. Nucleic acid amplification test (NAAT) like Line Probe Assays have been recently endorsed for use in low-income settings and can be used to screen smear-positive sputum specimens for diagnosing of resistance to rifampicin and isoniazid in 1–2days. ObjectiveOverall objective of the study was to evaluate performances of Line Probe Assay GenoType® MTBDRplus (Hain Life science, GmbH, Nehren, Germany) to move forward its introduction into routine diagnostics. Secondary objective was to determine the type of mutations in rpoB, katG and inhA genes from culture specimens isolated from Iraqi selected MDR-TB patients. Materials and methodsThe DNA was extracted by CTAB method from 51 clinical isolates which were previously characterized as MDR strains in reference laboratory/center of Chest and Respiratory Diseases obtained from patients living in Baghdad/Iraq (2010–2011). GenoType® MTBDRplus ver. 2.0 (HainLifescience, GmbH, Nehren, Germany) was performed at the Supra-national TB Reference Laboratory in Milan (Italy) as well as interpretation of mutations involved in drug resistance to rifampin and Isoniazide according to manufacturer’s instructions. Results and conclusionLine Probe Assays were performed on 51 MDR of Mycobacterium tuberculosis strains; 6 strains were excluded from the analysis due to un-interpretable results; 5 strains were identified as sensitive to both rifampicin (RIF) and isoniazid (INH) by MTBDR plus assay. The most common genetic mutation conferring RIF resistance was S531L of rpoB gene which was detected in 33 (82.5%) resistant strains. Other mutations in this gene were D516Vand H526Y which were detected in a single strain (2.5%) for each. Also, there were 5 (12.5%) RIF-resistant strains with mutations that could not be identified specifically in this assay.Isoniazid (INH) resistance due to katG mutation S315T1 was found in 17 (42.5%) strains of INH-resistant M. tuberculosis. The second most common site of mutation was in the upstream promoter sequence of inhA, which was found in 15 (37.5%) strains, 14 (35%) strains of which carried a C→T transition at the −15 position, while single strains (2.5%) carried a T→A mutation at the −8 position. The result showed 8 (20%) strains missing a band on the wild-type region of these strains, but no hybridization with mutation probes on this gene suggested resistance due to mutations other than those included in this assay.Regardless the type of mutations detected, GenoType® MTBDR plus ver. 2.0 showed an overall accuracy of 95.5% (C.I. 95% 89.9–98.1) in detecting MDR strains. Despite the relatively low number of MDR strains tested, they show very common mutations patterns being the majority mutated at the codon 531 and 315 of rpoB and katG gene. In such setting, GenoType® MTBDR plus ver. 2.0 could be a great tool to rapidly screen for MDR-TB and has the potential to substantially reduce the turnaround time of drug sensitivity test (DST) results.

Drug resistance of Mycobacterium tuberculosis in Malawi: a cross-sectional survey.

ObjectiveTo document the prevalence of multidrug resistance among people newly diagnosed with – and those retreated for – tuberculosis in Malawi.MethodsWe conducted a nationally representative survey of people with sputum-smear-positive tuberculosis between 2010 and 2011. For all consenting participants, we collected demographic and clinical data, two sputum samples and tested for human immunodeficiency virus (HIV).The samples underwent resistance testing at the Central Reference Laboratory in Lilongwe, Malawi. All Mycobacterium tuberculosis isolates found to be multidrug-resistant were retested for resistance to first-line drugs – and tested for resistance to second-line drugs – at a Supranational Tuberculosis Reference Laboratory in South Africa.FindingsOverall, M. tuberculosis was isolated from 1777 (83.8%) of the 2120 smear-positive tuberculosis patients. Multidrug resistance was identified in five (0.4%) of 1196 isolates from new cases and 28 (4.8%) of 581 isolates from people undergoing retreatment. Of the 31 isolates from retreatment cases who had previously failed treatment, nine (29.0%) showed multidrug resistance. Although resistance to second-line drugs was found, no cases of extensive drug-resistant tuberculosis were detected. HIV testing of people from whom M. tuberculosis isolates were obtained showed that 577 (48.2%) of people newly diagnosed and 386 (66.4%) of people undergoing retreatment were positive.ConclusionThe prevalence of multidrug resistance among people with smear-positive tuberculosis was low for sub-Saharan Africa – probably reflecting the strength of Malawi’s tuberculosis control programme. The relatively high prevalence of such resistance observed among those with previous treatment failure may highlight a need for a change in the national policy for retreating this subgroup of people with tuberculosis.