Multiplexed RT-qPCR Coupled with Whole-Genome Sequencing to Monitor a SARS-CoV-2 Omicron Variant of Concern in a Hospital Laboratory Setting in Latvia.

At the end of 2021, the SARS-CoV-2 Omicron variant of concern (VOC) displaced the previously dominant Delta VOC and enhanced diagnostic and therapeutic challenges worldwide. Respiratory specimens submitted to the Riga East University Hospital Laboratory Service by the central and regional hospitals of Latvia from January to March 2022 that were positive for SARS-CoV-2 RNA were tested by commercial multiplexed RT-qPCR targeting three of the Omicron VOC signature mutations: ΔH69/V70, E484A, and N501Y. Of the specimens tested and analyzed in parallel by whole-genome sequencing (WGS), 964 passed the internal quality criteria (genome coverage ≥90%, read depth ≥400×) and the Nextstrain's quality threshold for "good". We validated the detection accuracy of RT-qPCR for each target individually by using WGS as a control. The results were concordant with both approaches for 938 specimens, with the correct classification rate exceeding 96% for each target (CI 95%); however, the presumptive WHO label was misassigned for 21 specimens. The RT-qPCR genotyping provided an acceptable means to pre-monitor the prevalence of the two presumptive Omicron VOC sublineages, BA.1 and BA.2.

Abundance and prevalence of ESBL coding genes in patients undergoing first line eradication therapy for Helicobacter pylori.

The spread of extended-spectrum beta-lactamases (ESBLs) in nosocomial and community-acquired enterobacteria is an important challenge for clinicians due to the limited therapeutic options for infections that are caused by these organisms. Here, we developed a panel of ESBL coding genes, evaluated the abundance and prevalence of ESBL encoding genes in patients undergoing H. pylori eradication therapy, and summarized the effects of eradication therapy on functional profiles of the gut microbiome. To assess the repertoire of known beta lactamase (BL) genes, they were divided into clusters according to their evolutionary relation. Primers were designed for amplification of cluster marker regions, and the efficiency of this amplification panel was assessed in 120 fecal samples acquired from 60 patients undergoing H. pylori eradication therapy. In addition, fecal samples from an additional 30 patients were used to validate the detection efficiency of the developed ESBL panel. The presence for majority of targeted clusters was confirmed by NGS of amplification products. Metagenomic sequencing revealed that the abundance of ESBL genes within the pool of microorganisms was very low. The global relative abundances of the ESBL-coding gene clusters did not differ significantly among treatment states. However, at the level of each cluster, classical ESBL producers such as Klebsiella sp. for blaOXY (p = 0.0076), Acinetobacter sp. for blaADC (p = 0.02297) and others, differed significantly with a tendency to decrease compared to the pre- and post-eradication states. Only 13 clusters were common across all three datasets, suggesting a patient-specific distribution profile of ESBL-coding genes. The number of AMR genes detected in the post-eradication state was higher than that in the pre-eradication state, which could be attributed, at least in part, to the therapy. This study demonstrated that the ESBL screening panel was effective in targeting ESBL-coding gene clusters from bacterial DNA and that minor differences exist in the abundance and prevalence of ESBL-coding gene levels before and after eradication therapy.

Toll-Like Receptor 1 Locus Re-examined in a Genome-Wide Association Study Update on Anti-Helicobacter pylori IgG Titers.

BACKGROUND & AIMS: A genome-wide significant association between anti-Helicobacter pylori (H pylori) IgG titers and Toll-like receptor (TLR1/6/10) locus on 4p14 was demonstrated for individuals of European ancestry, but not uniformly replicated. We re-investigated this association in an updated genome-wide association study (GWAS) meta-analysis for populations with low gastric cancer incidence, address potential causes of cohort heterogeneity, and explore functional implications of genetic variation at the TLR1/6/10 locus. METHODS: The dichotomous GWAS (25% individuals exhibiting highest anti-H pylori IgG titers vs remaining 75%) included discovery and replication sampls of, respectively, n = 15,685 and n = 9676, all of European ancestry. Longitudinal analysis of serologic data was performed on H pylori-eradicated subjects (n = 132) and patients under surveillance for premalignant gastric lesions (n = 107). TLR1/6/10 surface expression, TLR1 mRNA, and cytokine levels were measured in leukocyte subsets of healthy subjects (n = 26) genotyped for TLR1/6/10 variants. RESULTS: The association of the TLR1/6/10 locus with anti-H pylori IgG titers (rs12233670; ß = -0.267 ± SE 0.034; P = 4.42 × 10-15) presented with high heterogeneity and failed replication. Anti-H pylori IgG titers declined within 2-4 years after eradication treatment (P = 0.004), and decreased over time in patients with premalignant gastric lesions (P < 0.001). Variation at the TLR1/6/10 locus affected TLR1-mediated cytokine production and TLR1 surface expression on monocytes (P = 0.016) and neutrophils (P = 0.030), but not mRNA levels. CONCLUSIONS: The association between anti-H pylori IgG titers and TLR1/6/10 locus was not replicated across cohorts, possibly owing to dependency of anti-H pylori IgG titers on therapy, clearance, and antibody decay. H pylori-mediated immune cell activation is partly mediated via TLR1 signaling, which in turn is affected by genetic variation.

Profiles of Volatile Biomarkers Detect Tuberculosis from Skin.

Tuberculosis (TB) is an infectious disease that threatens >10 million people annually. Despite advances in TB diagnostics, patients continue to receive an insufficient diagnosis as TB symptoms are not specific. Many existing biodiagnostic tests are slow, have low clinical performance, and can be unsuitable for resource-limited settings. According to the World Health Organization (WHO), a rapid, sputum-free, and cost-effective triage test for real-time detection of TB is urgently needed. This article reports on a new diagnostic pathway enabling a noninvasive, fast, and highly accurate way of detecting TB. The approach relies on TB-specific volatile organic compounds (VOCs) that are detected and quantified from the skin headspace. A specifically designed nanomaterial-based sensors array translates these findings into a point-of-care diagnosis by discriminating between active pulmonary TB patients and controls with sensitivity above 90%. This fulfills the WHO's triage test requirements and poses the potential to become a TB triage test.

Biomarkers of treatment success in fully sensitive pulmonary tuberculosis patients: a multicenter longitudinal study.

Aim: Novel biomarkers that are able to accurately monitor tuberculosis (TB) treatment effectiveness are needed to adjust therapy and identify a need for a regimen change. Materials & methods: In our study, conducted on a cohort comprising 100 pulmonary TB patients, we analyzed the role of plasma cytokines and Toll-like receptors expression as biomarkers of treatment response. Results: Changes in toll-interacting protein (TOLLIP) and lymphocyte antigen 96 (LY96) gene expression as well as nine cytokine levels over the first 2 months were significantly associated with successful treatment outcome. Successful treatment was associated with higher serum concentration of Toll-like receptor-2. Conclusion: Our results suggest that differential expression of specific effector molecules and dynamics of selected cytokines may help to identify those responding to TB treatment early.

Lack of significant differences between gastrointestinal tract microbial population structure of Helicobacter pylori-infected subjects before and 2 years after a single eradication event.

BACKGROUND: According to recent estimates 80% of Latvian population is infected with Helicobacter pylori thus their susceptibility to numerous gastric tract diseases is increased. The 1st line H. pylori eradication therapy includes treatment with clarithromycin in combination with amoxicillin or metronidazole and a proton pump inhibitor. However, potential adverse events caused by such therapies to microbiome are insufficiently studied. OBJECTIVE: This study aimed to evaluate the long-term effect of H. pylori eradication on human gastrointestinal tract (GIT) microbiome. METHODS: The assessment of H pylori eradication impact on GIT microbiome was done by analyzing 120 samples acquired from 60 subjects. Each individual was prescribed the following 10-day eradication regimen: Esomeprazolum 40 mg, Clarithromycinum 500 mg, and Amoxicillinum 1000 mg, BID. Samples from each individual were collected before starting H pylori eradication therapy, and 2 years after the completion of the therapy in OC-Sensor (Eiken Chemical Co.) sample collection containers and stored at -86°C. Prior to DNA extraction, the samples were lyophilized, and total DNA was extracted using FastDNA Spin Kit for Soil. 16S V3 rRNA gene sequencing was done employing Ion Torrent PGM, and the obtained raw sequences were analyzed using vsearch and R (phyloseq, cluster packages). RESULTS: Alpha diversity measurements-observed OTUs, Chao1 and Shannon index did not differ significantly between the pre- and post-eradication states (two-tailed paired t test: P = .95; P = .71, P = .24, respectively). Unweighted and weighted UniFrac distances of beta diversity analysis indicated a non-specific pattern of sample clustering. Enterotype shift was observed for the majority of individuals comparing pre- and post-eradication study groups. Association analysis revealed that certain bacterial genera significantly correlated with age (eg, Dialister, Paraprevotella, Bifidobacterium), individual (eg, Thermotunica, Streptomyces, Faecalibacterium), and history of respiratory and/or allergic diseases (eg, Colinsella, Faecalibacterium). Redundancy analysis confirmed that the individual was a significant determinant of the subject's microbial community composition (ANOVA, 999 perm., P = .001) with the further lower impact of subject-specific medical history (eg, medication used as prescribed: P = .005, history of cardiovascular diseases: P = .005, history of respiratory, and/or allergic diseases: P = .015) and physiological (eg, age: P = .005, gender: P = .02) parameters. In the post-eradication study group, number of influential genera (n = 260) was increased compared to the pre-eradication study group (n = 209). CONCLUSION: Modest global differences at the community level exist between individuals before and after the eradication therapy; however, the microbiome structure is more related to the subject-specific parameters rather than by the eradication therapy itself.

Analysis of Mycobacterium tuberculosis genetic lineages circulating in Riga and Riga region, Latvia, isolated between 2008 and 2012.

Although the number of new tuberculosis (TB) cases registered per year has decreased by 3-fold between 2001 and 2017 in Latvia, the TB incidence and rates of multidrug resistant TB in this Baltic country remain substantially higher than in most other European countries. Molecular typing methods of Mycobacterium tuberculosis (MTB) play an important role both in clinical studies of the disease and the epidemiological investigations, allowing to describe and characterize the pathogen's population structure and spread of particular genotypes. Aim of this study was to examine the prevalence of MTB lineages in Riga and Riga region of Latvia within a five-year period (2008-2012), and to evaluate the discriminatory power (DP) of spoligotyping, standard 24-locus MIRU-VNTR and IS6110-RFLP methods in this setting. The results showed that the main MTB spoligotype families were Beijing (25.3%) and LAM (24.3%), followed by T (22.1%), Ural (11.2%), Haarlem (6.6%) and X superfamily (3.4%). This distribution remained stable over the five consecutive years. 67.6% of MTB isolates were pan-susceptible, and 32.4% were resistant to any drug; multi-drug resistance was found in 5.8% of MTB strains, and 7.6% of MTB isolates were extensively drug-resistant. Drug resistance was associated with SIT1, SIT283 and SIT42 genotypes, while SIT1 and SIT42 were overrepresented among multi drug-resistant MTB strains. Overall, DP of spoligotyping method alone was 0.8953, while DP of both 24-locus MIRU-VNTR analysis and IS6110 RFLP was higher (DP = 0.9846 and 0.9927, respectively), mainly due to the improvement of the resolution for the Beijing strains. In conclusion, this work represents the first comprehensive molecular epidemiological description of TB in Latvia, highlighting the high genetic diversity of MTB strains circulating in Riga and Riga region. In combination with detailed epidemiological data this approach was helpful for the in-depth understanding of epidemiological processes in settings where the Next-Gen sequencing is not available as a routine method.

Generation of a novel next-generation sequencing-based method for the isolation of new human papillomavirus types.

With the advent of new molecular tools, the discovery of new papillomaviruses (PVs) has accelerated during the past decade, enabling the expansion of knowledge about the viral populations that inhabit the human body. Human PVs (HPVs) are etiologically linked to benign or malignant lesions of the skin and mucosa. The detection of HPV types can vary widely, depending mainly on the methodology and the quality of the biological sample. Next-generation sequencing is one of the most powerful tools, enabling the discovery of novel viruses in a wide range of biological material. Here, we report a novel protocol for the detection of known and unknown HPV types in human skin and oral gargle samples using improved PCR protocols combined with next-generation sequencing. We identified 105 putative new PV types in addition to 296 known types, thus providing important information about the viral distribution in the oral cavity and skin.

Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study.

BACKGROUND: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis. METHODS: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing. FINDINGS: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87-94) for rpoB (rifampicin resistance), 86% (74-93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39-68) for pncA (pyrazinamide resistance), 85% (77-91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81-92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing. INTERPRETATION: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation. FUNDING: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.

Tuberculosis cases caused by heterogeneous infection in Eastern Europe and their influence on outcomes.

INTRODUCTION: Mycobacterium tuberculosis superinfection is known to occur in areas with high rates of tuberculosis (TB) and has a significant impact on overall clinical TB management. AIM: We aimed to estimate the superinfection rate in cohorts of drug sensitive and multi-drug resistant tuberculosis (MDR TB) patients from Eastern Europe and the potential role of a second MDR TB strain infecting a patient with active non-MDR TB in treatment outcome. METHODS: The study population included 512 serial M. tuberculosis isolates obtained from 84 MDR- and 136 non-MDR TB patients recruited sequentially at sites in Lithuania, Latvia and Russia in 2011-2013. Strains were genotyped using standardized 24-loci Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing. RESULTS: Changes in two or more MIRU-VNTR loci suggesting superinfection were detected in 13 patients (5.9%). We found 4 initially non-MDR TB patients superinfected with an MDR TB strain during treatment and 3 of them had an unsuccessful outcome. CONCLUSIONS: An unsuccessful treatment outcome in patients initially diagnosed with drug sensitive TB might be explained by superinfection with an MDR TB strain. Bacteriological reversion could be indicative of superinfection with another strain. Archiving of all serial isolates and their genotyping in case of culture reversion could support therapeutic strategies in high MDR TB burden settings if resources are available.

MIRU-VNTR genotype diversity and indications of homoplasy in M. avium strains isolated from humans and slaughter pigs in Latvia.

Diseases which are caused by non-tuberculous mycobacteria (NTM) are an increasing problem in the developed countries. In Latvia, one of the most clinically important members of NTM is Mycobacterium avium (M. avium), an opportunistic pathogen which has been isolated from several lung disease patients and tissue samples of slaughter pigs. This study was designed to characterize the genetic diversity of the M. avium isolates in Latvia and to compare the distribution of genotypic patterns among humans and pigs. Eleven (Hall and Salipante, 2010) clinical M. avium samples, isolated from patients of Center of Tuberculosis and Lung Diseases (years 2003-2010), and 32 isolates from pig necrotic mesenterial lymph nodes in different regions (years 2003-2007) were analyzed. The majority (42 of 43) of samples were identified as M. avium subsp. hominissuis; one porcine isolate belonged to M. avium subsp. avium. MIRU-VNTR genotyping revealed 13 distinct genotypes, among which nine genotype patterns, including M. avium subsp. avium isolate, were newly identified. IS1245 RFLP fingerprinting of 25 M. avium subsp. hominissuis samples yielded 17 different IS1245 RFLP patterns, allowing an efficient discrimination of isolates. Clusters of identical RFLP profiles were observed within host species, geographical locations and time frame of several years. Additional in silico analysis on simulated MIRU-VNTR genotype population datasets showed that the MIRU-VNTR pattern similarity could partly arise due to probabilistic increase of acquiring homoplasy among subpopulations, thus the similar MIRU-VNTR profiles of M. avium strains even in close geographical proximity should be interpreted with caution.

Decreased Time to Treatment Initiation for Multidrug-Resistant Tuberculosis Patients after Use of Xpert MTB/RIF Test, Latvia.

Few studies have examined whether the Xpert MTB/RIF test improves time to treatment initiation for persons with multidrug-resistant tuberculosis (MDR TB). We determined the impact of this test in Latvia, where it was introduced in 2010. After descriptive analyses of pulmonary MDR TB patients in Latvia during 2009-2012, time to treatment initiation was calculated, and univariate and multivariable accelerated failure time models were constructed. Univariate results showed strong evidence of an association between having rifampin-resistant TB detected by Xpert MTB/RIF and reduced time to treatment initiation versus the test not being used. A multivariable model stratifying by previous TB showed similar results. Our finding that in Latvia, time to treatment initiation was decreased for MDR TB cases that were rifampin-resistant TB by XpertMTB/RIF has implications for the use of this test in other settings with a high burden of MDR TB in which rifampin resistance is highly predictive of MDR TB.

Multidrug-Resistant Tuberculosis Treatment Outcomes in Relation to Treatment and Initial Versus Acquired Second-Line Drug Resistance.

BACKGROUND: Resistance to second-line drugs develops during treatment of multidrug-resistant (MDR) tuberculosis, but the impact on treatment outcome has not been determined. METHODS: Patients with MDR tuberculosis starting second-line drug treatment were enrolled in a prospective cohort study. Sputum cultures were analyzed at a central reference laboratory. We compared subjects with successful and poor treatment outcomes in terms of (1) initial and acquired resistance to fluoroquinolones and second-line injectable drugs (SLIs) and (2) treatment regimens. RESULTS: Of 1244 patients with MDR tuberculosis, 973 (78.2%) had known outcomes and 232 (18.6%) were lost to follow-up. Among those with known outcomes, treatment succeeded in 85.8% with plain MDR tuberculosis, 69.7% with initial resistance to either a fluoroquinolone or an SLI, 37.5% with acquired resistance to a fluoroquinolone or SLI, 29.3% with initial and 13.0% with acquired extensively drug-resistant tuberculosis (P < .001 for trend). In contrast, among those with known outcomes, treatment success increased stepwise from 41.6% to 92.3% as the number of drugs proven effective increased from ≤1 to ≥5 (P < .001 for trend), while acquired drug resistance decreased from 12% to 16% range, depending on the drug, down to 0%-2% (P < .001 for trend). In multivariable analysis, the adjusted odds of treatment success decreased 0.62-fold (95% confidence interval, .56-.69) for each increment in drug resistance and increased 2.1-fold (1.40-3.18) for each additional effective drug, controlling for differences between programs and patients. Specific treatment, patient, and program variables were also associated with treatment outcome. CONCLUSIONS: Increasing drug resistance was associated in a logical stepwise manner with poor treatment outcomes. Acquired resistance was worse than initial resistance to the same drugs. Increasing numbers of effective drugs, specific drugs, and specific program characteristics were associated with better outcomes and less acquired resistance.

Genotypic and phenotypic characteristics of aminoglycoside-resistant Mycobacterium tuberculosis isolates in Latvia.

Mutations causing resistance to aminoglycosides, such as kanamycin (KAN), amikacin (AMK), and streptomycin, are not completely understood. In this study, polymorphisms of aminoglycoside resistance influencing genes such as rrs, eis, rpsL, and gidB in 41 drug-resistant and 17 pan-sensitive Mycobacterium tuberculosis clinical isolates in Latvia were analyzed. Mutation A1400G in rrs gene was detected in 92% isolates with high resistance level to KAN and diverse MIC level to AMK. Mutations in promoter region of eis were detected in 80% isolates with low-level MIC of KAN. The association of K43R mutation in rpsL gene, a mutation in the rrs gene at position 513, and various polymorphisms in gidB gene with distinct genetic lineages of M. tuberculosis was observed. The results of this study suggest that association of different controversial mutations of M. tuberculosis genes to the drug resistance phenotype should be done in respect to genetic lineages.

Utility of propidium monoazide viability assay as a biomarker for a tuberculosis disease.

Reliable laboratory diagnosis of tuberculosis (TB), including laboratory biomarkers of cure, remains a challenge. In our study we evaluated the performance of a Propidium Monoazide (PMA) assay for the detection of viable TB bacilli in sputum specimens during anti-TB chemotherapy and its potential use as a TB biomarker. The study was conducted at three centres on 1937 sputum specimens from 310 adult bacteriologically confirmed pulmonary TB patients obtained before commencing anti-TB treatment and at regular intervals afterwards. Performance of the PMA assay was assessed using various readout assays with bacteriology culture results and time to positivity on liquid media used as reference standards. Treatment of sputum with N-acetyl-cysteine was found to be fully compatible with the PMA assay. Good sensitivity and specificity (97.5% and 70.7-80.0%) for detection of live TB bacilli was achieved using the Xpert(®) MTB/RIF test as a readout assay. Tentative Ct and ΔCt thresholds for the Xpert(®) MTB/RIF system were proposed. Good correlation (r = 0.61) between Ct values and time to positivity of TB cultures on liquid media was demonstrated. The PMA method has potential in monitoring bacterial load in sputum specimens and so may have a role as a biomarker of cure in TB treatment.

Delamanid improves outcomes and reduces mortality in multidrug-resistant tuberculosis.

Multidrug-resistant and extensively drug-resistant tuberculosis (TB) are associated with worse treatment outcomes for patients, including higher mortality, than for drug-sensitive tuberculosis. Delamanid (OPC-67683) is a novel anti-TB medication with demonstrated activity against multidrug-resistant disease. Patients who participated in the previously reported randomised, placebo-controlled trial of delamanid and the subsequent open-label extension trial were eligible to participate in a 24-month observational study designed to capture treatment outcomes. Treatment outcomes, as assessed by clinicians and defined by the World Health Organization, were categorised as favourable and unfavourable. Delamanid treatment groups were combined for analysis, based on their duration of treatment. In total, for 421 (87.5%) out of 481 patients from the original randomised controlled trial, consent was granted for follow-up assessments. Favourable outcomes were observed in 143 (74.5%) out of 192 patients who received delamanid for ≥6 months, compared to 126 (55%) out of 229 patients who received delamanid for ≤2 months. Mortality was reduced to 1.0% among those receiving long-term delamanid versus short-term/no delamanid (8.3%; p<0.001). Treatment benefit was also seen among patients with extensively drug-resistant TB. This analysis suggests that treatment with delamanid for 6 months in combination with an optimised background regimen can improve outcomes and reduce mortality among patients with both multidrug-resistant and extensively drug-resistant TB.

Multicentre laboratory validation of the colorimetric redox indicator (CRI) assay for the rapid detection of extensively drug-resistant (XDR) Mycobacterium tuberculosis.

OBJECTIVES: To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin. METHODS: The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method. RESULTS: Phase I: a strain was considered resistant by the CRI assay if the MIC was ≥0.5 mg/L for rifampicin, ≥0.25 mg/L for isoniazid, ≥4.0 mg/L for ofloxacin and ≥5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively. CONCLUSIONS: Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.