Risk factors for tuberculosis treatment failure, default, or relapse and outcomes of retreatment in Morocco.

BACKGROUND: Patients with tuberculosis require retreatment if they fail or default from initial treatment or if they relapse following initial treatment success. Outcomes among patients receiving a standard World Health Organization Category II retreatment regimen are suboptimal, resulting in increased risk of morbidity, drug resistance, and transmission.. In this study, we evaluated the risk factors for initial treatment failure, default, or early relapse leading to the need for tuberculosis retreatment in Morocco. We also assessed retreatment outcomes and drug susceptibility testing use for retreatment patients in urban centers in Morocco, where tuberculosis incidence is stubbornly high. METHODS: Patients with smear- or culture-positive pulmonary tuberculosis presenting for retreatment were identified using clinic registries in nine urban public clinics in Morocco. Demographic and outcomes data were collected from clinical charts and reference laboratories. To identify factors that had put these individuals at risk for failure, default, or early relapse in the first place, initial treatment records were also abstracted (if retreatment began within two years of initial treatment), and patient characteristics were compared with controls who successfully completed initial treatment without early relapse. RESULTS: 291 patients presenting for retreatment were included; 93% received a standard Category II regimen. Retreatment was successful in 74% of relapse patients, 48% of failure patients, and 41% of default patients. 25% of retreatment patients defaulted, higher than previous estimates. Retreatment failure was most common among patients who had failed initial treatment (24%), and default from retreatment was most frequent among patients with initial treatment default (57%). Drug susceptibility testing was performed in only 10% of retreatment patients. Independent risk factors for failure, default, or early relapse after initial treatment included male gender (aOR = 2.29, 95% CI 1.10-4.77), positive sputum smear after 3 months of treatment (OR 7.14, 95% CI 4.04-13.2), and hospitalization (OR 2.09, 95% CI 1.01-4.34). Higher weight at treatment initiation was protective. Male sex, substance use, missed doses, and hospitalization appeared to be risk factors for default, but subgroup analyses were limited by small numbers. CONCLUSIONS: Outcomes of retreatment with a Category II regimen are suboptimal and vary by subgroup. Default among patients receiving tuberculosis retreatment is unacceptably high in urban areas in Morocco, and patients who fail initial tuberculosis treatment are at especially high risk of retreatment failure. Strategies to address risk factors for initial treatment default and to identify patients at risk for failure (including expanded use of drug susceptibility testing) are important given suboptimal retreatment outcomes in these groups.

Evaluation of the Yield of Histopathology in the Diagnosis of Lymph Node Tuberculosis in Morocco, 2017: Cross-Sectional Study.

BACKGROUND: The frequency of occurrence of extrapulmonary tuberculosis (EPTB) has been increasing globally over the last two decades. In Morocco, EPTB cases account for 46% of the patients reported with a new episode of tuberculosis (TB). Lymph node TB (LNTB) is the most common form of EPTB. In line with the guidelines of the National TB Program, the diagnosis is mainly based on clinical evidence, including histopathology. OBJECTIVE: This study aimed to evaluate the yield of histopathology testing in the diagnosis of LNTB. METHODS: This cross-sectional, prospective study was conducted among patients with cervical lymph node who were enrolled in the study from November 2016 to May 2017 in three regions of Morocco. We compared the outcomes of histopathological testing with those of bacteriology. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) of histopathology testing were calculated. Culture and Xpert tests were used as the gold standard Laboratoty Testing. RESULTS: A total of 262 patients were enrolled in this study. The Se, Sp, PPV, and NPV of histopathology testing were 95.6% (129/135), 64.6% (82/127), 74.1% (129/174), and 93.2% (82/88), respectively, in the presence of granuloma with or without caseous necrosis and were 84.4% (114/135), 74.8% (95/127), 78.1% (114/146), and 81.9% (95/116), respectively, in the presence of granuloma with caseous necrosis. The granuloma with caseous necrosis was associated with increased PPV and Sp of histopathology testing (P<.05). CONCLUSIONS: The presence of the granuloma with caseous necrosis in the histopathological examination had significantly improved the yield of histopathology testing for the diagnosis of LNTB. The findings recommend to maintain histopathology testing in establishing the LNTB diagnosis and to explore other techniques to improve it.

Analysis of isoniazid and rifampicin resistance in Mycobacterium tuberculosis isolates in Morocco using GenoType® MTBDRplus assay.

OBJECTIVES: The number of multidrug-resistant tuberculosis (MDR-TB) cases is rising worldwide. The present investigation aimed to evaluate, using the GenoType® MTBDRplus assay, the most common mutations associated with rifampicin (RIF) and isoniazid (INH) resistance among resistant strains in Morocco. METHODS: A total of 319 Mycobacterium tuberculosis isolates sent to the National Tuberculosis Reference Laboratory between 2013 and 2015 were subjected to GenoType® MTBDRplus for detecting M. tuberculosis and determination of drug susceptibility. Correlation of the minimum inhibitory concentrations (MICs) of INH with genotypic assay results was carried out for 97 MDR-TB strains. Various concentrations of INH were tested. RESULTS: The most frequent mutations observed were rpoBS531L (67.2%) and katGS315T1/2 (66.5%). Isolates with inhA gene mutation, katG gene mutation, and dual mutations in katG and inhA had MICs ranging from 0.5-1µg/mL, 2-10µg/mL and ≥12µg/mL, respectively. CONCLUSION: In Morocco, 66.5% and 76.7% of M. tuberculosis strains carried mutations causing high-level resistance to INH and RIF, respectively.

Extensively drug-resistant tuberculosis (XDR-TB) in Morocco.

OBJECTIVES: Extensively drug-resistant tuberculosis (XDR-TB) has recently been identified as a major global health threat. The aim of this study was to evaluate the presence of XDR-TB among Mycobacterium tuberculosis isolates in Morocco and its association with demographic, clinical and epidemiological features. METHODS: A total of 524 patients from the Moroccan National Tuberculosis Reference Laboratory, representative of all of the geographic regions, were subject to first-line drug susceptibility testing (DST). Subsequently, 155 isolates found to be multidrug-resistant tuberculosis (MDR-TB) underwent second-line DST. Moreover, to enhance our understanding of the genetic basis of these drug-resistant strains, drug resistance-associated mutations were investigated in isolates either identified as pre-XDR- and XDR-TB or suspected resistant using the GenoType® MTBDRsl V1.0 assay. RESULTS: In this study, 4 (2.6%) XDR-TB and 18 (11.6%) pre-XDR-TB isolates were identified. Agreement between the MTBDRsl assay results and phenotypic DST was 95.2% for ofloxacin, 81.0% for kanamycin and 95.2% for amikacin. CONCLUSIONS: To the best of our knowledge, this is the first study to evaluate the frequency of XDR-TB in Morocco. These results highlight the need to reinforce the TB management policy in Morocco with regard to control and detection strategies in order to prevent further spread of XDR-TB isolates.

Molecular Typing of Mycobacterium Tuberculosis Complex by 24-Locus Based MIRU-VNTR Typing in Conjunction with Spoligotyping to Assess Genetic Diversity of Strains Circulating in Morocco.

BACKGROUND: Standard 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing allows to get an improved resolution power for tracing TB transmission and predicting different strain (sub) lineages in a community. METHODOLOGY: During 2010-2012, a total of 168 Mycobacterium tuberculosis Complex (MTBC) isolates were collected by cluster sampling from 10 different Moroccan cities, and centralized by the National Reference Laboratory of Tuberculosis over the study period. All isolates were genotyped using spoligotyping, and a subset of 75 was genotyped using 24-locus based MIRU-VNTR typing, followed by first line drug susceptibility testing. Corresponding strain lineages were predicted using MIRU-VNTRplus database. PRINCIPAL FINDINGS: Spoligotyping resulted in 137 isolates in 18 clusters (2-50 isolates per cluster: clustering rate of 81.54%) corresponding to a SIT number in the SITVIT database, while 31(18.45%) patterns were unique of which 10 were labelled as "unknown" according to the same database. The most prevalent spoligotype family was LAM; (n = 81 or 48.24% of isolates, dominated by SIT42, n = 49), followed by Haarlem (23.80%), T superfamily (15.47%), >Beijing (2.97%), > U clade (2.38%) and S clade (1.19%). Subsequent 24-Locus MIRU-VNTR typing identified 64 unique types and 11 isolates in 5 clusters (2 to 3isolates per cluster), substantially reducing clusters defined by spoligotyping only. The single cluster of three isolates corresponded to two previously treated MDR-TB cases and one new MDR-TB case known to be contact a same index case and belonging to a same family, albeit residing in 3 different administrative regions. MIRU-VNTR loci 4052, 802, 2996, 2163b, 3690, 1955, 424, 2531, 2401 and 960 were highly discriminative in our setting (HGDI >0.6). CONCLUSIONS: 24-locus MIRU-VNTR typing can substantially improve the resolution of large clusters initially defined by spoligotyping alone and predominating in Morocco, and could therefore be used to better study tuberculosis transmission in a population-based, multi-year sample context.

The CYP7A1 gene rs3808607 variant is associated with susceptibility of tuberculosis in Moroccan population.

INTRODUCTION: Despite the medical progress in treatment. Tuberculosis (TB) continues to be a serious global health problem. A genome-wide linkage study identified a major susceptibility locus on chromosomal region 8q12-q13 in Moroccan TB patients. The CYP7A1 gene is located in this region and codes for cholesterol 7a-hydroxylase, an enzyme involved in cholesterol catabolism. METHODS: We selected three SNPs (rs3808607, rs8192875 and rs8192879) and studied their genotype and allele frequencies distribution in patients with pulmonary (PTB) or pleural TB (pTB), and compared them to Healthy Controls (HC). Genotyping of rs8192875 and rs8192879 SNPs was carried out using the Taq Man SNP genotyping Assay while rs3808607 was investigated by PCR-RFLP. RESULTS: We reported here for the first time a statistically significant increase in the AA homozygote genotype frequency of rs3808607 in PTB patients compared to HC (p=0.02, OR=1.93, 95% CI: 1.93 (1.07;3.49). The increased risk of developing TB was maintained when we combined the groups of patients (PTB-pTB) (p=0.01, OR=1.91, 95% CI=(1.07-3.42). In contrast, no genetic association was observed between the rs8192875 or rs8192879 polymorphisms and TB. CONCLUSION: Our investigations suggest that rs3808607 may play a role in susceptibility to TB in a Moroccan population.

Contribution of spoligotyping and MIRU-VNTRs to characterize prevalent Mycobacterium tuberculosis genotypes infecting tuberculosis patients in Morocco.

In the present study, Mycobacterium tuberculosis complex (MTBC) clinical isolates from culture-positive TB patients in Morocco were studied by spoligotyping and 12-loci MIRU-VNTR typing methods to characterize prevalent genotypes (n = 219 isolates from 208 patients). Spoligotyping resulted in 39 unique patterns and 167 strains in 30 clusters (2-50 strains per cluster). Comparison with international database showed that 29 of 39 unique patterns matched existing shared spoligotype international types (SITs). Nine shared types containing 10 strains were newly created (SIT 2891 to SIT 2899); this led to the description of 69 SITs with 206 strains and two orphan patterns. The most prevalent spoligotype was SIT42 (LAM; n = 50 or 24% of isolates). The repartition of strains according to major MTBC clades was as follows LAM (46.1%)> Haarlem (26%) >ill-defined T superfamily (22.6%) and S clade (0.96%). On the other hand, Beijing, CAS (Central Asian) and EAI (East-African Indian) strains were absent in this setting. Subsequent 12-Loci MIRU typing resulted in a total of 25 SIT/MIT clusters (n = 66 isolates, 2-6 isolates per cluster), with a resulting recent transmission rate of 22.3%. The MIRU-VNTR patterns corresponded to 69 MITs for 138 strains and 46 orphan patterns. The most frequent patterns were MIT43 (n = 8), MIT9 (n = 7) and MIT42 (n = 7). HGDI analysis of the 12 MIRU loci showed that loci 10, 23 and 40 were highly discriminative in our setting. The results also underlined the usefulness of spoligotyping and MIRU-VNTR to detect mixed infections among certain of our TB patients. Globally, the results obtained showed that TB is almost exclusively transmitted in Morocco through evolutionary-modern MTBC lineages belonging to principal genetic groups 2/3 strains (Haarlem, LAM, T), with a high level of biodiversity seen by MIRU typing. This study provides with a 1st global snapshot of MTBC population structure in Morocco, and validates the potential use of spoligotyping in conjunction with minisatellites for future investigations in Morocco that should in future ideally include optimized 15- or 24-loci MIRU-VNTRs.

Treatment default amongst patients with tuberculosis in urban Morocco: predicting and explaining default and post-default sputum smear and drug susceptibility results.

SETTING: Public tuberculosis (TB) clinics in urban Morocco. OBJECTIVE: Explore risk factors for TB treatment default and develop a prediction tool. Assess consequences of default, specifically risk for transmission or development of drug resistance. DESIGN: Case-control study comparing patients who defaulted from TB treatment and patients who completed it using quantitative methods and open-ended questions. Results were interpreted in light of health professionals' perspectives from a parallel study. A predictive model and simple tool to identify patients at high risk of default were developed. Sputum from cases with pulmonary TB was collected for smear and drug susceptibility testing. RESULTS: 91 cases and 186 controls enrolled. Independent risk factors for default included current smoking, retreatment, work interference with adherence, daily directly observed therapy, side effects, quick symptom resolution, and not knowing one's treatment duration. Age >50 years, never smoking, and having friends who knew one's diagnosis were protective. A simple scoring tool incorporating these factors was 82.4% sensitive and 87.6% specific for predicting default in this population. Clinicians and patients described additional contributors to default and suggested locally-relevant intervention targets. Among 89 cases with pulmonary TB, 71% had sputum that was smear positive for TB. Drug resistance was rare. CONCLUSION: The causes of default from TB treatment were explored through synthesis of qualitative and quantitative data from patients and health professionals. A scoring tool with high sensitivity and specificity to predict default was developed. Prospective evaluation of this tool coupled with targeted interventions based on our findings is warranted. Of note, the risk of TB transmission from patients who default treatment to others is likely to be high. The commonly-feared risk of drug resistance, though, may be low; a larger study is required to confirm these findings.

The genotypic population structure of Mycobacterium tuberculosis complex from Moroccan patients reveals a predominance of Euro-American lineages.

BACKGROUND: Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level. METHODOLOGY/PRINCIPAL FINDINGS: Spoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004-2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1- ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2- modern/TbD1-/PGG1 group (Beijing, CAS), group 3- evolutionary recent/TbD1-/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%. CONCLUSION: The M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the "evolutionary recent" TbD1-/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco.

Evaluation of conventional molecular diagnosis of Mycobacterium tuberculosis in clinical specimens from Morocco.

INTRODUCTION: Tuberculosis is a major public health threat, annually affecting new individuals worldwide, especially those in developing countries. Rapid detection of the agent and effective treatment are two important factors in controlling this disease. METHODOLOGY: The present study aimed to evaluate polymerase chain reaction (PCR) as a rapid and direct molecular method for the diagnosis of Mycobacterium tuberculosis (MTB) in 70 clinical specimens (62 sputum samples, six cerebrospinal fluids, and two biopsies) using heat shock protein (hsp65) as the gene target. Automated sequencing of the same gene was used for the identification of MTB to the species level. RESULTS: The sensitivity of PCR was 81.13%, with specificity of 88.24%; the positive and negative predictive values were 95.56% and 60%, respectively. CONCLUSION: Based on these results, the hsp65 gene sequence can be used to differentiate the members of MTB complex from non-tuberculosis mycobacteria (NTM).

The MCP-1 (CCL2) -2518 GG genotype is associated with protection against pulmonary tuberculosis in Moroccan patients.

INTRODUCTION: Both monocyte chemoattractant protein-1 (MCP-1), also designated officially as chemokine (C-C motif) ligand 2 (CCL2), and interleukin-12 p40 (IL-12 p40) molecules, encoded by polymorphic genes, are central components of the immune response to infection by Mycobacterium tuberculosis (Mtb). Their genetic diversity has previously been associated with the outcome of tuberculosis (TB) infection. We investigated whether the MCP-1 -2518 A/G and the IL-12B (p40) +1188 A/C polymorphisms influence susceptibility to or resistance against pulmonary tuberculosis (PTB) in a Moroccan population group. METHODOLOGY: Genomic DNA from 337 patients along with 204 healthy controls were genotyped for the above-mentioned genetic variations using polymerase chain reaction-based restriction fragment length polymorphism assay. RESULTS: We found a higher prevalence of homozygous MCP-1 -2518 G allele in healthy individuals than in patients (pc = 0.04; odds ratio  = 0.35; 95% confidence interval  = 0.13 - 0.86), suggesting a potential protective effect, whereas analysis of IL-12B +1188 variation failed to reveal any such association. CONCLUSION: Our results are in agreement with recent findings in Ghanaian patients, complying with the known genetic admixture of the Moroccan population.

Analysis of isoniazid, streptomycin and ethambutol resistance in Mycobacterium tuberculosis isolates from Morocco.

BACKGROUND: Drug-resistant tuberculosis is a major problem worldwide. Based on the knowledge of specific mutations occurring in Mycobacterium tuberculosis genome, drug resistance can be detected earlier. The aim of this study was to determine the prevalence of the most common mutations associated with resistance to Isoniazid (INH), Streptomycin (SM) and Ethambutol (EMB) in Mycobacterium tuberculosis isolates from Morocco in order to select target mutations to develop tests for rapid detection of drug-resistant Mycobacterium tuberculosis Moroccan isolates. METHODOLOGY: A total of 199 M. tuberculosis isolates collected from the National Tuberculosis Reference Laboratory in Morocco were subject to katG, inhA, rrs, rpsL and emb mutation analysis by PCR probe-based assay. The genotypic results were then compared to drug susceptibility testing results for the corresponding drugs. RESULTS: Among 66 phenotypically INH resistant isolates, 80.3% (53/66) were found to be genotypically INH resistant from which 77.3% (51/66) and 3% (2/66) had respective mutations in katG315 and inhp-15 codons. Of the 58 phenotypically SM resistant isolates, genotypic SM resistance was confirmed in 17.2% (10/58) cases. Nucleotide mutations at codons 43 and 88 of rpsL gene and at codon 512 of rrs gene were found respectively in 12.1% (7/58); 1.7% (1/58) and 3.4% (2/58) of the phenotypically SM resistant Mycobacterium tuberculosis isolates. Finally, mutations at codon 306 of embB gene were identified in 42.3% (11/26) of Mycobacterium tuberculosis isolates phenotypically EMB resistant. CONCLUSION: This study showed that a large proportion of Mycobacterium tuberculosis resistant isolates from Morocco carry a large number of mutations in different codons (especially katG315, embB306 and rpsL43) of the corresponding genes associated with drug resistance. Thus, molecular analysis based on the identification of such mutations is useful but not fully sufficient to predict all drug resistance cases. Based on these results, rapid drug resistance genotyping can be used as an adjunct to the traditional culture based methods in reference laboratories.