Transmission of drug-resistant Mycobacterium tuberculosis isolates between Finnish- and foreign-born cases, 2014-2021: A molecular epidemiological study.

BACKGROUND: Data on the molecular epidemiology and transmission of drug-resistant Mycobacterium tuberculosis (MTB) in low-incidence settings with immigration from high-incidence settings is limited. METHOD: We included 115 drug-resistant (DR) MTB isolates with whole-genome sequencing data isolated in Finland between 2014 and 2021. Potential transmission clusters were identified using a threshold of 12 single-nucleotide polymorphisms (SNPs). Highly related clusters were identified using a threshold of 5 SNPs. RESULT: Of the 115 DR MTB isolates, 31 (27.0%) isolates were from Finnish-born cases and 84 (73.0%) were from foreign-born cases. The proportion of multidrug-resistant (MDR) MTB isolates (30/84, 35.7%) from foreign-born cases was higher than that of MDR MTB isolates from Finnish-born cases (8/31, 25.8%). Lineage 2 (40/115, 34.8%) and lineage 4 (40/115, 34.8%) were the most prevalent lineages. A total of 25 (21.7%) isolates were classified into eight potential transmission clusters (≤12 SNPs). Furthermore, five highly related clusters (≤5 SNPs) were identified, including three DR MTB isolates from Finnish-born cases and 14 DR isolates from foreign-born cases. CONCLUSION: The risk of DR MTB transmission between Finnish- and foreign-born persons is not negligible. Further research on clustering analysis in drug-susceptible MTB is worth to inform tuberculosis management and control in low-incidence settings with increasing immigration.

Understanding knowledge, attitudes and practices on Ebola Virus Disease: a multi-site mixed methods survey on preparedness in Rwanda.

BACKGROUND: The overall goal of this survey was to understand the knowledge, attitudes, and practices related to the Ebola Virus Disease (EVD) in Rwanda. METHODS: This mixed-method cross-sectional survey was conducted in five selected districts of Rwanda. Quantitative data were collected from 1,010 participants using Kobo Collect Software and the analysis was performed using SPSS and Python software. Qualitative data were specifically collected from 98 participants through Key Informant Interviews (KIIs) and Focus Group Discussion (FGDs). Interview transcripts were imported into NVIVO 8 for coding and subsequent analysis. RESULTS: As per our quantitative findings, we report that from the 1,010 respondents, 99.6% reported having previously heard of Ebola, 97.2% believed that vaccination is important in combatting the disease and 93.3% of individuals reported a willingness to receive vaccination should one become available. Around 54% of the respondents were correct in identifying that the disease is of a viral origin which originates from wild animals (42.1%). When asked if they believed that Rwanda is at risk of an EVD outbreak, 90% of the respondents believe that the country is at risk of an EVD outbreak, and the cofactors *gender* and *whether people dwell in Rubavu/Rusizi* were found to significantly impact their perception of threat. As per our qualitative findings, the respondents mentioned that both geographical proximity and relations with the Democratic Republic of Congo place Rwanda at risk of developing an internal outbreak. Although the respondents seemed to be aware of the Ebola prevention behaviours, it was noted that some of them will require significant time before reintegrating into the community an EVD survivor, as they will first need assurance that the patient has fully recovered. Therefore, the qualitative findings reinforce what we originally reported in the quantitative approach to this study. CONCLUSION: Our results show that there was high EVD-related knowledge and awareness among the general population in Rwanda. However, for strong public health awareness, preparedness, and protection, a massive investment should always be made in education about EVD with a special focus on districts neighboring countries where the disease is consistently being reported.

Predicting the distribution of Ixodes ricinus and Dermacentor reticulatus in Europe: a comparison of climate niche modelling approaches.

BACKGROUND: The ticks Ixodes ricinus and Dermacentor reticulatus are two of the most important vectors in Europe. Climate niche modelling has been used in many studies to attempt to explain their distribution and to predict changes under a range of climate change scenarios. The aim of this study was to assess the ability of different climate niche modelling approaches to explain the known distribution of I. ricinus and D. reticulatus in Europe. METHODS: A series of climate niche models, using different combinations of input data, were constructed and assessed. Species occurrence records obtained from systematic literature searches and Global Biodiversity Information Facility data were thinned to different degrees to remove sampling spatial bias. Four sources of climate data were used: bioclimatic variables, WorldClim, TerraClimate and MODIS satellite-derived data. Eight different model training extents were examined and three modelling frameworks were used: maximum entropy, generalised additive models and random forest models. The results were validated through internal cross-validation, comparison with an external independent dataset and expert opinion. RESULTS: The performance metrics and predictive ability of the different modelling approaches varied significantly within and between each species. Different combinations were better able to define the distribution of each of the two species. However, no single approach was considered fully able to capture the known distribution of the species. When considering the mean of the performance metrics of internal and external validation, 24 models for I. ricinus and 11 models for D. reticulatus of the 96 constructed were considered adequate according to the following criteria: area under the receiver-operating characteristic curve > 0.7; true skill statistic > 0.4; Miller's calibration slope 0.25 above or below 1; Boyce index > 0.9; omission rate < 0.15. CONCLUSIONS: This comprehensive analysis suggests that there is no single 'best practice' climate modelling approach to account for the distribution of these tick species. This has important implications for attempts to predict climate-mediated impacts on future tick distribution. It is suggested here that climate variables alone are not sufficient; habitat type, host availability and anthropogenic impacts, not included in current modelling approaches, could contribute to determining tick presence or absence at the local or regional scale.

Molecular detection of pathogens from ticks collected from dogs and cats at veterinary clinics in Finland.

BACKGROUND: Ticks carry microbes, some of which are pathogenic for humans and animals. To assess this One Health challenge, 342 ticks were collected from pet dogs and cats at 10 veterinary clinics in Finland as part of the European project "Protect Our Future Too". METHODS: The tick species were identified, and ticks were screened with quantitative PCR (qPCR) for tick-borne pathogens, including Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Ehrlichia canis, Anaplasma spp., Candidatus Neoehrlichia mikurensis, tick-borne encephalitis virus (TBEV), and Babesia spp. For comparison, a subset of tick DNA (20 qPCR-positive samples) was analysed with 16S next-generation sequencing (NGS). RESULTS: Most ticks were Ixodes ricinus (289, 84.5%), followed by Ixodes persulcatus (51, 14.9%). One hybrid tick (I. ricinus/I. persulcatus, 0.3%) and one Rhipicephalus sanguineus tick (0.3%) were identified. We found one or more of the analysed pathogens in 17% (59/342) of the ticks. The most prevalent pathogen was B. burgdorferi s.l. (36, 10.5%), followed by Anaplasma phagocytophilum (12, 3.5%), B. miyamotoi (5, 1.5%), Babesia venatorum (4, 1.2%), and TBEV (1, 0.3%). Candidatus Neoehrlichia mikurensis DNA was amplified from three (0.9%) ticks. Ehrlichia canis was not detected. In the 16S NGS, six samples produced enough reads for the analysis. In these six samples, we confirmed all the positive qPCR findings of Borrelia spp. and Ca. N. mikurensis. CONCLUSIONS: The high prevalence of pathogenic microorganisms in the ticks of this study emphasizes the importance of awareness of ticks and tick-borne diseases and prevention. Furthermore, the results show that veterinary surveillance can facilitate early detection of tick-borne pathogens and new tick species and draw attention to possible co-infections that should be considered both in symptomatic humans and animals after tick bites.

Activity of lipid-loaded lectin against co-infection of Candida albicans and Staphylococcus aureus using the zebrafish model.

AIM: Nosocomial infection caused by mixed species of methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans (CA) is difficult to manage with existing antimicrobials, particularly in the presence of mixed-species biofilm. This study evaluates the activity of cationic lipid, specifically functionalized with lectin, against mixed biofilms of MRSA and CA and their effectiveness in vivo using the zebrafish model. METHODS AND RESULTS: The present study demonstrates for the first time the antimicrobial activity of 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) against MRSA and mixed species of MRSA + CA. The cN16E functionalized with Butea monosperma seed lectin (BMSL) showed a lower minimum inhibitory concentration (MIC) as compared with cN16E. BMSL-cN16E (BcN16E) exhibited strong membrane-damaging activity at a lower concentration than cN16E. Crystal violet assay showed that BcN16E inhibits mixed-species biofilm at the concentration of 15.63 µM, which is four-fold lower than the MIC. Especially, BcN16E was found to be effective in disturbing mature mixed biofilm at 31.25 µM, which is two-fold lower than the MIC, suggesting true antibiofilm activity without pressurizing the microorganisms. The treatment with BcN16E significantly reduced the exopolysaccharide synthesis (> 78%), cell surface hydrophobicity (> 70 %), hyphae formation, staphyloxanthin biosynthesis (> 41 %), and antioxidant enzyme and hemolysin activity (> 70 %). Notably, BcN16E was efficient in reducing the in vivo colonization of bacterial and fungal burden in the blood and muscle tissues of zebrafish. CONCLUSION: Antimicrobial and antibiofilm efficacy of BcN16E against MRSA, and mixed species of MRSA + CA were demonstrated. Importantly, BcN16E treatment rescued Zebrafish coinfected with mixed species of MRSA + CA. Significance and Impact of the study: The results highlight that antimicrobial loaded on lectin provides an additional advantage to recognize microorganism surface glycans and maximize drug delivery to treat polymicrobial infections caused by MRSA and CA.

Clinical and Serological Findings of COVID-19 Participants in the Region of Makkah, Saudi Arabia.

Makkah in Saudi Arabia hosts the largest annual religious event in the world. Despite the many strict rules enacted, including Hajj cancellation, city lockdowns, and social distancing, the region has the second highest number of new COVID-19 cases in Saudi Arabia. Public health interventions that identify, isolate, and manage new cases could slow the infection rate. While RT-PCR is the current gold standard in SARS-CoV-2 identification, it yields false positive and negative results, which mandates the use of complementary serological tests. Here, we report the utility of serological assays during the acute phase of individuals with moderate and severe clinical manifestations of SARS-CoV-2 (COVID19). Fifty participants with positive RT-PCR results for SARS-CoV-2 were enrolled in this study. Following RT-PCR diagnosis, serum samples from the same participants were analyzed using in-house ELISA (IgM, IgA, and IgG) and microneutralization test (MNT) for the presence of antibodies. Of the 50 individuals analyzed, 43 (86%) showed a neutralizing antibody titer of ≥20. Univariate analysis with neutralizing antibodies as a dependent variable and the degree of disease severity and underlying medical conditions as fixed factors revealed that patients with no previous history of non-communicable diseases and moderate clinical manifestation had the strongest neutralizing antibody response "Mean: 561.11". Participants with severe symptoms and other underlying disorders, including deceased individuals, demonstrated the lowest neutralizing antibody response. Anti-spike protein antibody responses, as measured by ELISA, showed a statistically significant correlation with neutralizing antibodies. This reinforces the speculation that serological assays complement molecular testing for diagnostics; however, patients' previous medical history (anamnesis) should be considered in interpreting serological results.

Incidence Trends for SARS-CoV-2 Alpha and Beta Variants, Finland, Spring 2021.

Severe acute respiratory syndrome coronavirus 2 Alpha and Beta variants became dominant in Finland in spring 2021 but had diminished by summer. We used phylogenetic clustering to identify sources of spreading. We found that outbreaks were mostly seeded by a few introductions, highlighting the importance of surveillance and prevention policies.

Viral RNA Metagenomics of Hyalomma Ticks Collected from Dromedary Camels in Makkah Province, Saudi Arabia.

Arthropod-borne infections are a medical and economic threat to humans and livestock. Over the last three decades, several unprecedented viral outbreaks have been recorded in the Western part of the Arabian Peninsula. However, little is known about the circulation and diversity of arthropod-borne viruses in this region. To prepare for new outbreaks of vector-borne diseases, it is important to detect which viruses circulate in each vector population. In this study, we used a metagenomics approach to characterize the RNA virome of ticks infesting dromedary camels (Camelus dromedaries) in Makkah province, Saudi Arabia. Two hundred ticks of species Hyalomma dromedarii (n = 196) and Hyalomma impeltatum (n = 4) were collected from the Alkhurma district in Jeddah and Al-Taif city. Virome analysis showed the presence of several tick-specific viruses and tick-borne viruses associated with severe illness in humans. Some were identified for the first time in the Arabian Peninsula. The human disease-associated viruses detected included Crimean Congo Hemorrhagic fever virus and Tamdy virus (family Nairoviridae), Guertu virus (family Phenuiviridae), and a novel coltivirus that shares similarities with Tarumizu virus, Tai forest reovirus and Kundal virus (family Reoviridae). Furthermore, Alkhurma hemorrhagic virus (Flaviviridae) was detected in two tick pools by specific qPCR. In addition, tick-specific viruses in families Phenuiviridae (phleboviruses), Iflaviridae, Chuviridae, Totiviridae and Flaviviridae (Pestivirus) were detected. The presence of human pathogenetic viruses warrants further efforts in tick surveillance, xenosurveillence, vector control, and sero-epidemiological investigations in human and animal populations to predict, contain and mitigate future outbreaks in the region.

Pan and Core Genome Analysis of 183 Mycobacterium tuberculosis Strains Revealed a High Inter-Species Diversity among the Human Adapted Strains.

Tuberculosis (TB) is an airborne communicable disease with high morbidity and mortality rates, especially in developing countries. The causal agents of TB belong to the complex Mycobacterium tuberculosis (MTBc), which is composed of different human and animal TB associated species. Some animal associated species have zoonotic potential and add to the burden of TB management. The BCG ("Bacillus Calmette-Guérin") vaccine is widely used for the prevention against TB, but its use is limited in immunocompromised patients and animals due to the adverse effects and disseminated life-threatening complications. In this study, we aimed to carry out a comparative genome analysis between the human adapted species including BCG vaccine strains to identify and pinpoint the conserved genes related to the virulence across all the species, which could add a new value for vaccine development. For this purpose, the sequences of 183 Mycobacterium tuberculosis (MTB) strains were retrieved from the freely available WGS dataset at NCBI. The species included: 168 sensu stricto MTB species with other human MTB complex associated strains: M. tuberculosis var. africanum (3), M. tuberculosis var. bovis (2 draft genomes) and 10 BCG species, which enabled the analysis of core genome which contains the conserved genes and some virulence factor determinants. Further, a phylogenetic tree was constructed including the genomes of human (183); animals MTB adapted strains (6) and the environmental Mycobacterium strain "M. canettii". Our results showed that the core genome consists of 1166 conserved genes among these species, which represents a small portion of the pangenome (7036 genes). The remaining genes in the pangenome (5870) are accessory genes, adding a high inter-species diversity. Further, the core genome includes several virulence-associated genes and this could explain the rare infectiousness potential of some attenuated vaccine strains in some patients. This study reveals that low number of conserved genes in human adapted MTBc species and high inter-species diversity of the pan-genome could be considered for vaccine candidate development.

Molecular detection and phylogenetic analysis of Borrelia miyamotoi strains from ticks collected in the capital region of Finland.

Borrelia miyamotoi is an emerging pathogen that shares high similarity with relapsing fever Borrelia, but has an atypical clinical presentation. Within the framework of tick-borne disease surveillance in Finland, human serum samples suspected for tick-borne encephalitis (n=974) and questing ticks (n=739) were collected from the capital region in Finland to determine the prevalence of B. miyamotoi. All tested human samples were negative and 5 (0.68 %) Ixodes ricinus ticks were positive for B. miyamotoi. Partial sequencing of the flagellin (flaB) gene of 3 positive samples and 27 B. miyamotoi-positive tick samples obtained from previous studies across Finland were amplified, sequenced, and included in the phylogenetic analysis. The phylogenetic tree revealed that most B. miyamotoi strains isolated from ticks in Finland share high similarity with other European strains, including strains related to human infection. Possible disease transmission may occur during exposure to tick bites. A single strain collected from an I. persulcatus tick in Pajujärvi grouped with an outlier of B. miyamotoi strains isolated from Russia and Far East Asian countries. Further studies should investigate the pathogen's role in human infection in Finland. Another important finding is the occurrence of I. persulcatus ticks (8%) collected by crowdsourcing from the coastal southern part of Finland. This suggests a regular introduction and a possible wide expansion of this tick species in the country. This could be associated with transmission of new pathogens.

Challenges and coping strategies faced by female scientists-A multicentric cross sectional study.

Women can play a pivotal role in the progress and sustainability of the world if they are empowered through education and employment opportunities in Science, technology, innovation and through changing the social stereotypes that restrain them in certain workplaces. In the literature, few recently published studies exist that document the challenges faced by female scientists in their workplaces. The purpose of this study was to understand the challenges and coping strategies faced by female scientists around the world today, in order to contribute to their improved performance. A multi-centre electronic cross-sectional survey across 55 countries was conducted to profile female scientists and to identify the challenges that they experience throughout their career as well as the coping mechanisms that they use to overcome the barriers. A total of 263 female scientists from different countries across the world participated in our study and most participants were from the South East Asian Region. Most female scientists in our study belong to the middle and junior level career category and earning around 1250 USD per month. Most of the scientists reported availability of maternity leave at their workplace but less than a third reported presence of a creche at work. Workplace sexual harassment was reported by 24% of the study population. Work related stress (71.5%) and work life imbalance (46%) are also major challenges faced by female scientists. Self-confidence, dedication and hard work are the most commonly adopted coping strategy. Flexible work timings, woman-friendly management policies, fair appraisal and mentorship appear to reduce the work-related stress and improve work-life balance among female scientists. In conclusion, female scientists face numerous challenges, which can greatly affect both their individual and career growth. Intrinsic (personal) and extrinsic (institutional) factors are important for improving female scientists' wellbeing and productivity.

Genome sequencing of Mycobacterium tuberculosis clinical isolates revealed isoniazid resistance mechanisms undetected by conventional molecular methods.

A combination of targeted molecular methods and phenotypic drug-susceptibility testing is the most widely used approach to detect drug resistance in Mycobacterium tuberculosis isolates. We report the delay in the introduction of an efficient anti-tuberculous drug regimen because of a M. tuberculosis strain displaying a high level of resistance to isoniazid, in the absence of the common mutations associated with isoniazid-resistance, including katG mutations and inhA promoter mutations. Whole-genome sequencing (WGS) identified a large loss-of-function insertion (>1000 pb) at the end of katG in the isolate together with a -57C>T ahpC mutation, a resistance mechanism that would have remained undetected by a conventional molecular targeted approach. A retrospective search using publicly available WGS data of more than 1200 isoniazid-resistant isolates and a similar sized control dataset of isoniazid-susceptible isolates revealed that most (22/31) isoniazid-resistant, KatG loss-of-function mutants had an associated rare ahpC promoter mutation. In contrast, only 7 of 1411 isoniazid-susceptible strains carried a rare ahpC promoter mutation, including shared mutations with the 31 isoniazid-resistant KatG loss-of-function mutants. These results indicate that rare ahpC promoter mutations could be used as a proxy for investigating simultaneous KatG loss-of-function or missense mutations. In addition, WGS in routine diagnosis would improve drug susceptibility testing in M. tuberculosis clinical isolates and is an efficient tool for detecting resistance mechanisms undetected by conventional molecular methods.

Detection of novel tick-borne pathogen, Alongshan virus, in Ixodes ricinus ticks, south-eastern Finland, 2019.

The newly identified tick-borne Alongshan virus (ALSV), a segmented Jingmen virus group flavivirus, was recently associated with human disease in China. We report the detection of ALSV RNA in Ixodes ricinus ticks in south-eastern Finland. Screening of sera from patients suspected for tick-borne encephalitis for Jingmen tick virus-like virus RNA and antibodies revealed no human cases. The presence of ALSV in common European ticks warrants further investigations on its role as a human pathogen.

Molecular diagnosis and enrichment culture identified a septic pseudoarthrosis due to an infection with Erysipelatoclostridium ramosum.

We describe here a rare case of septic pseudarthrosis due to Erysipelatoclostridium ramosum in a female young patient. The patient, currently in remission from Ewing's sarcoma treated by a bone resection and allograft combined with chemotherapy, suffered from a chronic femoral pseudarthrosis in a context of bone insufficiency and graft resorption. A broad range 16S PCR followed by sequencing, as well as an enrichment culture of a bone biopsy revealed the presence of E. ramosum, an anaerobic firmicute with a low Gram-positive affinity staining and low GC content, that was further characterized by whole genome sequencing (WGS).

Added Value of Xpert MTB/RIF Ultra for Diagnosis of Pulmonary Tuberculosis in a Low-Prevalence Setting.

Xpert MTB/RIF (Xpert) for direct molecular detection of Mycobacterium tuberculosis and rifampin resistance from clinical specimens has dramatically improved the diagnosis of tuberculosis (TB). Xpert MTB/RIF Ultra (Ultra) is proposed as a substitute of Xpert with increased sensitivity and improved rifampin resistance detection. We evaluated the diagnostic performance of Ultra and Xpert for pulmonary TB diagnosis in a low-TB-burden setting. Performance of Ultra and Xpert were compared to culture on respiratory specimens from patients with suspected pulmonary TB (November 2016 to August 2018; n = 196) in Lausanne (Switzerland). Clinical data were used to investigate discrepant results. Correlation between semiquantitative result of Ultra and smear microscopy status for the detection of acid-fast bacilli (AFB) was established. The sensitivities of Xpert and Ultra were 82.9% (39/47) and 95.8% (45/47), respectively, when considering all culture-positive specimens, 100% (23/23) for both assays on smear-positive specimens, and 66.7% (16/24) and 91.7% (22/24) on smear-negative specimens. Using culture as gold standard, the specificities of Xpert and Ultra were 97.3% (145/149) and 96.64% (144/149), respectively. All the patients with Ultra-positive results with the new category "trace" were diagnosed with active TB based on clinical findings and microbiological culture. The semiquantitative results of both Xpert and of Ultra positively correlated with the semiquantitative result of AFB detection. Our data support an increased sensitivity of Ultra compared to Xpert in a low-prevalence setting. Correlation between the Ultra semiquantitative result and AFB burden can help in evaluating a patient's transmission potential.

CD4+ regulatory T cells and CD4+ activated T cells in new active and relapse tuberculosis.

The aim of the present study was to examine characteristics of tuberculosis (TB) patients with different clinical forms and to study the frequency of Regulatory T cells (Treg cells) and Activated T cells in patients with new active and relapse TB. Forty-five pulmonary TB patients and a control group of 15 healthy individuals were enrolled in this study. Of the 45 TB patients, 15 were new cases with drug-susceptible active TB and 30 were relapsed cases (15 drug-susceptible and 15 multidrug resistant-TB). The age of study participants ranged from 21 to 68 years old. According to sex presentation, males were appreciably highly affected than females with a sex ratio of 2. The patients reported a mean recent weight loss of 8.9 kg. The Erythrocyte Sedimentation Rate was high in TB group, far exceeding the normal value. The results revealed that the number of CD3+ CD4+ T-cells significantly decreased whereas the level of blood Treg cells and expression of activation markers CD38 and HLA-DR on CD4+ T-cells significantly increased in TB group compared with the control group (p<0.05). The frequency of Treg cells was significantly higher in the TB group than the control group. Both the patients with new active TB and relapse TB demonstrated significantly higher levels of CD4+FoxP3+ Treg compared to healthy subjects (p<0.05). A high and significant percentage of Treg cells were found in patients with DS active TB than patients with MDR relapse TB. Interestingly, the frequency of CD4+FoxP3+ cells also differs according to the sputum smear microscopy status. The presence of high numbers of Treg cells and corresponding high immune activation may be an unfavourable factor that can predispose individuals to different clinical forms of TB, including relapse TB.

Molecular characterization of mutations associated with resistance to second-line tuberculosis drug among multidrug-resistant tuberculosis patients from high prevalence tuberculosis city in Morocco.

BACKGROUND: The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global TB control. Although multi drug-resistant tuberculosis (MDR- TB) prevalence and associated genetic mutations in Morocco are well documented, scarce information on XDR TB is available. Hence, the evaluation of pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drugs, is of great value for better management of M/XDR TB in Morocco. OBJECTIVES: To evaluate pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drug resistance, in 703 clinical isolates from TB patients recruited in Casablanca, and to assess the usefulness of molecular tools in clinical laboratories for better management of M/XDR TB in Morocco. METHODS: Drug susceptibility testing (DST) was performed by the proportional method for first line drugs, and then the selected MDR isolates were tested for second line drugs (Ofloxacin, Kanamycin, Amikacin and Capreomycin). Along with DST, all samples were subjected to rpoB, katG and p-inhA mutation analysis by PCR and DNA sequencing. MDR isolates as well as 30 pan-susceptible strains were subjected to PCR and DNA sequencing of gyrA, gyrB, rrs, tlyA genes and eis promoter, associated with resistance to fluoroquinolones and injectable drugs. RESULTS: Among the 703 analysed strains, 12.8% were MDR; Ser531Leu and Ser315Thr being the most common recorded mutations within rpoB and katG genes associated with RIF and INH resistance respectively. Drug susceptibility testing for second line drugs showed that among the 90 MDR strains, 22.2% (20/90) were resistant to OFX, 2.22% (2/90) to KAN, 3.33% (3/90) to AMK and 1.11% (1/90) to CAP. Genotypic analysis revealed that 19 MDR strains harbored mutations in the gyrA gene; the most recorded mutation being Asp91Ala accounting for 47.6% (10/21), and 2 isolates harbored mutations in the promoter region of eis gene. No mutation was found in gyrB, rrs and tlyA genes. Moreover, none of the pan-susceptible isolates displayed mutations in targeted genes. CONCLUSION: Most of mutations associated with SLD resistance occurred in gyrA gene (codons 90-94) and eis promoter region. These findings highlight the impact of mutations in gyrA on the development of fluroquinolones resistance and provide the first estimates of the proportion of pre-XDR-TB among MDR-TB cases in Morocco.