title: Direct sequencing for rapid detection of multidrug resistant Mycobacterium tuberculosis strains in Morocco.
abstract: BACKGROUND
  Tuberculosis (TB) is a major public health problem with high mortality and morbidity rates, especially in low-income countries. Disturbingly, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) TB cases has worsened the situation, raising concerns of a future epidemic of virtually untreatable TB. Indeed, the rapid diagnosis of MDR TB is a critical issue for TB management. This study is an attempt to establish a rapid diagnosis of MDR TB by sequencing the target fragments of the rpoB gene which linked to resistance against rifampicin and the katG gene and inhA promoter region, which are associated with resistance to isoniazid.
  
  
  METHODS
  For this purpose, 133 sputum samples of TB patients from Morocco were enrolled in this study. One hundred samples were collected from new cases, and the remaining 33 were from previously treated patients (drug relapse or failure, chronic cases) and did not respond to anti-TB drugs after a sufficient duration of treatment. All samples were subjected to rpoB, katG and pinhA mutation analysis by polymerase chain reaction and DNA sequencing.
  
  
  RESULTS
  Molecular analysis showed that seven strains were isoniazid-monoresistant and 17 were rifampicin-monoresistant. MDR TB strains were identified in nine cases (6.8%). Among them, eight were traditionally diagnosed as critical cases, comprising four chronic and four drug-relapse cases. The last strain was isolated from a new case. The most recorded mutation in the rpoB gene was the substitution TCG > TTG at codon 531 (Ser531 Leu), accounting for 46.15%. Significantly, the only mutation found in the katG gene was at codon 315 (AGC to ACC) with a Ser315Thr amino acid change. Only one sample harbored mutation in the inhA promoter region and was a point mutation at the -15p position (C > T).
  
  
  CONCLUSIONS
  The polymerase chain reaction sequencing approach is an accurate and rapid method for detection of drug-resistant TB in clinical specimens, and could be of great interest in the management of TB in critical cases to adjust the treatment regimen and limit the emergence of MDR and XDR strains.
authors.affiliation: Unité de Biologie et Recherché Médicale, Centre National de l'Energie, des Sciences et des Techniques Nucléaires (CNESTEN), Rabat, Morocco ; Laboratoire de Microbiologie, Hygiène et Virologie, Faculté des Sciences et Techniques, Mohammedia, Morocco.
authors.forename: Fathiah
authors.initials: F
authors.lastname: Zakham
authors.affiliation: Unité de Biologie et Recherché Médicale, Centre National de l'Energie, des Sciences et des Techniques Nucléaires (CNESTEN), Rabat, Morocco.
authors.forename: Imane
authors.initials: I
authors.lastname: Chaoui
authors.affiliation: Laboratoire de Génétique Mycobacterienne, Institut Pasteur, Tangier, Morocco.
authors.forename: Amina Hadbae
authors.initials: AH
authors.lastname: Echchaoui
authors.affiliation: Laboratoire de Tuberculose Institut Pasteur, Casablanca, Morocco.
authors.forename: Fouad
authors.initials: F
authors.lastname: Chetioui
authors.affiliation: Laboratoire de Tuberculose Institut Pasteur, Casablanca, Morocco.
authors.forename: My Driss
authors.initials: MD
authors.lastname: Elmessaoudi
authors.affiliation: Laboratoire de Microbiologie, Hygiène et Virologie, Faculté des Sciences et Techniques, Mohammedia, Morocco.
authors.forename: My Mustapha
authors.initials: MM
authors.lastname: Ennaji
authors.affiliation: Laboratoire de Génétique Mycobacterienne, Institut Pasteur, Tangier, Morocco.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Abid
authors.affiliation: Unité de Biologie et Recherché Médicale, Centre National de l'Energie, des Sciences et des Techniques Nucléaires (CNESTEN), Rabat, Morocco.
authors.forename: Mohammed El
authors.initials: ME
authors.lastname: Mzibri
keywords: Morocco; Mycobacterium tuberculosis; inhA promoter; katG; multidrug resistance; rpoB