INTERNATIONAL ONLINE ONE-ON-ONE BIOMEDICAL RESEARCH TRAINING ON MYCOBACTERIUM TUBERCULOSIS GENOTYPING: GENOTYPE ANALYSIS OF MYCOBACTERIUM TUBERCULOSIS ISOLATES.

Hello,

My name is Philip Anochie. I am a Research Scientist/ISID/ESCMID Fellow, President/CEO of Philip Nelson Institute of Medical  Research and Author of many books and publications in international medical scientific peer-reviewed journals on Mycobacterium tuberculosis.

I am the First recipient of the International Society of Infectious Diseases (ISID) and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Joint Fellowship Award in 2010 on Genotype analysis of Mycobacterium tuberculosis isolates in Milan , Italy under Professor Daniela Maria Cirillo the Head of Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, WHO Tuberculosis Suparanational Reference Laboratory, San Raffaele Hospital, Milan, Italy.

I am here to introduce you to an international online one-on-one Biomedical Research Training Course in Mycobacterium tuberculosis Genotyping.

This  three months’ course   on principles and protocols of Mycobacterium tuberculosis genotyping – genotype analysis of Mycobacterium tuberculosis isolates , consists of  three parts  - Part 1,  Part 2, and Part 3 with 46  lectures, assignment submission on the last date of the third month , examination and award of an  International Professional Proficiency Certificate on Principles and Protocols of Mycobacterium tuberculosis Genotyping – Genotype analysis of Mycobacterium tuberculosis isolates by the institute.

Part 1 of the course describes the conventional cultural methods for the clinical laboratory diagnosis, monitoring and evaluation  of tuberculosis (TB), multi-drug resistant tuberculosis (MDR-TB) and extensive drug resistant tuberculosis (XDR-TB).

Part 2  explains fully  the essential molecular laboratory preparations required for the clinical molecular analysis , monitoring and evaluation of TB, MDR-TB and XDR-TB in a TB reference laboratory.

Part 3  gave an account of the molecular genetic assays for the genotyping of Mycobacterium tuberculosis isolates in a TB reference laboratory.

This course contains essentially the laboratory techniques  used in conducting and performing the following duties very competently as well as the preparation and storage of the reagents for them- Diagnosis for the presence of Mycobacterium strains in sputa and other clinical specimens by light microscopy, culture and isolation of Mycobacterium strains from sputum and other specimens using essentially the Lowenstein-Jensen (LJ) medium, characterization and identification of isolated strains by both cultural , biochemical and molecular methods like Hain assay, spoligotyping, MIRU etc, establishment of the sensitivity profiles of the isolates to various anti-TB drugs using LJ slopes ,the proportion method, the BACTEC method and Hain’s assay:

•      GenoQuick ®MTB: Rapid molecular genetic assay for the direct detection of the Mycobacterium tuberculosis complex from patient specimens.

•      GenoType ® MTBC: Molecular genetic assay for the differentiation of the Mycobacterium tuberculosis complex from cultured material.

•      GenoType ®Mycobacterium CM: Molecular genetic assay for the identification of the clinically most relevant mycobacterial species from cultured material.

•      GenoType® MTBDRplus: Molecular genetic assay for the identification of resistance to rifampicin and/or isoniazid of the Mycobacterium tuberculosis complex.

•      GenoType® MTBDRsl: Molecular genetic assay for the identification of resistances to fluoroquinolones, aminoglycosides/ cyclic peptides, and ethambutol of the Mycobacterium tuberculosis complex.

•      Spoligotyping: A PCR-based method to simultaneously detect and type Mycobacterium tuberculosis complex bacteria for epidemiological studies..

•      BACTEC TM MGIT TM 960 SIRE. Test for the antimycobacterial susceptibility testing of Mycobacterium tuberculosis for first and second line drugs.

•      BD BBL TM MGIT TM : Mycobacteria growth indicators.

•      BACTEC 460 TB radiometric method uses a liquid medium containing anti-tuberculosis drugs at different concentrations to cultivate resistant bacilli.

•      Capilia TB-Neo for the detection of Mycobacterium tuberculosis complex from patient specimens.

•      PCR and reverse hybridization assays.

•      DNA inactivation and extraction from positive MGIT tubes.

•      DNA inactivation and extraction from positive LJ tubes.

•      DNA inactivation and extraction from sediments.

•      Chemical DNA extraction.

•      MIRU-VNTR : Mycobacterial interspersed repetitive units –Variable number tandem repeats; a strain identification and differentiation protocol for the epidemiological study of Mycobacterium tuberculosis.

•      GT-BLOT 20 automated equipment for reverse hybridization assays.

•      DsRNA genome extraction, gel electrophoresis and RT-PCR of  dsRNA.

•      And many more.

 At the end if this training, the participants are expected to acquire the following skills : PCR, MIRU-VNTR, Genotyping, Spoligotyping, Genome extraction and purification, BACTEC, Gel electrophoresis, GT-Blot 20 hybridization assay, Hain assay, Capilia TB-neo, and Microscopy.

 

                            COURSE INTRODUCTION

The ability to discern the molecular “fingerprint” (genotype) of Mycobacterium tuberculosis isolates has revolutionized the understanding of the transmission of tuberculosis. Increasing levels of drug resistance are threatening to erode the medical advances of recent decades.

The social causes contributing to the spread of anti-tuberculosis resistance are somehow paradoxical. In some settings – especially in resource limited countries – the under-use of drugs or the use of low quality drugs encourages the development of resistance. In wealthy countries, resistance is the opposite reason – the over use of drugs, that is driving the emergence of resistance.

 Regardless of where drug resistance originates, globalization, increased travel and trade ensure that these strains quickly travel elsewhere. The DNA

fingerprinting technology (genotyping) is useful to track down the worldwide spreading of resistant strains.

Health authorities in many countries are confronted with a serious challenge of Mycobacterium tuberculosis that are resistant to anti-mycobacterial drugs. Dramatic outbreaks of multi-drug resistant tuberculosis (TB) among institutionalized HIV-infected patients have focused international attention on this problem. There is therefore an urgent need to  generate reliable and internationally comparable data on TB drug resistance in these countries. This comparable data will not be obtained without the genotypic analysis of the Mycobacterium tuberculosis isolates, which will ensure the elimination of nosocomial transmission and laboratory error.

Mycobacterium tuberculosis genotyping is essential to investigate and confirm transmission, and to confirm and exclude laboratory contamination by genotyping isolates for every new TB case to improve the efficiency of tuberculosis control, decrease the length of unnecessary treatment among patients with false-positive cultures, identify previously unknown links

among  genotypically clustered patients and unidentified clusters of transmission. Genotyping will also more rapidly and efficiently determine the extent and dynamics of ongoing transmission to focus programme interventions for specific areas and populations, assess tuberculosis transmission in outbreaks to refine contact investigations, identify nosocomial

transmission not identified by conventional methods and also identify false-positive cultures so that clinicians could be notified on diagnostic errors quickly and prevent unnecessary tuberculosis treatment.

With an ISID/ESCMID Fellowship, I  concluded a study on the genotype analysis of Mycobacterium tuberculosis isolates at the Emerging Bacteria Pathogens Laboratory of the San Raffaele Scientific Institute, Vita – salute San Raffaele University, Milan, Italy under  Professor  Daniela Maria Cirillo, the Head of the laboratory. The laboratory focuses its activities on the study of major problems related to the issue of drug resistance in Mycobacterium tuberculosis strains: the biology and resistance mechanisms of multi drug

resistance (MDR). The laboratory is recognized by WHO, jointly with the ISS in Rome, as a Supranational Reference Laboratory for tuberculosis control for the international activities performed in this field.  Professor  Cirillo is the coordinator of two EU funded projects (2008- 2013): “TB PAN – Net – Pan-European network for the study and clinical management of drug resistant tuberculosis”, and “TM – Rest – A new platform for fast molecular detection of multi drug resistant (MDR) and extensive drug resistant (XDR) strains of Mycobacterium tuberculosis.”

The ISID and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) are co-sponsoring the ISID/ESCMID Fellowship to enable young researchers from developing countries to come to Europe for training.

More info can be found at www.isid.org/grants/grants.shtml

http://www.isid.org/wpcontent/uploads/2018/03/2010_ESCMIDFellow_ANOCHIE.pdf

ISID NEWS • May 2011

 

                               COURSE OUTLINE

Section A: First Month: Conventional Cultural Methods for the Clinical Laboratory Diagnosis ,  Monitoring and Evaluation of Tuberculosis (TB), Multi-drug Resistant Tuberculosis (MDR-TB) and Extensive Drug Resistant Tuberculosis (XDR-TB).

LECTURE 1: Introduction

LECTURE  2: Specimen collection

LECTURE 3: Sample collection and study patients.

LECTURE 4: Media and methods for bacteriological analysis.

LECTURE 5: Quantitative microscopy of sputum smears.

LECTURE 6: Preparation of Lowenstein – Jensen (LJ) Medium .

LECTURE 7: Biochemical tests for identification.

LECTURE 8: The microscope and its applications.

LECTURE 9: Surveillance of anti-drug resistance using proportion method.

LECTURE 10: Tuberculosis stains and reagents.

Section B: Second  Month: The Essential Molecular Laboratory Preparations Required for the Clinical Molecular analysis and Monitoring of TB, MDR-TB and XDR-TB in a TB Reference Laboratory.

LECTURE 11:  Structure and functions of a Biosafety  level 3 (P3) Suparanational Tuberculosis Reference Laboratory.

LECTURE 12: Types of pipettes used in a Biosafety  level 3 (P3) Suparanational Tuberculosis Reference Laboratory.

LECTURE 13: Equipment , materials and other laboratory products used in a Biosafety  level 3 (P3) Suparanational Tuberculosis Reference Laboratory.

LECTURE 14: Emergency procedures in case of major biohazard incident outside the Biological Safety Cabinet in a Tuberculosis Reference Laboratory.

LECTURE 15: Preparation of Buffers and Solutions in a Tuberculosis Reference Laboratory.

LECTURE 16: DNA inactivation and extraction.

LECTURE 17: PCR and reverse hybridization assays.

LECTURE 18: DNA inactivation and extraction from positive MGIT tubes.

LECTURE  19: DNA inactivation and extraction from positive LJ tubes.

LECTURE  20: DNA inactivation and extraction from sediments.

LECTURE  21: Chemical DNA extraction.

LECTURE 22: PCR using Hotstar Taq DNA polymerase.

LECTURE 23:  PCR using Hotstar Taq DNA polymerase and Q- solution.

LECTURE 24: PCR using Hotstar Taq Master mix.

LECTURE 25: Starting template.

LECTURE 26:  Primer design, concentration and storage.

LECTURE  27:  Number of PCR cycles.

LECTURE  28:  Sensitive PCR assays.

LECTURE  29:  RT-PCR

LECTURE  30: Touchdown PCR.

LECTURE  31: Purification of PCR products.

LECTURE 32: Control of  contamination.

LECTURE  33. Laboratory Safety

Section C: Third  month:  Molecular genetic assays for the genotyping of Mycobacterium tuberculosis isolates in a TB reference laboratory.

LECTURE  34: GenoQuick ®MTB: Rapid molecular genetic assay for the direct detection of the Mycobacterium tuberculosis complex from patient specimens.

LECTURE  35: GenoType ® MTBC: Molecular genetic assay for the differentiation of the Mycobacterium tuberculosis complex from cultured material.

LECTURE 36: GenoType ®Mycobacterium CM: Molecular genetic assay for the identification of the clinically most relevant mycobacterial species from cultured material.

LECTURE  37: GenoType® MTBDRplus: Molecular genetic assay for the identification of resistance to rifampicin and/or isoniazid of the Mycobacterium tuberculosis complex.

LECTURE  38: GenoType® MTBDRsl: Molecular genetic assay for the identification of resistances to fluoroquinolones, aminoglycosides/ cyclic peptides, and ethambutol of the Mycobacterium tuberculosis complex.

LECTURE 39: Spoligotyping: A PCR-based method to simultaneously detect and type Mycobacterium tuberculosis complex bacteria for epidemiological studies.

LECTURE  40: BACTEC TM MGIT TM 960 SIRE. Test for the antimycobacterial susceptibility testing of Mycobacterium tuberculosis for first and second line drugs.

LECTURE  41: Capilia TB-Neo for the detection of Mycobacterium tuberculosis complex from patient specimens.

LECTURE 42: MIRU-VNTR : Mycobacterial interspersed repetitive units –Variable number tandem repeats; a strain identification and differentiation protocol for the epidemiological study of Mycobacterium tuberculosis.

LECTURE  43: GT-BLOT 20 automated equipment for reverse hybridization assays.

LECTURE 44: DsRNA genome extraction, gel electrophoresis and RT-PCR of dsRNA.

LECTURE 45: BD BBL TM MGIT TM : Mycobacteria growth indicators.

LECTURE 46:  BACTEC 460 TB radiometric method.

LECTURE 47: Tuberculosis Reference Laboratory Decontamination Procedures.

And many, many , more.

For further information on how to enrol in this one-on-one online biomedical research training course on Mycobacterium tuberculosis genotyping, please contact:

THE INTERNATIONAL ONLINE TRAINING ADMINISTRATOR,

PHILIP NELSON INSTITUTE OF MEDICAL RESEARCH.

E-MAIL:  philipnelsoninstitute@yahoo.com

             philipanochie@gmail.com

             philipanochie@yahoo.co.uk.