Seminario Biología Molecular José David Arrieta Sibaja

1. JOSE DAVID ARRIETA SIBAJA
MEDICINE III SEMESTER
UPB

2. TUBERCULOSIS
 Highly variable transmittable
disease
 Caused by Mycobacterium
tuberculosis.
 Affects especially the lungs
 Is characterized by:
 Fever
 Cough and difficulty in breathing
 Formation of tubercles
 Pleural effusions
 Fibrosis

3. DRUG RESISTANCE
 Drug resistance: microorganisms
change when they are exposed to
antimicrobial drugs; as a result,
the medicines become ineffective.
 OFLOXACIN: Antiobiotic-
Fluoroquinolone class.
 KANAMYCIN: Aminoglycoside
bacteriocidal antibiotic.
Fluoroquinolones and
kanamycin are core
compounds in current MDR-TB
treatment regimens

4. OBJECTIVE
 To investigate the diagnostic
utility of multiplex allele
specific PCR for detection of
resistance against ofloxacin
and kanamycin of second
line anti tuberculosis
treatment.

5. MATERIALS AND METHODS
 Such patients whose 4th month
culture result was positive were
considered as XDR suspects and
were included in the study.
Ethics statement
This study has been ethically
approved by the Institute ethical
committee of Institute of Medical
Sciences (ECR/526/Inst/UP/2014),
Banaras Hindu University, Varanasi.
-150 patients.
-Tertiary care center in
Varanasi in 2017.
-150 non duplicate MDR
strains isolated.
- BACTERIAL ISOLATES

6. - GENOMIC DNA ISOLATION
 DNA extraction from the
cultures of Mycobacterium
tuberculosis.
 The quality and quantity of
DNA was analyzed by using a
spectrophotometer.
MATERIALS AND METHODS

7. - MULTIPLEX ALLELE SPECIFIC PCR
Basis
Process by which different
DNA target sequences are
amplified simultaneously and
in a single tube, allowing the
detection and simultaneous
identification of different
genes
Objective
Identify the genes that
produce resistance of M.
Tuberculosis to ofloxacin
and kanamycin
MATERIALS AND METHODS
Electrophoresis is used in
laboratories to separate
macromolecules based on size.

9. RESULTADOS
Fig.1 Electroforesis en gel
de agarosa de ensayos
MAS-PCR para la
identificación de
mutaciones gyrA D94G.

10. RESULTADOS
Fig.1 Electroforesis en gel
de agarosa de ensayos
MAS-PCR para la
identificación de
mutaciones rrs A1401G.

11. RESULTADOS

12. DISCUSSION
AUTHOR SAID APPLIES OR NO
Ajbani K et al. The most frequently observed
mutations for ofloxacin and
kanamycin resistance are gyrA94G
and rrs1401G
YES
Evans Joanna et al. The sensitivity obtained by MAS-
PCR was 88.66%, 93.55% and 86%
for OFL, KAN and XDR-TB
respectively.
YES
Vadwai Viral et al. MAS-PCR presents a specificity of
100% for the identification of
resistance against KAN, OFL and
XDR-TB
YES

13. CONCLUSIONS
1. MAS-PCR could help in the fast detection of MDR and XDR Tuberculosis for the specifity to identify genes
related whit it.
2. The methods to identify the mutations studied in this article related to drug resistance can reduce the
diagnostic delay to tuberculosis and could be of great help for the implementation of effective drugs at the
right time to prevent the symptoms procreated by the spread of strains resistant to medications.