it discuss pulmonary tuberculosis and MDR TB & antiTB drug and laboratory diagnosis of TB and host genomics esp NRAMP ,NAT2 ,TNF&LTA ,MBL Snps

1. Impact of Host Genomics on
Susceptibility and Treatment of
Tuberculosis
Abd Alla Ibrahim Ahmed Shady
Assistant lecturer of Clinical Pathology
Faculty of Medicine-Mansoura University

2. Supervisors
Prof. Dr. Mohamed Hossam Eldeen Zaghloul
( Principal supervisor )
Professor of Clinical Pathology
Faculty of Medicine -Mansoura University
Prof. Dr. Maysaa El sayed Zaki
Professor of Clinical Pathology
Faculty of Medicine-Mansoura University
Prof. Dr.Douaa Raafat El Deeb
Professor of Clinical Pathology
Faculty of Medicine -Mansoura University

3. Introduction
&
Aim of work

4. • Tuberculosis remains a major global health problem
world wide .it is ranked as the second leading cause of
death from an infectious disease .
• Geographically in 2019 ,most people who developed TB
were in South-East Asia (44%), Africa (25%) of total
population according to WHO ., 2020 .
• The case detection rate of positive cases in Egypt was
72% (global target is 70%) and treatment success rate
was 87% (global target is 85%) ( WHO 2012 ).

5. Epidemology of tubeculosis
• Total TB incidence in Egypt is 12,000 (10,000-13,000) of
population . ( MDR/RR-TB ) incidence of total incidence is
about 270 (190-360) of population .
• TB case notifications in 2019 is considered ( for total new and
relapse ) about 8,046 & 20% tested with rapid methods at
time of diagnosis and 95% bacteriologically confirmed .
• TB case fatality ratio (estimated mortality/estimated incidence) is
4% (3-5) . However In 2019, Egypt had a low incidence of TB
(<10 cases per 100 000 population per year) .(WHO ., 2020)

6. Anti tuberculous drug
The American Thoracic Society ., 2000 guidelines
recommend an initial phase for TB treatment which consists of ;
- Rifampicin 10 mg/kg , Isoniazid 5 mg/kg , Pyrazinamide 15–30
mg/kg , and Ethambutol 15–20 mg/kg , given daily for 8
weeks,
- followed by a continuous phase of Isoniazid 15 mg/kg and
Rifampicin 10 mg/kg administered 2–3 times/week for 18
weeks

7. Multi drug resistant tuberculosis
• Worldwide in 2019, close to half a million people developed (RR-TB)
where 78% of them had (MDR-TB). Globally in 2019, 3.3% of new
TB cases and 17.7% of previously treated cases had MDR/RR-TB.
• According to Drug-resistant TB care in Egypt in 2019 , Laboratory-
confirmed cases MDR/RR-TB were considered 264 . 91% of new cases
of MDR-TB were bacteriologically confirmed for rifampicin resistance
. 59% of previously treated cases were bacteriologically confirmed for
rifampicin resistance .(WHO ., 2020) .
• The frequency of resistance to multiple drugs varies geographically, and
acquired (secondary) resistance is more common than primary
resistance. ( Mansour et al., 2010 ) .

8. Laboratory
dignosis of TB
Direct
Conventional
(stain & culture)
Zn stain
AR stain
LJ culture
MODS
ESP culture system
Bactec 460 TB
MB /Bact
Bactec / MGIT
Molecular
methods
Phage amplification assay
PCR
LCR
TB PNA FISH
Xpert MTB/RIF assay
DNA fingerprinting
Line probe assay
Indirect

9. Laboratory
diagnosis of
TB
Direct
Indirect
Ag –ab assay ELIZA
ALS
Ag detection assay
quantiferon TB gold test
‘’’’’’’’’’’’’’’’’’’ in tube
MPB 64 Patch test
Ab detection assay
SOD
Biochemical assay
Chromatography
MDR-XDR TB
colorimetric test
ADA
Recent automated
Tb spot test
Others
TST

10. Microscopic appearance for M tuberculosis with ZN stain for
acid fast bacilli

11. Host genomics polymorphism
• Approximately 90% of TB-infected individuals will remain
asymptomatic with latent infection and only 10% will develop
active disease, suggesting that host genetic factors play an
important role to regulate the progression of tuberculosis infection .
• Complex interactions of MTB with environmental and host
genetic factors play a critical role in tuberculosis infection ( Azad
et al ., 2012 ).
• Assessing the contributions and functional consequences of human
genetic polymorphisms to tuberculosis susceptibility or disease
progression remains amajor challenge ( Harapan , et al ., 2013)

12. 1-Natural resistance-associated macrophage protein
1 (NRAMP1)
• NRAMP1 is a critical mediator in the innate immune
response to tuberculosis infection which leads to
decreased DNA replication and respiratory chain function
in MTB ( Blackwell et al ., 2003 ).
• NRAMP1 30-UTR and D543N polymorphisms appeared
to be associated with an increased susceptibility or
protection against the risk of developing active PTB (Mo¨
ller and Hoal ., 2010 ).

13. 2-N-acetyltransferase 2 (NAT2)
The determination of the acetylator phenotype mediating NAT2
SNPs would permit adequate drug therapy to be administered to
the population with TB (Salazar-Gonza et al ., 2014).
3-Tumor necrosis factor alpha (TNF-a) &
leukotriene A4 hydrolase ( LTA)
TNF-a is likely involved in host protective responses against M.
tuberculosis infection (Azad et al ., 2012) .

14. The importance of the high LTA4H/TNF state in human is to
understand TB severity and treatment responsiveness ( Tobin et al
., 2010).
4-Mannose-binding lectin (MBL)
Mbl2 polymorphisms have been associated with low levels of
MBL in serum . On other hand, MBL deficiency was also
associated with susceptibility to TB infection ( Capparelli et al .,
2009 ).

15. • Is to through a light on the impact of host
genomics such as NRAMP1, TNF-a , LTA ,
MBL and NAT2 on susceptibility and
treatment of tuberculosis
Aim of work

16. Subject
& Method

17. 150 patient &
control
100 pulmonary TB
cases
( TB cases group )
subjected to hx taking ,
CXR evaluation , anti TB
treatment
50 MDR TB cases
subgroup
50 Cured TB cases
subgroup
50 apparently healthy
People ( control group )

18. DNA
extraction
from 5 ml bl
sample then
do PCR-
RFLP
NRAMP
AvaII D543N
FokI 30 UTR
NAT2
BamHI NAT2*7
TaqI NAT2*6
TNF & LTA
Nco1 TNF-308
Nco1 LTA+252
MBL
BanI Allele A
MboII Allele C

19. NRAMP
D543N
Allele G/G
Allele A/A
Genotype
A/A ,A/G, G/G
NRAMP
30 UTR
Allele TGTG+/+
Allele Del+/+
Genotype TGTG+/+,
Del+/+, TGTG/Del
NAT2*7
Allele G/G
Allele A/A
Genotype
A/A ,A/G,G/G
NAT2*6
Allele G/G
Allele A/A
Genotype
A/A ,A/G ,G/G0
TNF-308
Allele A/A
Allele G/G
Genotype
A/A ,A/G , G/G
LTA+252
Allele A/A
Allele G/G
Genotype
A/A, A/G ,G/G
MBL.A
Allele A/A
Allele G/G
Genotype
G/G ,G/A ,A/A
MBL.C
Allele A/A
Allele G/G
Genotype
G/G ,G/A ,A/A

20. Results

29. Photo PCR-RFLP profile of NRAMP 30-UTR genotype/allele . There are 2 rows
of 50 wells/lanes contained (45) samples of cured TB cases where There are ;
Lanes ( 16 &37) molecular size marker
Lanes ( 9- 11-12- 14-17-18-19-23 to 29- 30-31-33-35-38 to 43)
undigested fragment of allele TGTG/TGTG
The remaining lanes digested fragment of 2 band ( 211 bp –
240bp ) of genotype Del/TGTG

30. Photo PCR-RFLP profile of NAT2 , NAT2*6 & NAT2*7 genotype/allele There
are 2 rows of 40 wells/lanes contained samples of cured TB cases where there are ;
Lanes ( 24-50) molecular size marker - Lanes ( 21) digested fragment 2 band ( 276 bp
-380bp) NAT2*6 genotype A/G , lanes ( 20) undigested fragment allele A/A ,
remaining lanes are allele G/G - Lanes ( 28-29-34-35-37-38-44-45 to 53) digested
fragment 2 band (282bp - 810 bp) NAT2*7 genotype A/G , lanes ( 31-42-43)
undigested fragment allele A/A .the remaining lanes are allele G/G

31. Photo PCR-RFLP profile of TNF-308 genotype . There are 2 rows of 40
wells/lanes contained samples of cured TB cases where there are ;
Lanes ( 14& 35) molecular size marker
Lanes ( 8-10-16) digested fragment genotype G/G , lanes (18-
19- 24 to 27-29-33) undigested fragment allele G/G
The remaining lanes digested fragment of 2 band ( 87-107 bp )
genotype A/G

32. Photo PCR-RFLP profile of LTA+252 genotype . There are 2 rows of 35
well/lanes contained samples MDR/cured TB cases where there are ;
Lanes (11-25-18-33) molecular size marker
Lanes ( 7-8-17-20-23-27-31-32) digested fragment 2 band ( 586
bp -782bp) genotype A/G , lanes (13-15-16-21-22-28) undigested fragment allele
G/GThe remaining lanes & (1st 25 ) lanes digested fragment 2 band
(586-196 bp) genotype G/G

33. Photo PCR-RFLP profile of MBL.A genotype/allele . There are row of
20 wells/lanes contained samples of MDRTB cases where there are ;
Lanes (14) molecular size marker
Lanes ( 1-2-3-5-6-7-12-13-15 ) digested fragment of 2 band (89 bp - 260bp )
genotype G/A , lanes (20 ) undigested fragment allele A/A
The remaining lanes digested fragment genotype G/G

34. Conclusion

35. • There are no significant difference in age , complication (CXR)
between MDR-Cured cases group except bilateral cavitation more
in relation to host genomics predictors to MDR TB
• NRAMP D543N genotype A/G & & allele G/G , 30-UTR genotype
Del/TGTG & allele TGTG/TGTG , NAT2*6 genotype A/G ,
TNFA _308 genotype A/G and MBL.C genotype G/G
polymorphisms were protective against occurrence of MDR TB
• NAT2*6 genotype G/G & TNFA _308 genotype G/G and MBL.C
genotype G/A polymorphism were predictors of MDR TB .NAT2 (
NAT2*7 ) & LTA+252 and MBL .A polymorphism genotyping
not either predictors or protective .