UJMR, Volume 5 Number 1, June, 2020, pp 24 - 30 ISSN: 2616 – 0668

https://doi.org/10.47430/ujmr.2051.004

Received: 17th June, 2020 Accepted: 22nd June, 2020

Phenotypic and Genotypic Characterization of Multi-Drug Resistant

Mycobacterium tuberculosis
1
Aliyu, M. S., 2Garba, I., 1Tijjani, M. B., 1Doko, M. H .I., 3Mukhtar G. L., 1Musa, B. and
1
Madika, A.
1
Department of Microbiology, Faculty of Life Sciences, Ahmadu Bello University, Zaria
2
Department of Medical Microbiology, Faculty of Medical Laboratory Science, Usmanu Danfodiyo
University, Sokoto
3
Department of Microbiology, Faculty of Natural and Applied Sciences, Umaru Musa Yar’adua
University, Katsina
Abstract
This study characterized multi-drug resistant M. tuberculosis phenotypically by LJ-proportion
method and genotypically by Geno Type MTBDR plus LPA. Out of the forty M. tuberculosis
isolates tested, two (5.0%) were found to be multi-drug resistant by LJ proportion method and
one (2.5%) was MDR by LPA. None was found to be mono-resistant to any of the drugs by LJ
however, one isolate was mono resistant to RIF and one was mono resistant to INH by LPA.
Comparison of Geno Type MTBDRplus LPA and phenotypic LJ-proportion methods showed that
one isolate was mono resistant to RIF and one was mono resistant to INH by LPA, one and two
MDR-TB isolates respectively were characterized by genotypic and phenotypic methods. The
remaining isolates were found to be pan susceptible by both methods. One isolate was
characterized as MDR with bands at rpoβ MUT2A region and ihnA MUT2 corresponding to H526Y
and A16G mutations respectively. Rifampicin mono resistance with band at rpoβ MUT3
corresponding to S531L was found in one isolate. Also, isoniazid mono resistance was observed
in one isolate with ihnA MUT2 band corresponding to A16G mutation. This study has shown an
overall high prevalence of MDR-TB in the study area which needs to be urgently addressed.
Laboratory facilities for rapid drug resistance detection are needed across the country for
early and accurate diagnosis of TB and drug resistant cases. This remains an important step in
managing TB drug resistance in Nigeria.
Key words: Multi-drug resistance, M. tuberculosis, phenotypic, genotypic, LJ-proportion, Geno
Type MTBDRplus LPA, RIF, INH

INTRODUCTION of primary resistant organisms varies for each

Multidrug-resistant (MDR) strains of drug; however, it is usually 1 in 10 6 to 1 in 108
Mycobacterium tuberculosis have emerged (Iseman, 1993).
worldwide. In many countries and regions, Spontaneous resistance to isoniazid is
these resistant strains constitute a serious estimated to occur once in every 106 organisms,
threat to the efficacy of tuberculosis control and to rifampicin once in every 108 organisms.
programs. An important element in gaining The probability of spontaneous mutants being
control of this epidemic is developing an simultaneously resistant to two or more drugs is
understanding of the molecular basis of the product of the individual mutants. The
resistance to the most important development of drug resistance is a man made
antituberculosis drugs; isoniazid and rifampicin. amplification of a naturally occurring
On the basis of this information, more exacting phenomenon. Previous treatment for
laboratory testing, and ultimately more tuberculosis predisposes to the selection of
appropriate and timely treatment regimens, multi drug resistant organisms. Non compliance
can be developed (Somoskovi et al., 2001). is a major factor in allowing the resistant
Drug resistance in M. tuberculosis occurs as a organisms to survive (Iseman, 1993). Multi drug
result of random spontaneous chromosomal therapy is used to prevent the emergence of
mutations during natural cell replication. These drug resistant mutants during the long duration
mutations are not drug induced and are not of treatment.
linked. The probability of a drug-resistant Resistance can be classified as single-drug,
mutant occurring is directly proportional to the multi-drug, or poly-drug resistance depending
size of the bacterial population. The frequency
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 UJMR, Volume 5 Number 1, June, 2020, pp 24 - 30 ISSN: 2616 – 0668
on the number of drugs and/or which drugs are
involved (Rieder, 1999).
Although an unequal global distribution of drug and staining by the Ziehl-Neelsen procedure.
resistance exists between poor and rich Typical acid fast bacilli were further subjected
countries, the problem is global. The regions to test according to the manufacturer’s
where drug-resistant TB is more prevalent lack instructions by SD BIOLINE TB Ag MPT64 Rapid;
the resources to implement adequate measures a rapid immuno chromatographic identification
to control even the susceptible types of the test for the M. tuberculosis complex (MTBC)
disease (Espinal et al., 2001; Cohen et al., that uses mouse monoclonal anti-MPT64. The
2003). kit has sensitivity and specificity of 98.6% and
Molecular methods for MDR-TB detect the 100% respectively. The test cassette consists of
common mutations conferring resistance to RIF a sample pad, a gold conjugate pad, a
and INH, rather than the resistance phenotype. nitrocellulose membrane and an absorbent pad.
The commercially available line probe assays Mouse monoclonal anti-MPT64 was immobilized
involve DNA extraction, polymerase chain on the nitrocellulose membrane as the capture
reaction (PCR), and solid phase reverse material (test line). Another antibody which
hybridization of amplified DNA to probes recognized another epitope of MPT64,
covering the core region of the target gene, conjugated with colloidal gold particles was
immobilized on a nitrocellulose strip. These used for antigen capture and detection in a
tests can be applied on MTB isolates or on sandwich type assay.
smear positive sputum (De Beenhouwer et al., Four colonies were suspended in 200 µl of the
1995; Rossau et al., 1997). extraction buffer prior to the test. The cassette
The GenoType® MTBDR assay (Hain Life was removed from the foil pouch and placed on
sciences, Nehren, Germany) simultaneously a flat dry surface. One hundred micro litres
detects the common mutations in the rpoβ and (100 µl) of the suspended colonies in buffer was
katG gene (Hillemann et al., 2005). The added into the sample well. As the test began
GenoType® MTBDRplus, a newer version of the to run, a purple colour moved across the result
genotype MTBDR detects more of the common window in the centre of the device. After 15
mutations in the rpoβ and katG genes, and also minutes of sample application, the appearance
mutations in the inhA promoter region, making of two colour bands (“T” test band and “C”
it the most sensitive line probe assay for control band) within the result window was
detection of resistance (Hillemann et al., considered a positive result. Confirmed MTBC
2007). isolates were stored at -20oC for further use.
Evaluation studies of these assays have Drug susceptibility assays
reported sensitivity and specificity of 98-100% Susceptibility to isoniazid (INH) and rifampicin
for rifampicin, and of 70-100% for isoniazid, (RIF) was determined by the proportion
with results in 1-3 days (Ling et al., 2008). method on Lowenstein Jensen egg based slopes
Most of these studies were performed in containing different concentrations of INH and
developed countries and there is limited data RIF (0.2 μg/ml and 40 μg/ml respectively)
on the performance of the tests in developing (Canetti et al., 1969). Standard antibiotic
countries (Ling et al., 2008). A major limitation powders (INH and RIF) were obtained from
of these assays in developing countries could be Sigma-Aldrich (Lot. No. SLBC 3024V and SLBD
the expertise in molecular biology required to respectively).
perform them correctly, the unidirectional The inoculum was prepared by directly
work flow laboratory infrastructure and the suspending colonies grown for approximately
cost of molecular assays. three weeks on Lowenstein Jensen drug free
This study therefore, was aimed at slopes to a turbidity equivalent to a 1.0
characterizing multi-drug resistant M. MacFarland standard. The 1.0 MacFarland
tuberculosis by phenotypic and genotypic standardized suspension was further diluted to
techniques in Zaria, Kaduna State. 10-1, 10-2 and 10-3. The 10-1 suspension was
subsequently inoculated on the drug-containing
MATERIALS AND METHODS medium. Three drug-free LJ slopes were
Mycobacterial Isolates inoculated with 1:10, 1:100 and 1:1000 diluted
The test isolates M. tuberculosis were suspensions of a 1.0 MacFarland standardized
obtained from National Tuberculosis and inoculum. This was done for each sample
Leprosy Training Centre, Saye, Zaria, Nigeria. tested. The drug-susceptible MTB reference
The isolates were confirmed by standard strain ATCC 27294 (H37Rv) was used as a
Microbiological techniques. They were susceptible control and known resistant strains
examined for morphology by making a smear (ATCC 35825 H37Rv for INH and ATCC35838
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 UJMR, Volume 5 Number 1, June, 2020, pp 24 - 30 ISSN: 2616 – 0668
H37Rv for RIF) were used as resistant controls. resistant if the proportion of bacilli resistant to
The slopes were incubated at 37°C and read the critical concentration of a drug exceeded
after 4 and 6 weeks. An isolate was considered 1% (Canetti et al., 1963; Canetti et al., 1969).
Genotypic assays were carried out by the 120 seconds; followed by an additional 20
GenoType® MTBDRplus molecular line probe cycles of denaturation at 95 oC for 25 seconds,
assay according to the manufacturer’s annealing at 53 oC for 40 seconds, elongation at
specifications as follows: 70 oC for 40 seconds and final extension at 70 oC
DNA extraction for 8 minutes. The amplicons were used for
Sterile plastic loops (one per sample) were used further analysis.
to suspend colonies from the LJ slants into 2ml Denaturation of DNA
micro centrifuge tubes containing 100 µl The TwinCubator® (shaking water bath) was
molecular grade water and centrifuged at pre-warmed to 45 oC and 20μl of denaturation
10,000xg for 15 min. The supernatant was solution (NaOH) was added to each labeled well
discarded and the pellet re-suspended in of the TwinCubator® tray followed by the
molecular grade water, to ensure that the addition of 20μl of the amplicons respectively.
suspension of extracted DNA was free of The mixture was mixed gently by pipetting up
impurities that might inhibit the PCR. The re- and down five times and then incubated at
suspended solutions were vortexed until they room temperature for 5mins.
appeared slightly opaque (milky). The tubes Hybridization and detection
were arranged in floating racks, placed in One (1) ml of the pre-warmed hybridization
water bath and heated at 95 oC for 20 min. This buffer (HYB) was carefully added to the wells
was to kill the bacilli and partially lyse the using a pipette. The tray was then carefully
cells, thereby rendering the solution non tilted back and forth so that the purple
infectious and to obtain a higher yield of DNA. denaturation solution and green hybridization
The tubes were placed in ultrasonic bath and buffer were thoroughly mixed. The tray was
incubated for 15 min. The powerful ultrasonic placed on the TwinCubator® and the labeled
shockwaves created by the sonicator disrupted strips added to each well ensuring that the
the cell walls of the tubercle bacilli, causing strips were completely covered by the liquid.
further cell lyses, and releasing the DNA and This was incubated at 45 oC for 30mins. After
other cell debris into the molecular grade incubation, the glass panel lid was opened and
water. Finally, the tubes were centrifuged at the condensate that formed during the
maximum speed (10,000xg) to separate the incubation wiped off. The HYB buffer was
impure cell debris (containing the cell wall, aspirated completely from each well using a
proteins and other macromolecules) from the Pasteur pipette. One (1) ml of the pre-warmed
DNA. The heavier debris formed the pellet and red stringent wash buffer (STR) was dispensed
the lighter DNA (free from impurities) was into the tray using a multi-channel pipette
suspended in the supernatant. The avoiding the contact of the strips with the tips.
supernatants were transferred into clean After 15 minute incubation at 45 oC in the
labeled micro centrifuge tubes for further use. TwinCubator®, STR buffer was aspirated and
PCR amplification of the extracted DNA disposed of with a Pasteur pipette. The STR
The master mix was prepared in clean DNA-free buffer was washed off with 1 ml of Rinse
room. The master mix was made of five solution (RIN) for 1 minute. One (1) ml of the
components with a total volume of 45μl for Conjugate (CON) solution was dispensed into
each PCR reaction (35μl of the primer each well and the glass panel lid was closed
nucleotide mix (PNM), 5μl of buffer, 2μl of and incubated for 30 minutes on the
MgCl2, 3μl of molecular grade H2O and 0.2 μl of TwinCubator®. The strips were washed twice
Taq polymerase). The master mix was then well with 1 ml of Rinse solution (RIN) for 1 minute in
mixed and 45μl required for each specimen was the TwinCubator® to wash off the excess CON
transferred into the PCR tubes and 5μl of each solution. One (1) ml of sterile distilled water
sample was added to the corresponding tube was added to each strip-containing well, and a
containing 45 μl of master mix and then mixed 1 minute wash performed on the TwinCubator®
by gently pipetting up and down a few times. to wash off the RIN solution after which the
The tubes were mixed slightly and spun down distilled water was completely decanted. One
for 5 - 10 seconds in a mini-centrifuge before (1) ml of the Substrate solution was dispensed
they were placed into the thermal cycler for into each well and incubated for 15 minutes on
amplification. A 30 cycle (10 + 20) thermal the TwinCubator® after which the Substrate
cycler program was used for the amplification. solution was aspirated and the strips washed
This involved ten cycles of denaturation at 95 twice with sterile distilled water. A pair of
o
C for 30 seconds and elongation at 58 oC for clean tweezers was used to remove the strips
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 UJMR, Volume 5 Number 1, June, 2020, pp 24 - 30 ISSN: 2616 – 0668
from the TwinCubator® tray and placed onto partially dried and transferred to the
absorbent paper. The developed strips were GenoType® MTBDRplus score sheet.

RESULTS 26 mono-resistant to any of the drugs by LJ-

Out of the forty M. tuberculosis isolates tested, proportion method, however, one isolate was
two (5.0%) were found to be multi-drug mono resistant to RIF and one was mono
resistant by LJ-proportion method and one resistant to INH by LPA (Figure I).
(2.5%) was MDR by LPA. None was found to be

Genotypic
MDR,
Phenotypic
2.50%
MDR,
5.00%

Figure I: Phenotypic and Genotypic Multi drug resistant M. tuberculosis

Table 1 shows the comparison of Geno Type such by the LJ Proportion method, one and two
MTBDRplus LPA and phenotypic LJ-proportion MDR-TB isolates respectively were
methods. The comparison showed that one characterized by genotypic and phenotypic
isolate was mono resistant to RIF and one was methods. The remaining isolates were found to
mono resistant to INH by LPA only and none of be pan susceptible by both methods.

Table 1: Comparison of Geno Type MTBDRplus LPA and phenotypic LJ-proportion methods
Susceptibility Geno Type MTBDRplus phenotypic LJ-proportion

RIF mono-resistance 1 0
INH mono-resistance 1 0
MDR-TB 1 2
Pan susceptible 37 38
Total 40 40

The banding patterns of mutations associated respectively. Rifampicin mono resistance with
with rifampicin and isoniazid resistance in band at rpoβ MUT3 corresponding to S531L was
multidrug resistant and mono-resistant strains found in one isolate. Also, isoniazid mono
detected by MTBDR plus is shown in Table 2. resistance was observed in one isolate with
One isolate was characterized as MDR with ihnA MUT2 band corresponding to A16G
bands at rpoβ MUT2A region and ihnA MUT2 mutation.
corresponding to H526Yand A16G mutations

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 UJMR, Volume 5 Number 1, June, 2020, pp 24 - 30 ISSN: 2616 – 0668
Table 2: Patterns of gene mutations detected by Geno Type MTBDRplus assay in drug resistant
M. tuberculosis isolates
Gene Band Gene region or MDR strains(n RIF mono resistant INH mono resistant
mutation =1) strains(n = 1) strains(n = 1)
rpoβ
WT1 506-509
WT2 510-513
WT3 513-517
WT4 516-519
WT5 518-522
WT6 521-525
WT7 526-529
WT8 530-533
MUT1 D516V
MUT2A H526Y 1
MUT2B H526D
MUT3 S531L 1
katG
WT 315
MUT1 S315T1
MUT2 S315T2
inhA
WT1 -15/-16
WT2 -8
MUT1 C15T
MUT2 A16G 1 1
MUT3A T8C
MUT3B T8A
Key: WT= Wild Type, MUT= Mutant, D=Aspatate, V=Valine, H=Histadine, Y=Tyrosine, S=Serine,
L=Leucine, T=Threonine, C=Cysteine, A=Alanine, G=Glycine
DISCUSSION In patients infected with a fully susceptible
The most important measure of TB drug strain, drug resistance can develop gradually
resistance is the number of new cases that are during inadequate treatment due to selection 28
MDR-TB (Dye et al., 2002). This study showed a of cells with random mutations in sites
prevalence rate of 5.0% (2/40). The associated with drug resistance (i.e. secondary
development of drug resistance is a man made resistance). In this case, as the proportion of
amplification of a naturally occurring susceptible cells decrease and resistant cells
phenomenon. Previous treatment for increase, a hetero-resistant population of cells
tuberculosis predisposes to the selection of will be present. These cells are primarily
multi drug resistant organisms. Non compliance identical throughout the genome but a
is a major factor in allowing the resistant proportion of the population differs in sites
organisms to survive. The availability of drugs associated with drug resistance (Foundation for
in the open market and a private sector that innovative new diagnosis, 2012).
delivers drugs to the population in an In contrast, patients infected with fully
unregulated fashion in Nigeria could also be susceptible strains may develop mixed
factors that might favour development of MDR- infections if they are co-infected with another
TB. strain that is drug resistant, resulting in a
The genotypic drug resistance assay revealed mixed population of two genetically distinct
that all the resistant isolates were hetero- strains, one drug susceptible, and the other
resistant; a phenomenon where some cells drug resistant.
within a population may remain susceptible to Also, hetero-resistance likely represents a
the antibiotic, whereas other cells display natural variation in the population of cells of
varying degrees of drug resistance. This was M. tuberculosis (Foundation for innovative new
determined by the simultaneous detection of diagnosis, 2012). Prescription of inadequate
the wild type and mutant molecular treatment regimen, irregular drug supply, poor
susceptibility (Foundation for innovative new drug quality with low bioavailability, and poor
diagnosis, 2012). compliance among the study population could

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 UJMR, Volume 5 Number 1, June, 2020, pp 24 - 30 ISSN: 2616 – 0668
be attributed to the development of hetero- resistance observed in this study.
Discordance between genotypic and phenotypic level resistance and 0.125µg/ml for high-level
assays was observed in one isolate with RIF resistance were reported by Gumbo, (2010).
mono resistance. The isolate was classified as
MDR by the phenotypic assay. This could be due CONCLUSION AND RECOMMENDATION
to silent mutation or synonymous single The findings showed that one isolate each was
nucleotide polymorphism (sSNP) at the target mono resistant to RIF and INH by LPA, one and
site. Silent mutations do not result in structural two MDR-TB isolates respectively were
changes in the inhA and so do not interfere characterized by genotypic and phenotypic
with its inhibition by INH. Moreover, findings of methods. The remaining isolates were found to
silent mutations in inhA are not surprising as be pan susceptible by both methods. One
SNPs occur every 3 kb of MTB genome (Comas isolate was characterized as MDR with bands at
et al., 2011). Ando, et al. (2014) reported a rpoβ MUT2A region and ihnA MUT2
silent mutation in a significant number of INH corresponding to H526Yand A16G mutations
resistant M. tuberculosis clinical isolates. respectively. Rifampicin mono resistance with
Mutations conferring INH resistance in other band at rpoβ MUT3 corresponding to S531L was
genes such as mabA (G609A) (Ando et al., 2014) found in one isolate. Also, isoniazid mono
aphC (alkyl hydroperoxide reductase), kasA (ß- resistance was observed in one isolate with
ketoacyl-ACP synthase) and nadh (NADH ihnA MUT2 band corresponding to A16G
dehydrogenase) (Cohn et al., 1997; mutation.
Balasubramanian et al., 2012) not included in The use of robust molecular techniques such as
Geno Type MTBDRplus have also been reported. DNA sequencing employed for the detection of
The critical concentration of INH on LJ medium occult cases with low-level resistance and other
which is being used for over 50 years is resistance not included in the Geno Type
0.2µg/ml (Jamieson et al., 2014). Lower MTBDRplus kit is recommended.
critical concentrations of 0.0312µg/ml for low-

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