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American Journal of  Medical 
Science and Innovation (AJMSI) 

Role of  HIV Infection in Multi-Drug Resistant Tuberculosis in 
Parts of  Benue State, Nigeria  

Lan, Abraham Ternaa1*, Amuta U Elizabeth2, Terzungue Sarc1

Volume 1 Issue 1, Year 2022
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Article Information ABSTRACT

Received: August 15, 2022

Accepted: September 02, 2022

Published: September 06, 2022

The pathogenesis of  Tuberculosis shows that M. tuberculosis target and persist within 
phagocytes including T-lymphocytes in blood circulation. As a result, the possibility of  cel-
lular interaction between M. tuberculosis and HIV, especially for patients that are co-infected 
with HIV and TB, and subsequent exchange of  genetic material via transduction needs to 
be investigated. Three hundred and eighty sputum samples mostly from suspected rifampi-
cin-resistance patients were collected from Nigerian Airforce (NAF) Hospital Makurdi, and 
Federal Medical Centre (FMC) Makurdi. In vitro culture of  sputum samples, Drugs Suscep-
tibility Testing (DST) of  M. tuberculosis isolates, and transduction protocol were carried out 
at the National Tuberculosis and Leprosy Training Centre (NTBLTC) Zaria, Nigeria. Statis-
tical analysis was carried out using Student’s t-test. Minitab version 14.0 statistical software 
was used for data analysis. P-values < 0.05 were considered significant. Twenty-six (9.7%) 
cases of  Multi-drug resistant tuberculosis (MDR-TB) were detected (retreated cases 7.1%; 
treatment naive 2.6%). Twenty-one (80.8%) were males and 5(19.2%) were females. There 
was statistical difference in MDR-TB between male and female in Benue State (P<0.05). 
The mean age group 35-45 years had the highest cases of  MDR-TB accounting for 35% of  
MDR-TB. Human-immunodeficiency virus and Tuberculosis co-infected patients (category 
I) had the highest MDR-TB incidence of  10(38.5%). There was no significant difference be-
tween category-1 and category-III [patient with only TB disease (P>0.05)]. However, there 
were elevated cases of  MDR-TB in category-III patients from 7(26.9%) to 10(38.5%) fol-
lowing transduction protocol. Multi-drugs resistant tuberculosis is prevalent in Benue State, 
affecting the most economically active youths within the age group of  35-45 years, as a result 
the need to direct more attention on molecular basis for M. tuberculosis drugs resistance is 

Keywords
Bacteriophage, Isoniazid, Multi-
Drug Resistance, Mycobacterium 
Tuberculosis, Rifampicin, 

1 Department of  Microbiology, Federal University of  Agriculture, Makurdi Nigeria
2 Department of  Zoology, Federal University of  Agriculture, Makurdi, Nigeria 
* Corresponding author’s e-mail: abrahamlanterna@gmail.com

INTRODUCTION
The genus Mycobacterium are non-motile, non-sporulating, 
weakly Gram-positive, acid-fast bacilli that appear 
microscopically as straight or slightly curved rods 
measuring 0.2 to 0.4 µm in length (Willey et al., 2011).
Mycobacteria are within the order Actinomycetales, 
which it shares with bacteria such as Corynebacterium, 
Norcadia and Rhodococcus. Mycobacteria have been 
divided into two major groups based on fundamental 
differences in epidemiology and association with disease. 
Those belonging to the Mycobacterium tuberculosis complex 
(MTBC) (M. tuberculosis, M. bovis, M. africanun, M. canetti, 
and M. microti, with M. laprae and M. pinnipedii considered 
variants of  M. bovis), and those referred to as the non-
tuberculous mycobacteria (NTM) such as M. avium complex, 
M. haemophilum, M. ulcerans, M. leprae (nonculturable), and 
the potentially pathogenic species such as M. smegmatis 
and M. abscessus (Forbes et al.,2002).
A very important and unique characteristic of  
Mycobacteria is that the organisms grow more slowly 
than most other human pathogenic bacteria because of  
their hydrophobic cell surfaces (Forbes et al.,2002).
Tuberculosis is a common, and in many cases fatal, 
infectious disease caused by various strains of  
mycobacteria, usually M. tuberculosis (Kumar et al., 2007). 
Tuberculosis is an airborne disease that affects the lungs 
(pulmonary TB), but can also affect other parts of  the 

body (extra pulmonary TB) such as the larynx, the lymph 
nodes, the pleura, the brain, the kidneys, or the bones and 
joints (Bardarov et al., 2002).
Based on clinical presentation, TB can be categorized 
into active TB disease characterized by chronic cough 
with blood-tinged sputum, fever, night sweats and weight 
loss (the latter giving rise to the formerly common term 
consumption), most infections do not have classical 
symptoms and are thus referred to as latent TB infection 
(LTBI). Persons with LTBI have M. tuberculosis in their 
bodies, but do not have TB disease and cannot spread the 
infection to other people (Bardarov et al., 2002).
It is estimated that one-third (at least 2 billion people) 
of  the world’s human population is infected (Willey et al., 
2011). Of  the 212 countries and territories in the world, 
202, (99.6% of  the world’s population) reported TB cases 
in 2007; the World Health Organization (WHO) reported 
9.2 million new TB cases, with approximately 7.7% being 
HIV positive (Willey et al., 2011).
Today, TB is second only to HIV/AIDS as the greatest 
killer worldwide due to a single infectious agent, in 2012 
for instance, 8.6 million people fell ill with TB and 1.3 
million died from TB, with over 95% of  TB deaths 
reported in low and middle-income countries (WHO, 
2014), tuberculosis is still among the top three causes of  
death for women aged 15 to 44 years (WHO, 2014).
The emergence of  MDR-TB, first reported in the late 

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1980s (Cegielski, 2010), and present in virtually all the 
countries surveyed has posed a great obstacle to effective 
TB control at both national and global levels.
In 2010, the World Health Organization (WHO) 
estimated that globally there were 290, 000 cases of  MDR-
TB among reported cases of  pulmonary TB (WHO, 
2011). The World Health Organization also reported an 
estimated 650, 000 cases of  MDR-TB among the world’s 
12 million prevalent cases of  TB with Nigeria alone 
accounting for 95 reported cases of  MDR-TB (WHO, 
2011). Resistance to TB-drugs is said to arise from both 
service and patient related factors in the management of  
Tuberculosis ranging from poor compliance, inadequate 
supervision, inadequate dosing, wrong drug combination, 
lengthy duration of  treatment and poor training of  health 
personnel (Federal Ministry of  Health FMOH, 2005).
Despite reasonable degree of  successes and achievements 
recorded in the stop TB targets of  the millennium 
Development Goals (MDGs) number-6, target-8 of  
reducing the global burden of  TB disease (death and 
prevalence) by 50% by 2015 (WHO, 2011), the second 
component of  the stop TB strategy to address TB/HIV 
and MDR-TB remains a global concern. 
The pathogenesis of  Tuberculosis shows that M. 
tuberculosis is often acquired early in life with acute infection 
and with developing immunity, granuloma formation, 
and calcification. This is followed by a long latent period, 
which continues until reactivation occurs in a proportion 
of  individuals. At this period, the organism target and 
persist within phagocytic monocytes, macrophages, 
polymorphonuclear neutrophils and T-lymphocytes in 
blood circulation (Van Crevel et al., 2002). As a result, 
there is a possibility of  cellular interaction between M. 
tuberculosis and HIV, especially for patients that are co-
infected with HIV and TB, and subsequent exchange of  
genetic information via transduction. It has therefore 
become necessary to determine the role of  HIV in 
the emergence of  MDR-TB in HIV/TB co-infection 
dynamics. Considering the fact that HIV plays a major 
role in infectious diseases generally, and TB in particular 
(Bardarov et al., 2002), the need to focus attention on 
understanding the molecular basis of  TB pathogenesis 
especially MDR-TB with particular emphasis on HIV/
TB co-infection has become a top priority.

MATERIALS AND METHODS
A total of  380 sputum samples determined by Raosoft 
sample size calculator (Raosoft, 2015) were collected from 
a wide spectrum of  TB patients from two geographically 
distinct sites: three hundred and three sputum samples 
were collected from Nigerian Airforce (NAF) Hospital/
CDC laboratory, while the remaining seventy-seven 
sputum samples were collected from Federal Medical 
Centre (FMC)/APIN laboratory, in Makurdi Benue 
State. Between three to ten millilitres of  sputum samples 
were collected in cotylpyridinium chloride (Bromide) 
containing universal bottles, and immediately refrigerated 
at 2-8°C. Patients who test positive for Acid Fast Bacilli 

(AFB) after two months of  intensive phase TB DOTS 
therapy (suspected Rifampicin resistance) was enrollment 
criteria. Demographic information of  subjects was 
obtained from medical records. Data was collected all age 
groups. The study design was approved by Research and 
Ethics committee, Benue State Ministry of  Health.
Out of  380 sputum samples analysed, 130 (34.2%) were 
from TB patients co-infected with HIV, and who were 
positive for AFB after more than two months’ intensive 
phase of  TB DOTS therapy (Category I Patients). 
Another 130 (34.2%) were from HIV negative TB 
patients who were positive for AFB after more than 
two months of  intensive TB DOTS therapy (Category 
II Patients), while 120 (31.6%) sputum samples were 
collected from HIV negative TB patients who yet to 
commence TB DOTS therapy (treatment naïve)-Category 
III Patients). Detection of  Rifampicin resistance was by 
GeneXpert Technology. All Sputum samples were plated 
on Lowenstein-Jensen (LJ) medium for pure cultures of  
M. tuberculosis and confirmed by biochemical tests. Pure 
cultures of  M. tuberculosis were preserved by refrigeration 
as stock cultures at -20°C (David, 1970). Anti-TB Drugs 
Susceptibility Testing (DST) of  M. tuberculosis isolates was 
carried out using the proportion method on BACTEC 
MGIT 960 TB system (Becton and Dickinson, New Jersey 
USA). Stock cultures of  Human T-cell Lymphotrophic 
Virus-3 (HTLV-III) polyclonal unconjugated preparation 
procured from GENTAUR Molecular GenWay products, 
U.S.A, were used as specialized transducing phage in 
transduction protocols. Drugs Susceptibility testing 
of  transductants was also carried out by proportion 
method on BACTEC MGIT 960 TB system (Becton and 
Dickinson, New Jersey USA). All procedures were carried 
out at Biosafety Level two (BSL-11).

GeneXpertGeneXpert  Procedure 
Xpert MTB-RIF Assay G4 Version 5 (Cepheid, USA) 
was used. Briefly, 2.0 ml of  Sputum sample was added 
into 4.0 ml of  Xpert reagent in a ratio of  1:2. The 
closed specimen was manually agitated twenty times and 
incubated at room temperature (20-25°C) for 15 minutes. 
Two millilitres of  the reagent-sample mixture was 
transferred to Xpert test cartridges and inserted into the 
Xpert device. Results (for M. tb either detected or not, 
and with or without Rifampicin resistance) were obtained 
in exactly 110 minutes (Cepheid, 2014).

Processing of  Sputum for M. tuberculosis Culture
Modified Petroff ’s method 2012 for culture of  M. 
tuberculosis was used. Between 3-5 ml sputum was 
homogenized in a shaker using an equal volume of  4% 
NaOH and centrifuged at 3000rpm for 15 minutes. 
Then 0.067M phosphate buffer (pH 6.8) was added to 
the digested-decontaminated sample (deposit) up to the 
45.0ml mark to reduce the continued action of  NaOH, 
while the sediment was re-suspended in 2.0ml of  buffer. 
The sediment was now ready for inoculation unto LJ 
slants, or refrigerated at 2- 8°C (Joshua et al., 2013).

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Culture of  Processed Sputum Sample for M. tuberculosis
Lowenstein-Jensen medium (Oxoid Biologicals, Canada) 
was used. Lowenstein-Jensen slants (egg based) were 
prepared according to manufacturer’s instructions and 
stored at 2-8°C in the dark. Three drops of  processed 
sputum sediment were added to each Lowenstein-
Jensen tube using a sterile plastic pipette. The inoculum 
was spread over the surface of  the slant by gently 
rolling the liquid over the slant and incubated at 35-
37°/7 days in a slanted position with loose screw for 
even distribution and adsorption of  inoculum. After 
one week, inoculated slants were incubated at 35-37°C, 
and examined respectively at three and seven days of  
incubation to allow early detection of  contaminants (or 
rapidly growing mycobacteria). Thereafter cultures were 
examined weekly for growth of  M. tuberculosis. Negative 
cultures were discarded after 8 weeks of  incubation. 
Pale cream colonies which were granular, rough or dry 
were suggestive of  M. tuberculosis. Ziehl Neelsen (ZN) 
staining confirmed growth of  mycobacteria. Growth was 
reported as none (no visible growth), contamination (C), 
<50 colonies (actual count), 50-100 colonies (1+), 100-
200 colonies (2+) >200 colonies (3+), confluent growth 
(4+) (Joshua et al., 2013). Conventional biochemical tests 
such as catalase test, growth on P-Nitro Benzoate (PNB), 
and Nitrate reduction were used to distinguish and 
differentiate M. tuberculosis from other mycobacteria. Pure 
cultures of  M. tuberculosis were inoculated on Lowenstein-
Jensen broth and preserved as stock cultures in 2ml 
cryovials at -20°C for drugs susceptibility testing and 
transduction protocols.

Staining Mycobacterial Isolates from LJ Slants
Ziehl Neelson (ZN) staining method was used. One 
drop of  distilled water was placed in the middle of  a 
clean grease free slide. Growth from the LJ slope was 
scrapped off  and emulsified in saline on the slide using 
a sterile disposable loop. The smear was allowed to air 
dry thoroughly and was fixed by passing the reverse three 
times through a blue flame. A negative control slide was 
also prepared from a genexpert negative sample as earlier 
described. The slides were arranged on a staining rack, 
flooded with carbol fuschsin working solution and heated 
to steam for five minutes without drying or boiling. The 
slides were washed gently with tap running water to 
remove excess carbol fuschsin and flooded with 3% acid 
alcohol for three minutes to decolorize completely, and 
washed under running water for one minute. The slides 
were flooded with Methylene Blue and counterstained for 
1 minute. The slides were rinsed with tap running water, 
drained and air dried, examined under oil immersion 
magnification (x100). Acid Fast Bacilli appeared as pink 
or red bacilli while the negative control slide appeared 
blue (Joshua et al., 2013).

Preparation of  mycobacteria growth indicator tube 
(MGIT) 
Plastic caps from the Streptomycin Isoniazid Rifampicin 

and Ethambutol (SIRE) supplements were removed. Caps 
from MGIT were also removed, and 0.8ml supplements 
were aseptically dispensed into each MGIT using sterile 
pipette. The tubes were immediately recapped. The 
procedure was repeated using PZA supplement. Five 
MGIT (7.0ml) were labeled with SIRE supplement for 
each test isolate as (growth control), S(SM), I(INH), 
R(RIF), and E(EMB). Two MGIT (7.0ml) were labeled 
with PZA supplements for each test isolate as C (growth 
control), S(SM), I(INH), R(RIF), and E(EMB). Two 
MGIT (7.0ml) were labeled with PZA supplement for 
each test isolate as C (growth control), and PZA. 
Micropipette was used to aseptically pipette 100µL 
working drug concentrations into each of  the 
appropriately labeled MGIT. No antibiotics were 
added to MGIT control tubes. 0.5 mL of  the organism 
suspension was aseptically dispensed into each of  the five 
tubes containing drugs (SM, INH, RIF, EMB, PZA). 1:10 
growth control suspension was prepared by aseptically 
adding 0.5 ml of  the organism suspension into 4.5 mL of  
sterile saline. The 1:10 suspension was mixed thoroughly, 
and 0.5ml inoculated into MGIT-PZA control tube, 
0.5ml was further diluted with 4.5 ml sterile saline from 
the previous 1:10 growth control suspension to produce 
1:100 dilutions. The 1:100 suspensions were thoroughly 
mixed and 0.5 ml was inoculated into the MGIT control 
tube. 
The Tubes were tightly recapped, thoroughly mixed by 
gently inverting three to four times and were wiped with 
disinfectant, loaded into the appropriate DST carrier. 
SIRE was loaded into a five-carrier holder, while PZA 
was loaded into a two-carrier holder. One drop of  the 
organism suspension from the 1:100 control tube was 
streaked on a Blood Agar Plate (BAP), sealed with 
paraffin and incubated at 35-37°C for four days. The 
BAP was read daily, for up to four days, for bacterial 
contamination. The DST was allowed to proceed only 
for BAP that showed no growth during the four days 
monitoring (Joshua et al., 2013).

Drug Susceptibility Test (DST) for M. tuberculosis 
BACTEC MGIT 960 TB system (Becton and Dickinson, 
New Jersey USA) was used. The system monitors 
continuous growth of  microorganisms in both drug-
containing and control tubes to determine susceptibility 
or resistance, and automatically interprets and reports 
results of  tests. The following anti-TB drugs (SIRE) 
were reconstituted to the following concentrations: 
Streptomycin 1.0 μg/ml, Isoniazid 0.1 μg/ml, Rifampicin 
1.0 μg/ml, Ethambutol 5.0 μg/ml, Pyrazinamide 100 μg/
ml (PZA). Preserved stock cultures of  0.1ml M. tuberculosis 
were sub-culturedm to Mycobacterium Growth Indicator 
Tube (MGIT) McFarland standard (1.0 suspension) 
equivalent to 3.0 x 108 cfu/ml. Reconstituted 0.8 ml SIRE 
supplements were aseptically dispensed into each MGIT 
with a pipette and sterile tips. MGIT was immediately 
recapped Interpretations of  DST results were read 
between days four and thirteen SIRE, PZA test. Control 

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tubes that flagged positive before day four were repeated. 
Similarly, control tubes that remained negative after day 
thirteen were also repeated. The DST for the drugs 
under consideration was reported as “sensitive” when the 
control tubes reached growth unit (GU) of  400, while the 
drug tubes had growth units (GU) of  less than 100. The 
results were reported as resistant with a rise in GU equal 
to or greater than 100 and growth in the control tube 
equivalent to 400 GU (Siddiqui and Rusch-Gerdes 2006). 
M. tuberculosis strains that exhibited combined resistance 
to rifampicin and isoniazid were reported as MDR-TB 
(Willey et al., 2011).

Transduction Protocol
Transduction protocol was carried out as described by 
Bardarov and co-researchers (Bardarov et al., 2002), with 
slight modifications. Preserved cultures of  M. tuberculosis 
from category III patients were used. One milliliter of  
M. tuberculosis stock was inoculated into 10ml LJ broth in 
30ml plastic culture bottles, and incubated at 37°C in an 
incubator shaker. The M. tuberculosis strains were grown to 
optical density OD of  600 ~ 0.8-1.0(6.0 x 108 c.f.u ml-1). 
Ten milliliters of  the culture were centrifuged at 2500g 
for 5minutes and re-suspended in 10ml washing medium 
of  1% tween 80 phosphate buffered saline (PBS-TW pH 
7.0) and incubated as a standing culture (37°C; 24hr). 
After the preparation period, the cells were again 
centrifuged at 2500g for five minutes and re-suspended 
in 10ml LJ broth, pre-warmed at 37°C and mixed 
with a specialized transducing phage (Human T-cell 

Lymphotrophic Virus-3) in a 10ml: 1ml v/v ratio. The 
cell/phage mixture was inoculated into 50ml LJ broth 
and incubated at 37°C. Outgrowth of  the cultures was 
performed for 24hr at 37°C. Cells were then pelleted by 
centrifugation at 2500g for 15minutes and re-suspended 
in one millilitre PBS-TW (1.0% tween 80 in phosphate 
buffered saline). DST of  transductant was also carried out 
using BACTEC MGIT 960TB system earlier described. 
The results of  DST for transductants were interpreted 
and reported using the format for pure M. tuberculosis 
isolates earlier described.
 
Analysis of  Data
The results were analyzed using minitab version 14.0 
statistical softwares. Student’s t-test was used to compute 
frequencies and proportions, P-values <0.05 were 
considered significant at 95.0 % confidence level.

RESULTS
Out of  the 380 sputum samples collected and analyzed, 
268(70.5%) yielded positive Mycobacterium tuberculosis 
cultures. The results of  DST carried out on the 268 
M. tuberculosis strains showed that MDR-TB (combined 
resistance to Rifampicin and Isoniazid) was detected 
in 26(9.7%) strains. Twenty-one (80.8%) cases were 
male, while five (19.2%) cases were female. There was 
statistically significant difference in MDR-TB between 
males and females (P<0.05) in the study population. The 
Results of  DST for the three categories of  patients is 
shown in table 1.

Table 1: Overall Results M. tb of  Culture and Anti-TB Drugs Susceptibility Testing (DST)
Age Group
(Years)

M. tb + Cultures CAT I CAT II CAT III Total MDR-TB 
Detected

%
M F M F M F

<1 – 10 2 0 0 0 0 0 0 0 0
11 – 20 8 0 0 1 0 1 0 2 7.69
21 – 30 70 1 1 1 0 1 0 4 15.38
31 – 40 106 2 0 2 0 2 0 6 23.08
41 – 50 37 3 1 3 1 1 1 10 38.46
51 – 60 31 1 1 1 0 1 0 4 15.38
61 – 70 9 0 0 0 0 0 0 0 0
≥ 71 5 0 0 0 0 0 0 0 0
Total 268 7 3 8 1 6 1 26 100
Key: M. tb = Mycobacterium tuberculosis, MDR-TB= Multi-drug Resistant Tuberculosis, M=Total Male, F=Total Female, 
+=positive M. tb cultures, CAT I = category 1, CAT II = Category 2, CAT III = Category 3 

The results of  DST for Category I patients showed 
that MDR-TB was detected in 10(38.5%) cases while 

rifampicin resistance was detected in 32(11.9%) of  cases 
as shown in table 2.

Table 2: Sputum Culture and Anti-TB DST (Category I Patients)
Age 
(years)

No. of  
M. tb + 
Cultures

RIF INH S PZA EMB MDR-TB 
Detected

M F
R S R S R S R S R S

<1 – 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11 – 20 3 0 3 0 3 0 3 0 3 2 1 0 0 0
21 – 30 26 9 17 2 24 7 19 2 24 16 10 2 1 1

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Table 3: Sputum Culture and anti-TB DST (Category II Patients)
Age 
(years)

No. of  
M. tb + 
Cultures

RIF INH S PZA EMB MDR-TB 
Detected

M F
R S R S R S R S R S

<1 – 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11 – 20 1 1 0 1 0 0 1 0 1 0 1 1 1 0
21 – 30 18 6 12 1 17 2 15 5 12 8 11 1 1 0
31 – 40 31 11 20 2 29 3 28 6 25 11 20 2 2 0
41 – 50 7 4 3 4 3 1 6 0 7 2 5 4 3 1
51 – 60 14 2 12 1 12 1 13 1 15 2 12 1 1 0
61 – 70 6 2 4 0 6 0 6 0 6 1 5 0 0 0
≥ 71 2 0 3 0 3 0 3 0 3 0 3 0 0 0
Total 79 26 54 9 70 7 72 12 69 24 57 9 8 1
t = 885.44, df  = 10, p< 0.05
Key: EMB = Etambutol, M. tb = Mycobacterium tuberculosis, MDR-TB= Multi-drug Resistant Tuberculosis, R = Resistant, 
S = Susceptible, RIF = Rifampicin, INH = Isoniazid, S = Streptomycin, PZA = Parazinamide, M=Total Male, F=Total 
Female, +=positive M. tb cultures, DST= Drugs Susceptibility Testing

Table 4: Sputum Culture and anti-TB DST (Category III Patients)
Age 
(years)

No. of  
M. tb + 
Cultures

RIF INH S PZA EMB MDR-
TB 
Detected

M F
R S R S R S R S R S

<1 – 10 2 0 2 0 2 0 2 0 2 0 2 0 0 0
11 – 20 4 3 1 1 3 0 4 1 3 0 4 1 1 0
21 – 30 26 6 20 1 25 5 21 2 24 4 22 1 1 0
31 – 40 37 4 33 2 35 4 33 8 29 6 31 2 2 0
41 – 50 16 6 10 2 14 2 14 6 10 4 12 2 1 1
51 – 60 12 3 9 1 11 2 10 1 11 3 9 1 1 0
61 – 70 2 1 1 0 2 0 2 0 2 0 2 0 0 0
≥ 71 3 1 2 0 3 0 3 0 3 1 2 0 0 0
Total 102 24 78 7 95 13 89 18 84 18 84 7 6 1
t = 632.46, df  = 10, p< 0.05
Key: EMB = Etambutol, M. tb = Mycobacterium tuberculosis, MDR-TB= Multi-drug Resistant Tuberculosis, R = Resistant, 
S = Susceptible, RIF = Rifampicin, INH = Isoniazid , S = Streptomycin, PZA = Parazinamide, M=Total Male, F=Total 
Female, +=positive M. tb cultures, DST= Drugs Susceptibility Testing

31 – 40 38 15 23 2 36 7 31 1 37 17 21 2 2 0
41 – 50 14 5 9 4 10 2 12 0 14 8 6 4 3 1
51 – 60 5 2 3 2 3 0 5 0 5 5 0 2 1 1
61 – 70 1 1 0 1 0 0 1 0 1 1 0 0 0 0
≥ 71 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Total 87 32 55 11 76 16 71 3 84 49 38 10 7 3
t = 800.00, df  = 14, p< 0.05
Key: EMB = Etambutol, M. tb = Mycobacterium tuberculosis, MDR-TB= Multi-drug Resistant Tuberculosis, R = Resistant, 
S = Susceptible, RIF = Rifampicin, INH = Isoniazid,  S = Streptomycin, PZA = Parazinamide, M=Total Male, F=Total 
Female, +=positive M. tb cultures, DST= Drugs Susceptibility Testing. 

Results of  DST for Category II patients showed a slight 
decline in cases of  MDR-TB from 10(38.5%) to 9(34.6%) 
with a corresponding decrease in the total number of  
Rifampicin resistance of  26(9.7%) detected as shown in 
table 3

The least cases of  MDR-TB of  7(26.9%) were detected 
in category III patients while Rifampicin resistance was 
detected in 24(90%) of  cases as shown in table 4.
There was no statistical difference in MDR-TB between 
Category I and Category III (P>0.05). However, cases 

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of  MDR-TB were statistically higher in Category I than 
Category III patients.
The age group 41-50 years had the highest cases of  
MDR-TB followed by the age group 31-40 years (mean 
age group 35-45 years) both of  which accounted for 
10(38.5%) and 6(23.1%) cases respectively, thereby 
placing the two age groups at relatively higher risk of  
developing MDR-TB. Demographic characteristics of  
the 268 M. tuberculosis strains analyzed in Tables 2 and 3 
(re-treated cases) and Table 4 (treatment naive) recorded 
19(7.1%) and 6(2.6%) cases respectively.
Following in-vitro transduction (induced mutation) of  M. 

tuberculosis strains from Category III patients, there was 
an increase in the number of  MDR-TB detected in this 
Category of  patients from 7(26.9%) to 10(38.5%) cases 
as shown in Table 5. However, there was no statistical 
significance in the number of  MDR-TB detected in 
Category III before and after transduction protocols 
(P>0.05).
Overall results of  susceptibility for all the tested anti-TB 
drugs shows a cumulative susceptibility of  1012(84.3%) 
and resistance of  189(15.7%). Etambutol showed the 
highest resistance of  91(34.0%) followed by rifampicin 
with 82(30.6%) of  cases, the least cases of  resistance were 

Table 5: Anti-TB DST of  Category III (control group) Patients after HTLV-3 transduction protocol
Age 
(years)

No. of  M. tb 
+ Cultures

MDR-TB 
detected

RIF INH S PZA EMB
R S R S R S R S R S

<1 – 10 1 0 0 1 1 0 0 1 0 1 0 1
11 – 20 2 1 2 0 1 1 0 2 0 2 0 2
21 – 30 13 2 5 8 2 11 9 4 3 10 9 4
31 – 40 18 3 7 11 3 15 11 7 4 14 11 7
41 – 50 8 2 2 6 3 5 4 4 1 7 4 4
51 – 60 6 2 2 4 4 2 2 4 0 6 2 4
61 – 70 2 0 0 2 0 2 0 2 0 2 0 2
≥ 71 2 0 0 2 1 1 0 2 0 2 0 2
Total 79 26 54 9 70 7 72 12 69 24 57
t=-300.00, df  =14, p<0.05
Key: EMB = Etambutol, M. tb = Mycobacterium tuberculosis, MDR-TB= Multi-drug Resistant Tuberculosis, R = Resistant, 
S = Susceptible, RIF = Rifampicin, INH = Isoniazid, S = Streptomycin, PZA = Parazinamide, M=Total Male, F=Total 
Female, +=positive M. tb cultures, DST= Drugs Susceptibility Testing

produced by isoniazid which recorded 27(10.1%) making 
it the most effective anti-TB drug in the study population. 
266(99.3%) of  the M. tuberculosis isolates produced 
various degrees of  susceptibility and resistance to the five 
tested anti-TB drugs. However, 2(0.7%) showed 100% 
susceptibility to all the five tested anti-TB drugs with no 

trace of  drugs resistance. Meanwhile all the M. tuberculosis 
strains were resistant to at least one or more anti-TB 
drugs thereby implying complete absence of  mono-drug 
resistant tuberculosis in the study population. It is equally 
noteworthy that no strain was completely resistant to all 
the tested anti-TB drugs as summarized in Table 6. 

Table 6: Cumulative Results of  anti-TB DST
Category RIF No (%) INH No (%) S No (%) PZA No (%) EMB No (%)

R S R S R S R S R S
CAT I 32(11.9) 55(20.5) 11(4.1) 76(28.4) 16 (5.0) 71(26.5) 3(1.1) 84 (31.3) 49 (18.3) 38 (14.2)
CAT II 26(9.7) 54(20.1) 9(3.4) 70(26.1) 7 (2.6) 72(26.9) 12(4.5) 69 (25.7) 24 (9.0) 57 (21.3)
CAT III 24(9.0) 78(29.1) 7(2.6) 95(35.4) 13 (4.9) 89(33.2) 18(6.7) 84 (31.3) 18 (6.7) 84 (31.3)
Total
(%)

82 
(30.6%)

187 
(69.8%)

27
(10.1%)

241 
(89.9%)

36 
(13.4%)

168 
(62.7%)

33 
(12.3%)

237 
(88.4%)

91 
(34.0%)

179 
(66.8%)

Key: Cat I = Category I, Cat II = Category II, Cat II = Category III, RIF = Rifampicin, INH = Isoniazid, S = 
Streptomycin, PZA = Parazinamide, EMB = Etambutol, R = Resistant, S = Susceptible, No. = Number, % = 
Percentage, DST= Drugs Susceptibility Testing.

cases. Male accounted for 36(58.1%) while female 
accounted for 26(41.9%) of  the cases as contained in 
Table 7.

t = - 148.48, df  = 13, p< 0.05 
Results of  Genexpert for all the 380 sputum samples 
showed rifampicin resistance was detected in 62(16.3%) 

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DISCUSSION
The results obtained from this study attest to the fact 
that MDR-TB, which is an emerging epidemic, is not 
just prevalent in parts of  Benue State, but it is on the 
increase as compared to the estimated 5.3% rate of  global 
MDR-TB (WHO, 2011). The prevalence of  MDR-TB in 
the study population was 9.7% (retreated cases 7.1%; 
treatment naive 2.6%) out of  the 268 isolated strains of  
M. tuberculosis. There was a higher proportion of  MDR-TB 
within the age group 31 – 40 and 41 – 50 (mean age limit 
35 – 45). This age range represents the most economically 
productive and viable workforce both in the private and 
public sectors. As a result, the need to urgently attend to 
the menace of  MDR-TB in Benue State cannot be over 
emphasized. Other research groups also reported that 
anti-TB drugs resistance peaks within the age range of  
25 – 35 years (Lawson et al., 2011; Uzoewulu et al., 2014).
Results of  this study with strong, significant statistical 
differences between male and female MDR-TB resistance 
rates, with male patients having more resistant strains 
agree with the work by Uzoewulu and co-workers 
(Uzoewulu et al., 2014). This underscores the enormous 
role of  male patients in the epidemiology of  drug 
resistant tuberculosis in the study population. This may 
be due to the fact that most females are economically 
disadvantaged in seeking appropriate medical attention, 
in addition to social and cultural beliefs that place women 
under movement restrictions. Other studies reported 
stigma as a principal factor (Uzoewulu et al., 2014).
The rate of  MDR-TB 9.7% in this study is higher than the 
estimated 5.3% rate of  global MDR-TB (WHO, 2011). 
The rate of  MDR-TB 2.6% in newly diagnosed TB cases 
has no statistical difference between current estimated 
rate of  MDR-TB 2.2% for new cases in Nigeria and 2.9% 
new cases in current national survey, although slightly 
lower, but MDR-TB of  7.1% in previously treated cases 
is lower compared to the estimated rate of  MDR-TB 
9.4%(WHO, 2012) and even grossly lower than the 14% 
from the current national survey on MDR-TB in Nigeria 
(WHO, 2011), but is within the trend for African countries 
in which 3.9 – 5.0% was reported for new TB cases and 
16.7% in previously treated cases (WHO, 2006). These 
results closely agree with the report by Uzoewulu and co-
reseachers. (Uzoewulu et al., 2014), who reported MDR-

TB of  7.7%. Kolo, Idigbe and co-reseachers, Lawson and 
co-reseachers, and Akaninyene and co-reseachers (Kolo, 
1991; Idigbe et al.,1992; Lawson et al., 2011; Akaninyene et 
al., 2013) all reported similar findings.
Comparative analysis of  the three categories of  patients 
revealed that category I patients (patients with HIV-TB co-
infection that tested AFB positive after 2 months of  TB 
treatment) recorded higher cases of  MDR-TB compared 
to category II patients (patients with only TB infection, 
that still test sputum AFB positive after 2 months of  
TB treatment). Both categories account for 10 (38.5%) 
and 9 (34.6%) cases of  MDR-TB respectively. Statistical 
analysis shows no significant difference (P>0.05).
However, MDR-TB detected in category I is statistically 
higher than MDR-TB detected in category III patients 
(patients with only TB infection, and are yet to commence 
TB treatment), in which MDR-TB was detected in only 
7(26.9%) cases. The relatively higher number of  MDR-
TB detected in category I patients underscores the 
enormous impact of  HIV as one of  the key factors 
underlying an approximately 1% annual increase in the 
global TB incidence as reported by Lawn and Gavin, 
(2014) in a retrospective study on the epidemiology of  
HIV-associated tuberculosis (HIV-TB) from 2007-2008. 
Findings in this study are also consistent with the report 
by Dean and co-researchers (2014), who reported a 
positive association between HIV infection and MDR-
TB disease using data of  member states of  the World 
Health Organization (WHO, 2014). Eleven out of  the 24 
countries for which analysis was performed, HIV-positive 
TB patients had significantly higher odds (P<0.05) of  
MDR-TB disease than HIV negative TB patients. For 
almost all of  these 11 countries, the prevalence of  MDR-
TB among newly diagnosed TB cases was higher than the 
estimated global average of  3.6% (95% CI 2.1 – 5.1%).
Rifampicin resistance of  82(30.6%) cases detected by 
in-vitro culture and DST is statistically higher than 
the 62(16.3%) cases detected by Genexpert automated 
machine. Although this underscores the diagnostic 
advantage and higher sensitivity of  in-vitro culture over 
Genexpert machine, there was no statistically significant 
difference between the two testing methods (P>0.05), 
Genexpert is however faster and much easier to perform 
even in remote areas.

Table 7: Age and sex distribution for M. tuberculosis rifampicin Resistance by geneXpert
Age (years) Males   No. (%) Females   No. (%) Total No. (%)
<1 – 10 - - -
11 – 20 4   (6.5) 1   (1.6) 5   (8.1)
21 – 30 18 (29.0) 9   (14.5) 27 (43.5)
31 – 40 11 (17.7) 10 (16.1) 21 (33.9)
41 – 50 3  (4.8) 5   (8.1) 8   (12.9)
51 – 60 - - -
>61 – 70 - 1   (1.6) 1   (1.6)
Total 36 (58.1%) 26 (41.9%) 62 (16.3%)
t = - 148.48, df  = 13, p< 0.05

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Apart from various evidences suggesting HIV infection 
as a risk factor for MDR-TB, it has been specifically 
associated with acquired rifampicin resistance (Jenny-
Avital, 1997; Munsiff  et al., 1997). This is suggestive of  
a critical overlap between HIV and the global multi-
drug resistant TB (MDR-TB). Although, it is yet unclear 
whether HIV is driving a disproportionate increase in 
MDR-TB cases at a population level, results of  in-vitro 
Mycobacterium tuberculosis transduction protocol (induced 
mutation) in the current study recorded an increase 
from 7(26.9%) to 10(38.5%) cases in 50% of  the study 
population. This may be attributed to mutation in the 
nucleotide sequence of  the Mycobacterium tuberculosis, 
which may confer antibiotic resistance on the organism. 
If  such genetic changes are scientifically proven, 
they may be rightly attributed as contributing to the 
emergence of  mono and multiple drug resistance in the 
genome of  Mycobacterium tuberculosis. Several studies have 
reported that Mycobacterium tuberculosis does not exhibit 
an elevated mutation rate relative to most other bacteria 
under in-vitro conditions (David, 1970; SiddiMizrahi, 
and Andersen, 1998; Ford et al., 2011). It is not entirely 
clear, though, whether a relatively low mutation rate is 
sufficient to account for the elevated rates of  acquired 
drug resistance observed clinically (McGrath et al., 2013). 
Studies by Ford and co-researchers (2011), using whole 
genome sequencing (WGS) technology to estimate the 
mutation rate of  Mycobacterium tuberculosis during latent 
infections in non-human primates reported a slightly 
elevated but not significant increase in drug resistance 
in vivo. Sun and co-researchers (2012), in their research 
utilized more sensitive WGS technology to track genome 
changes in serial sputum samples obtained from three 
patients over the course of  anti-TB treatment and 
reported a higher degree of  diversity in the serial clinical 
specimens, an observation that is consistent with the idea 
that mutation rate in vivo might be higher than previously 
reported. Thus the levels of  genetic diversity identified 
in the studies above imply that M. tuberculosis might have 
an elevated mutation rate within the host compared to 
that calculated in vitro, thereby highlighting the need for 
further in vivo studies to truly ascertain the role of  HIV 
in the emergence of  MDR-TB especially for TB-patients 
co-infected with HIV/AIDS.

CONCLUSION
In conclusion, results of  this genetic study on the role 
of  HIV in the emergence of  MDR-TB, promise to offer 
useful, effective, and ground breaking molecular approach 
in the fight against mono, multiple, and extensively-
drug resistance (XDR). Drug resistance has hampered 
many public health targets and interventions such as 
“Stop TB”-target of  achieving 70% case detection and 
85% cure rate by 2005, and “Stop TB”-target of  50% 
reduction in the global burden of  TB disease (deaths and 
prevalence) by 2015 (WHO, 2011). Previous studies have 
shown that anti-TB drugs resistance arise from patient/
service related causes such as poor patient adherence/

compliance, wrong regimens, inadequate supervision, 
and lengthy duration of  treatment. The need to direct 
more attention on molecular aspects of  anti-TB drugs 
resistance however is fast becoming a top priority, 
especially since MDR-TB is an emerging public health 
epidemic, requiring novel TB drugs to adequately combat 
it. This is necessary if  the “Stop TB” targets of  less than 
one patient per million populations by year 2050 must be 
achieved.

Recommendations
Based on the results of  the present study, we hereby 
recommend that this genetic study should be stepped 
up under in vivo conditions using the actual Human 
Immunodeficiency Virus (HIV) as the transducing 
phage in order to ascertain the true nature of  mutation 
conferring antibiotics resistance. The nature of  mutation 
conferring anti-TB drugs resistance most especially 
MDR-TB should be well defined and documented 
especially that the DNA of  Mycobacterium tuberculosis has 
been fully sequenced. In-depth understanding of  the 
molecular biology of  Mycobacterium tuberculosis genetic 
dynamics can be very useful to pharmaceutical industry 
as targets in the design of  novel anti-TB drugs. Design 
of  newer automated systems in TB diagnosis and 
antibiotics susceptibility testing that includes other anti-
TB drugs such as isoniazid, parazinamide and ethambutol 
in addition to rifampicin is strongly recommended. 
Automated systems offer quicker turnaround time (TAT) 
and should be employed in routine clinical practice while 
in vitro sputum culture for TB that takes longer time 
should be restricted to research purposes.

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