Hindawi Publishing Corporation

Tuberculosis Research and Treatment

Volume 2015, Article ID 215015, 10 pages
http://dx.doi.org/10.1155/2015/215015

Research Article
Prevalence of Tuberculosis, Drug Susceptibility Testing,
and Genotyping of Mycobacterial Isolates from Pulmonary
Tuberculosis Patients in Dessie, Ethiopia
Minwuyelet Maru,1 Solomon H. Mariam,1,2 Tekle Airgecho,1
Endalamaw Gadissa,1 and Abraham Aseffa1
1

Armauer Hansen Research Institute, P.O. Box 1005, Jimma Road, Addis Ababa, Ethiopia
Aklilu Lemma Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia

Correspondence should be addressed to Solomon H. Mariam; [email protected]

Received 25 February 2015; Revised 14 May 2015; Accepted 18 May 2015
Academic Editor: Vincent Jarlier
Copyright 2015 Minwuyelet Maru et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Due to their initially seemingly high cost, timely diagnosis and effective treatment of tuberculosis (TB) are usually hampered by lack
or shortage of resources in many high TB burden countries. However, the benefits of effective treatment can eventually outweigh
those of empirical treatment. Here, a cross-sectional study was conducted on samples from smear-positive new and retreatment
TB patients. Data on sociodemographic and HIV status were collected. Samples were cultured for identification, conventional drug
sensitivity testing, and molecular typing by deletion typing and spoligotyping. The results showed the youth were disproportionately
affected. New cases were being treated following general treatment guidelines only. Monoresistance or multiple drug resistance was
found in 16.5% of new patients. Spoligotyping showed that there were 44 patterns with families H3 and T1 (lineage 4) and CAS-Delhi
(lineage 3) being dominant. Some rare patterns from lineage 7 were also found. Spoligotype pattern, HIV positivity, and previous
treatment were not associated with drug resistance. That the vast majority of the patients were new cases and young and the large
number of these patients with mono- or multiple drug resistance indicate that most TB cases are due to recent transmissions and
that urgent actions are needed to curb the transmissions.

1. Introduction
The latest World Health Organization (WHO) reports show
that there were 9.0 million new tuberculosis (TB) cases
and 1.5 million tuberculosis (TB) deaths, leaving TB as the
second leading cause of death from an infectious disease
worldwide, after the human immunodeficiency virus (HIV)
[1]. Coinfection with the HIV fuels the global TB crisis,
and successful TB treatment is further complicated and
hampered by the existence of multidrug-resistant (MDR)
TB and extensively drug-resistant (XDR) TB (MDR TB plus
additional resistance to a fluoroquinolone and an injectable
second-line drug). Nearly half a million cases of MDR TB
emerge every year worldwide, of which 50,000 are also
XDR TB [2]. The WHO report states that progress towards

targets for diagnosis and treatment of MDR TB is far offtrack, with less than 25% of MDR TB cases detected in most
MDR TB-burdened countries [3]. The estimated TB cases and
TB deaths in children were 6% and 8% of the global totals,
respectively, in 2012 [3].
Globally, drug susceptible TB is reported to be decreasing,
but MDR and XDR TB are on the rise mainly due to the excessively large number of MDR TB cases being left undiagnosed,
untreated, or inappropriately treated each year [46]. Thus,
the WHO declared MDR TB a public health crisis in 2012.
This indicates selection of the more severe forms of TB at
a global scale and subsequent transmissions generating primary MDR. An increase in MDR and XDR TB and increase
in childhood TB (i.e., recent transmissions) are strong indicators that there is schism in the TB control programs.

2
Inappropriate drug regimen, patient defaulting, previous
antituberculosis treatment, delays in diagnosis and initiation
of effective treatment, and primary infection with MDR TB
strains are among the risk factors leading to MDR/XDR TB
[7]. The global burden of MDR TB cases between 1994 and
2009 ranged from 0 to 28% in new cases and from 0 to 61%
in previously treated cases [8, 9]. Since the TB Bacillus is not
delimited by geographic boundaries and people have become
increasingly more mobile, TB strains, including those that
harbor drug resistance, spread globally. During the last couple
of decades, epidemiological studies of TB globally have been
facilitated following the introduction of several genotyping
methods, with applications including distinction whether
recurrent TB is due to reactivation, exogenous reinfection
or mixed infection, classification of clinical isolates into
phylogenetic lineages and strain levels, determination of the
population structures, development of drugs and vaccines,
and whencombined with drug susceptibility testing (DST)
and epidemiologic data, transmission of MDR and XDR
strains [10, 11].
According to the WHO report, Ethiopia had an estimated
incidence of 223, prevalence of 212, and TB deaths of 32
per 100,000 [1]. Ethiopia had one of the lowest estimated
rates of MDR TB in both new and retreatment cases (1.6%
and 12%, resp.) among 27 high-burden countries. This report
also showed that only 1% of new bacteriologically confirmed
TB cases and only 4.4% of retreatment cases had DST
coverage.
Prevalence of TB as well as levels of drug resistance
and treatment success reported from other areas of Ethiopia
varied greatly as shown by some recent reports [1215]. The
objectives of this study were (i) to study the prevalence of TB
in the study area (Dessie, Ethiopia), (ii) to characterize the
species of mycobacteria causing pulmonary TB among new
and retreatment cases, (iii) to determine the drug susceptibility patterns of the mycobacterial isolates, (iv) to type the
mycobacterial isolates molecularly, and (v) to assess the efficacy of treatment. These objectives emanated from the lack of
information regarding the TB situation in the study area.

Tuberculosis Research and Treatment

Morning sputum samples were collected using universal
sputum collection tubes and immediately stored at 20 C
following WHO guidelines [16] until they were transported
to the laboratory at the Armauer Hansen Research Institute
(AHRI). Then, sputum samples were first decontaminated
and concentrated following Petroff s method [17]. Each
specimen was inoculated into two Lowenstein-Jensen slants,
one containing 0.6% glycerol and the other 0.6% sodium
pyruvate.
2.3. Drug Susceptibility Testing. DST was performed for
isoniazid (INH), rifampicin (RMP), ethambutol (EMB), and
streptomycin (STR) (Sigma, St. Louis, USA) using modified
proportion Middlebrook 7H10 agar method [18]. Briefly,
twenty-four well plates were used for the DST. Each well
contained 2.5 mL complete medium supplemented with 10%
OADC and 0.5% glycerol. Drugs were added at the following
concentrations: INH 0.064, 0.125, 0.2, and 1.0 g/mL; RMP
at 1.0 g/mL; EMB at 4.0, 5.0, and 8.0 g/mL; and STR at
2.0 g/mL. Mycobacterial suspensions for inoculation into
wells were prepared by taking representative sample of 5
10 mg from primary culture with a sterile loop (diameter
0.7 mm and internal diameter of 3 mm) (Becton Dickinson, France) which delivers 0.01 mL. Then, it was placed
in a spherical, flat-bottomed tube containing glass beads
and drops of distilled water added slowly with continuous
shaking to adjust the turbidity of the bacterial suspension
to that of a McFarland standard 1. Two drug-free wells,
one containing a 1 : 100 dilution of the bacterial suspension
and another containing undiluted bacterial suspension, were
included as controls. Inoculated plates were incubated within
a 37 C incubator. Plates were read at 2128 days. The MIC
breakpoints were 0.2 g/mL, 1 g/mL, 5 g/mL, and 2 g/mL
for INH, RMP, EMB, and STR, respectively.

2.1. The Study Site and Duration of the Study. This study was
conducted in Dessie, northeast Ethiopia, on samples obtained
from pulmonary TB (PTB) patients at one government and
two private hospitals and three health centers. These patients
were obtained while they were seeking health care at their
own times. There were no culture and DST capabilities but
facilities for microscopic examination of acid-fast Bacilli and
radiological examination were available. Sputum samples
were collected from PTB patients from October 1, 2012, to
September 30, 2013.

2.4. DNA Extraction and Molecular Typing. To obtain DNA

for typing, two loops of colonies from LJ slants were resuspended in 50 L distilled water and heat-killed at 80 C for
one hour. The fluid portion excluding debris was transferred
to a new tube.
To differentiate M. tuberculosis from other species of
mycobacteria, PCR targeting region of difference 9 (RD9) was
conducted [19]. Spoligotyping [20] was performed to determine the presence or absence of the 43 spacers. SPOTCLUST
[21] was used to generate octal codes. SITVITWEB [10] was
utilized to assign SITs (spoligotype international types) and
families for the isolates.
Data were analyzed by SPSS software version 20 (IBM,
USA). The presence or absence of association between drug
resistance and spoligotype, HIV status, and treatments history was assessed. A value less than 0.05 was considered
statistically significant.

2.2. Study Design. A cross-sectional study was conducted on

samples from smear-positive newly diagnosed and retreatment PTB patients, age 10 years. Surveys focusing on
sociodemographic data were done using prestructured questionnaire.

2.5. Quality Control. M. tuberculosis H37Rv (ATCC 27294)

and M. bovis (AF 61/2122/97) were included for quality
control in DST, RD9 deletion typing, and spoligotyping. Laboratory procedures were done following standard operational
procedures.

2. Materials and Methods

Tuberculosis Research and Treatment

3. Results
3.1. Sociodemographic and Clinical Data. A total of 144
smear-positive PTB patients 10 years of age, consisting
of 128 (88.9%) new cases and 16 (11.1%) retreatment cases,
were enrolled in this study. The mean length of stay before
seeking health care was 6.49 6.1 weeks (range 248 weeks).
Sixty-four (44.4%) were females and 80 (55.6%) were males
(see Table S1, in Supplementary Material available online
at http://dx.doi.org/10.1155/2015/215015). The median age of
the patients was 27.5 years (range 1078 years). Twenty-five
(17.4%) of all patients were HIV-positive (consisting of 20
new cases (7 males and 13 females) and 5 retreatment cases
(3 males and 2 females)) (Table S1, Supplementary Material).
Of these, 17/25 (68%) were urban dwellers with 7/17 (41.2%)
being males and 10/17 (58.8%) being females. The rest (8/25,
32%) were rural dwellers with 3/8 (37.5%) and 5/8 (62.5%)
being males and females, respectively. With the caveat that the
sample size is small, we deduce that, overall, HIV positivity
was higher in both urban and rural dweller females (15/64,
23.4%) than in males (10/80, 12.5%).
When stratified by age group, a staggering > 67.4%
(97/144) were between 10 and 30 years of age, with 31/97
(32%) and 66/97 (68%) being in the age groups 1020 and
2130, respectively. Four patients in each of these two age
groups were retreatment cases while 89/97 (91.7%) were new
cases. Fifty-three (54.6%) of the 97 patients were males while
44 (45.4%) were females. Overall, the TB patients were split
50 : 50 between urban and rural residency. However, in the
age groups 1020 and 41 years, the rural PTB patients outnumbered the urban PTB patients by 2 : 1 (data not shown).
Among the 144 patients, there were 32 (22.2%) who were
3140 years of age (with 24 new cases and 8 retreatment
cases). Of these, 18 were males (12 rural and 6 urban residents)
and 14 were females (7 rural and 7 urban residents). The rest
(15, 10.4%) were 4178 years old and all were new cases, with
11 (73%) of them being rural residents.
Of the 144 patient samples, 26 samples (from 25 new cases
and 1 retreatment case) failed to grow in culture and DST and
spoligotyping were performed on 118 (103 new cases and 15
retreatment cases) samples.
3.2. Drug Resistance. Of 103 new cases, 86 were susceptible to
the four drugs and 17 showed resistance to one or more drugs.
Of 15 retreatment cases, 11 were susceptible to all four drugs
and 4 were resistant to one or more drugs, including 2 MDR
cases. Overall, 21 patients from both cases showed various
patterns of drug resistance (Table 1). RMP monoresistance
was observed in neither HIV-positive nor HIV-negative TB
patients.
In the age group 30 years, 4 patients had INH monoresistant TB, 2 each had STR or EMB monoresistant TB, and 3
had TB resistant to both INH and STR, all in new cases (Table
S2, Supplementary Material). There were 8 patients that
were retreatment cases in that age group; however, they did
not exhibit resistance to any drug (Table S3, Supplementary
Material).
Among the 24 new cases aged 3140 years, 1 was resistant
to INH alone, 1 was resistant to STR alone, and 4 were

3
Table 1: Number of drug susceptible or resistant isolates in new and
retreatment TB patients.
Drug(s)
INH
RMP
STM
EMB
INH + RMP
INH + STM
INH + EMB
INH + RMP +
STM
INH + RMP +
EMB
INH + RMP +
STM + EMB

New cases
Retreatment cases
Susceptible Resistant Susceptible Resistant
91
103
93
101
91
88
89

5 (0 : 5)
0
3 (0 : 3)
2 (0 : 2)
0
7 (1 : 6)
0

11
13
14
14
11
11
11

1
0
0
0
1
0
1

88

11

89

11

86

11

Ratio of HIV+ to HIV patients that exhibited the drug resistance. All
resistant retreatment cases except the INH-EMB resistant were HIV .

resistant to both INH and STR (Table S2, Supplementary

Material), while DST could not be performed for 3 other new
cases because of culture negativity. The other 15 new cases
aged 3140 years were all sensitive to the four tested drugs.
Among the 8 retreatment cases in that age group, 3 were fully
sensitive to all four drugs, 1 was resistant to INH alone, 1 was
resistant to both INH and EMB, and 2 were MDR cases, while
1 was culture negative (Table S3, Supplementary Material). No
drug resistance was observed in the 12 of 15 patients (with
positive cultures) above 40 years of age (all of them new cases)
(Table S2, Supplementary Material).
Cultures were negative for 8 (7 new cases and 1 retreatment case) of the 25 HIV-positive patients. In the remaining
17 HIV-positive patients, drug resistance was observed in
only 2, indicating there was no association between HIV positivity and drug resistance. The larger HIV-negative subgroup,
on the contrary, consisted of most (19/21) patients with resistance to one or more drugs (including 2 retreatment MDR
TB cases). Likewise, no association was observed between
any resistance to first-line drugs and previous treatment.
Most resistance was seen against INH, either alone or in
combination (Table 1); however, there was no association of
any resistance to INH with gender, HIV status, TB case, or age
(Table 2). Though the sample size is small, MDR TB appeared
to be strongly associated with previous history of treatment
( < 0.01) and lineage 3 M. tuberculosis (Table S3, Supplementary Material, and Table 3). Of all patients that were
resistant to one or more drugs (21 in total), 17 (81%) were in
new cases. Seventeen of 21 (81%) cases resistant to one or more
drugs were new cases, but none of them exhibited MDR TB.
3.3. Molecular Typing. RD9 deletion typing indicated that all
of the 118 isolates were M. tuberculosis (data not shown).
The spoligotype patterns of patient isolates with their
octal designations, spoligotype international types (SITs),
lineages, and family are shown in Table 4. There were a total

Tuberculosis Research and Treatment

Table 2: The risk of any resistance to INH by gender, HIV status, previous anti-TB treatment, and age group assessed using the Chi square
test.
Any resistance to INH
Susceptible (%)
Resistant (%)

Variable
Sex
Male
Female
HIV status
Positive
Negative
Previous history of anti-TB treatment
Yes
No
Age
1015
1630
3145
>45
Total

54 (84.4)
48 (88.8)

10 (15.6)
6 (11.2)

15 (88.2)
87 (86.1)

2 (11.8)
14 (13.9)

11 (73.3)
91 (88.3)

4 (26.7)
12 (11.7)

4 (100)
67 (90.5)
22 (70.9)
9 (100)
97 (82.2)

0 (0)
7 (9.4)
9 (29.0)
0 (0)
21 (17.8)

value

0.67 (0.221.94)

0.47

0.82 (0.174.02)

0.81

0.36 (0.101.32)

0.11

0.58

Table 3: Association of INH resistance or multidrug resistance with

lineages.

INH
Susceptible
Resistant
Total
MDR
Yes
No
Total

OR (95% CI)

Lineage 3

Lineage 4

Lineage 7

28
5
33

66
10
76

6
1
7

2
31
33

0
76
76

0
7
7

value
0.96

0.07

of 44 different patterns. Families H3 (with 18.2%), T1 (with

15.9%), and CAS-Delhi (with 13.6%) represented the majority
with clustered isolates in this study. SIT 25 (lineage 3) and SIT
53 (lineage 4) accounted for the largest number of isolates
with 17.8% and 16.95%, respectively. The patterns obtained
showed that lineage 4 was the most dominant with 78 isolates
in 30 diverse patterns, followed by lineage 3 with 33 isolates
in 11 diverse patterns and lineage 7 with 7 isolates in 3 diverse
patterns. SIT 53 (with 20 clustered isolates), SIT 25 (with 21
clustered isolates), and SIT 343 (with 5 clustered isolates) were
the most frequent in lineages 4, 3, and 7, respectively.
There were 14 isolates belonging to the T superfamily
(7T1, 4T3, 2T, and 1T3-ETH). CAS1-Delhi and CAS-Kili were
represented by 6 isolates and 1 isolate, respectively. The H3
clade had 6 isolates. LAM9 and LAM10 were represented
by 3 isolates and 1 isolate, respectively, and Manu2 and X1
clades were represented by 1 isolate each. Ten unknown or
orphan patterns not found in SITVITWEB were found in this
work. All except one were represented by 1 isolate and could
be called orphans according to the SITVIT designation [10].

Some patterns within lineage 7 rarely represented in the

database are also reported in this study.

4. Discussion
This study was conducted to assess the molecular diversity
and drug susceptibility pattern of mycobacterial isolates as
well as prevalence of TB in a sample of patients in Dessie,
Ethiopia. Sociodemographic characteristics, HIV status, and
previous TB treatment were compared to type of strains and
drug susceptibility patterns. Several issues of outstanding
clinical relevance that either corroborate previous findings
from Ethiopia or pinpoint major gap of knowledge that
require further studies are associated with this study.
A high percentage (17/103 (16.5%)) of new cases harbored
resistance to one or more of the tested drugs (most of which
were only little short of being MDR). Since these cases are
not generally suspects for MDR or any resistance because
they are new cases [1, 22], they remain undiagnosed and
are treated empirically with the standard first-line drugs
without any prior DST (assuming the capacity for DST is in
place). Thus, treatment efficacy is destined to be ineffective
because of the undetected resistance to those one or more
drugs, as also shown by other studies (e.g., [23, 24]). Such
empirical treatments of patients with undetected resistance
with single or mixed infections may lead to more severe
forms of drug resistance, including MDR TB [25, 26]. Studies
show that MDR TB can evolve into XDR TB over time and
during treatment (and from XDR to pan-resistant) [2730].
Furthermore, there is a high risk of transmission of this
primary resistance to new individuals. It is also likely we
could have found more resistance in the 25 other new case
samples, were they not culture negative.
In addition, most of the active cases of tuberculosis were
found to be children and young adults. This is a serious
concern for future TB control. Tuberculosis affecting people

Number
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

Spoligotype pattern

Octal code
777777477760771
777777777760771
777000377760771
777777777760731
777737777760771
777777775720771
777777777720771
777777743760771
777756777760771
737777777720771
777737377720771
617777777760771
077777777760771
007777707760771
377737777660760
717777777760771
777737777760671
776737737760771
777777607760771
777777607760761
777777407760771
777777377760731
777775777760731
776677677760771
777777777720671
777777777720471
677756777420731
777777777420771

SIT
451 (H37Rv)
53
149
52
37
121
50
61
302
871
Unknown
Orphan
751
1889
Orphan
358
565
Orphan
42
1074
1800
1077
584
Orphan
168
747
Orphan
777

Frequency (%)
20 (25.6)
10 (12.8)
7 (8.9)
6 (7.69)
3 (3.8)
3
3
2 (2.56)
2
2
1 (1.28)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

Family
T1
T3-ETH
T2
T3
H3
H3
LAM10
X1
H3
H3
T1
T
T1
T1
T1
T3
T1
LAM9
LAM9
LAM9
Ambiguous: T4
T2
T1
H3
H3
H3
H3

Table 4: Spoligotype pattern of M. tuberculosis isolates from pulmonary tuberculosis patients of Dessie and its surroundings, Ethiopia.

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4

Linage

Tuberculosis Research and Treatment

5

Number
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44

Spoligotype pattern

Total

Octal code
777777777760711
757000377760771
177000377760771
703777740003171
703777740000000
703777740002171
703777740003571
703777740000771
703777740003771
703377400001771
777777470000000
777737770000771
703777747177771
777777777763771
700000007175771
700000007177771
700000004177771

Table 4: Continued.
SIT
78
Orphan
Orphan
25
1264
1787
289
357
26
21
Orphan
2306
Orphan
54
343
910
1729
Manu2

Family
T
T3
T3
CAS1-Delhi
CAS1-Delhi
CAS1-Delhi
CAS1-Delhi
CAS1-Delhi
CAS1-Delhi
CAS1-Kili

Frequency (%)
1
1
1
21 (63.6)
3 (9.09)
1 (3.03)
1
1
1
1
1
1
1
1
5 (71.4)
1 (14.28)
1
118

Linage
4
4
4
3
3
3
3
3
3
3
3
3
3
3
7
7
7

6
Tuberculosis Research and Treatment

Tuberculosis Research and Treatment

of all ages is a concern, but it is even more so when the
vast majority of the patients are new cases, children, and the
youth (especially in a country with a population age structure
that is pyramidal). Children are more prone to exposure,
infection, and progression to disease than are adults. The
success of control of all forms of TB will be jeopardized unless
TB in children is controlled, since they will serve as future
reservoirs from which further amplification will occur [31].
A recent study from northwest Ethiopia showed that 82%
of PTB patients were both new cases and below 40 years of
age and 4.2% of these cases were MDR [32]. A recent report
from a nationwide study also indicated that 55% of TB cases
in a majority of newly diagnosed cases were in the young
age groups (1534 years) [33]. These findings are indicative
of the overall prevalence of TB in the general population
that is disproportionately affecting the young population that
are due to ongoing or recent transmissions of TB rather
than reactivation of latent infections. Recent transmissions
contribute to the majority of TB cases in both low and
high incidence countries [34, 35] and, in high incidence
countries, most such transmissions are believed to occur
outside of households [35, 36]. Thus, some transmissions or
epidemiological links can be difficult to trace.
Moreover, the findings of high rates of retreatment cases
that are drug susceptible as well as of new cases that are
drug-resistant (as described above) are serious concerns that
raise questions on the efficacy of treatment and TB transmission control strategies, respectively. In the 11 retreatment
cases that were susceptible to all 4 drugs (4 of them HIVpositive and most of them of young age), neither mixed
infection with both susceptible and resistant strains in the
first episodes nor the presence of clonal populations (that
are invariably susceptible to the drugs) serves as explanations
for the occurrence of the second episodes. Subtherapeutic
drug levels are known to cause treatment failure even with
treatment adherence, but the ensuing drug resistance that
usually follows [3739] was not seen in these patients, at
least until this study. Reinfection with drug susceptible strains
appears to be the most likely explanation. Since we did
not have serially collected samples from these patients, we
were unable to genotype the isolates from both episodes,
although this analysis may not always delineate reinfection
from relapse [40]. Drug susceptible TB in retreatment TB
patients has been reported from Ethiopia before [13, 41, 42].
For example, one of these studies [13] reported that 27%
of culture-positive retreatment cases were susceptible to all
4 first-line drugs tested. However, these studies apparently
regarded such form of TB as unimportant and gave no
emphasis on it except mentioning in passing. Further studies
are needed on the reason(s) for drug susceptible TB in
previously treated patients as this area of research has not
been addressed before in the Ethiopian context. This study
[13] also reported an alarmingly high level (46%) of MDR TB
in retreatment cases from a specialized TB referral hospital,
which is a huge increase from that reported previously [1].
In most resource-poor countries, new case MDR TB
patients are identified only after first-line therapy fails,
by which time these patients could have further disseminated the disease [8, 43]. This calls for a paradigm shift and

7
points to both the necessity for reevaluation of the belief
that associates being new case with a drug susceptible case
and the need for strain genotype-based individually tailored
drug regimen. The concept of primary resistance is not new,
but it is obviously overshadowed by this belief. Delayed
initiation of effective therapy, inappropriate therapy, and
absence of transmission control are reasons for amplification
of primary resistance [44, 45]. Assuming each infectious case
can transmit it to 1015 persons/year [46], the magnitude of
the problem cannot be underestimated.
The importance of individualized drug regimen for treatment of MDR and XDR TB cases is highlighted by diversity
in strain genotype and/or by treatment failure following
compliance to treatment that is based on general treatment
guidelines [28, 47, 48]. Moreover, the phenomenon of crossresistance (e.g., to both the first-line drug INH and the
second-line drug ethionamide due to a missense mutation
in the inhA promoter) [49, 50] renders both antibiotics
ineffective (ethionamide is a component of the drug regimen
for both MDR and XDR TB patients in Ethiopia [1, 22]).
This makes identifying the specific molecular mechanism
of resistance to INH important. The same can be said for
the aminoglycosides, for example, for both kanamycin and
STR [51] and amikacin, kanamycin, and capreomycin [52
54]. In this study, 11 isolates were STR resistant (including 3
monoresistance cases, 1 MDR case, and 7 INH-STR double
resistance cases), most (10/11) of which were in new cases.
Another study [41] also reported high STR resistance from a
region in which this study site is a part. Further information
is needed for the reasons for this high level of STR resistance.
Regulations and measures for better infection control
in all hospitals and other hot spots in communities (by
proper ventilation, avoiding congregated settings, raising
public TB awareness, etc.) should be instituted [55]. It is
critical that all public and private parties involved in the
management and treatment of TB work in concert and
streamline their activities [8]. Rapid DST capabilities for all
cases are critically needed. However, until those capabilities
are built, the spectrum of drug resistance circulating in the
communities must be known if standard drug regimens will
continue to be used. Regular follow-up of CD4+ levels of
HIV-TB patients with mono- or multiple drug resistance is
advisable. Collection and storage of isolates from each patient
as well as complete treatment record keeping are strongly
recommended for use in retrospective studies and further
analyses and to monitor progress of therapy. These measures
should be applied together; one or the other alone, or even all
but one, will not suffice. These recommendations have been
passed on to clinicians and authorities with jurisdiction over
the study area.
This study has some limitations. These include unavailability of treatment records for all patients, the use of only
one DST method and inability to test drug resistance for more
first- and second-line drugs, and the limited genotyping data.
Finally, the study period was limited to one year.
In conclusion, this study provided important findings
and recommendations that can be incorporated into the
current practices in the control of TB in the study area and
other areas with similar situations. The high rates of TB in

8
the vulnerable children and the youth require immediate
attention for proper protective measures, along with further
enhancement of case detections. The finding that more than
80% of the patients with resistance to one or more drugs
were new cases and that the vast majority were also 30
years of age is a strong indicator of recent transmissions with
primary resistant infections. Available reports on resistance
are usually for MDR and XDR cases and these usually focus
on retreatment cases. This work reports very high (16.5%)
monodrug and multiple drug resistance involving first-line
drugs in new cases. Undetected resistance represents a hidden
danger fueling more drug resistance. Even with accurate
diagnosis and effective treatment in place, unless ongoing
transmission is aggressively dealt with, the gains from the
former are likely to be elusive.

Ethical Approval
The study was approved by Aklilu Lemma Institute of Pathobiology, AHRI, and the Health Bureau Ethical Review Committees of the study site.

Consent
Informed consent was obtained from patients or their
guardians.

Conflict of Interests
The authors declare that there is no conflict of interests
regarding the publication of this paper.

Acknowledgments
The authors thank the hospitals, health centers, and the TB
patients for their assistance and participation in this study.
This work was supported by funds from the AHRI core
budget. Additional funding was provided by Addis Ababa
University.

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