African Journal of Microbiology Research Vol. 5(25), pp.

4291-4295, 9 November, 2011

Available online at http://www.academicjournals.org/AJMR
ISSN 1996-0808 ©2011 Academic Journals
DOI: 10.5897/AJMR11.037

Full Length Research Paper

Isoniazid resistance of Mycobacterium tuberculosis

isolated from human patients
Taha Nazir1*, Abdul Hameed2, Javed Anver Qureshik3 and Bashir Ahmad4
1
Riphah Institute of Pharmaceutical Sciences, Riphah International University 7th Avenue G/7-4, Islamabad, Pakistan.
2
Department of Microbiology, Quaid-I-Azam University, Islamabad, Pakistan.
3
National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
4
School of Pharmacy, University of the Punjab, Lahore, Pakistan.
Accepted 30 July, 2011

Isoniazid resistant Mycobacterium tuberculosis strains are a serious threat for successful tuberculosis
control programs. Therefore, present study was aimed to figure out the pattern and level of resistance
of M. tuberculosis against isoniazid. A total of 172 specimens of sputum, pus and bronchial washings;
70.9% males and 29.1% females with 84.30% pulmonary and 15.69% extrapulmonary tuberculosis were
collected from six different sources. The inoculums were prepared using 0.5 Mac Farland turbidity
standards. Five concentrations of isoniazid were used in Lowenstein Jensen (LJ) medium that is 3, 6, 9,
12 and + 12 µg/ml for sensitivity testing. Data showed 25 (14.5%) resistant and 147 (85.5%) sensitive M.
tuberculosis strains. The growth was not inhibited at first (3 µg/ml) and second (6 µg/ml) drug levels,
while 36% isolates inhibited at third level (9 µg/ml), 28% at forth level (12 µg/ml) and 24% at fifth level
(10 µg/ml) and 12% at higher than fifth Level (> 12 ug/ml). These incorporated drug concentrations are
higher than therapeutic index and not recommended in actual clinical practice. It is thus obvious to
explore some other effective chemotherapeutic agents, modify combinations or figure out more
effective procedures to stop morbidity and mortality due to isoniazide resistance of M. tuberculosis.

Key words: Isoniazid, mycobacterium tuberculosis, resistance, Lowenstein Jensen medium.

INTRODUCTION

The emergence of isoniazid resistance is alarming, the need to search for some more effective procedure to
problem of great importance concern to public health. control mortalities due to multidrug resistance (MDR)
Certain clinical, biological and epidemiological risk factors tuberculosis.
are constantly contributing to develop resistance. The major factors of emergence of isoniazid resistance
Primarily, it is induced because of the inaccurate treat- are erratic drug ingestion, monotherapy, omission of one
ment protocols, substandard socio-economic condition or more prescribed agents and suboptimal doses. The
and poor therapeutical surveillance (Koh et al., 2005). It half of all new TB cases are in 6 Asian countries –
may also be caused by genetic mutations, certain Bangladesh, India, Pakistan, Indonesia and Philippine
physiological conditions and therapeutical errors that and multidrug resistance (isoniazid and rifampicin
eventually proliferate the resistant strains (Nolan and resistance with or without resistance against other anti-
Goldberg, 2002). The recent increase in the incidence of TB drugs) present virtually in all 109 countries (WHO,
this disease and emergence of resistance has urged the 2005). In most developing countries, the disease has
always been endemic (Herendra and Shah, 1998) but in
industrialized countries the development of resistance,
suppressed immunity and contributed the disease's
*Corresponding author. E-mail: [email protected] or resurgence (Barnes et al., 1991). The anti TB drugs are
[email protected]. Tel: +92 51 282 9162-64, 282 9697-8, freely available in market that leads to self medication
Fax: +92 51 282 9238. and improper regimen. However, an optimal regimen and
4292 Afr. J. Microbiol. Res.

duration for this treatment remains a matter of some dilution inoculums 10 to 5 were used to culture for sensitivity
controversy. Here, we tied to share our experience in a testing.
case of pulmonary and extra-pulmonary TB with primary
and acquired isoniazid resistance. The major objective of
Susceptibility testing of M. tuberculosis
our investigation was to study the resistance pattern of M.
tuberculosis against isoniazid and explain certain
The drug sensitivity testing was performed within 1 to 2 weeks after
therapeutical issues to decide clinical credibitily of obtaining primary growth of M. tuberculosis. The sensitivity was
isoniazid in tuberculosis treatment programs. evaluated against isoniazid by drug proportion method as recom-
mended by world health organization (WHO) and International
Union against Tuberculosis and lung diseases (Richard et al.,
MATERIALS AND METHODS 2006). The patient’s samples were processed in batches of 10 to 15
specimens on drug containing LJ medium and drug free control LJ
This study was conducted at Pakistan Medical Research Centre medium. Five different concentration of isoniazid were used in LJ
(PMRC), King Edward Medical College, Lahore, National Institute of medium that is 3, 6, 9 and 12 µg/ml. Each LJ medium slant was
Biotechnology and Genetic Engineering (NIBGE), Faisalabad, inoculated by using the above inoculums prepared from primary
Pakistan and Department of Microbiology, Quaid-i-Azam University cultures of M. tuberculosis and incubated at 37°C for 4 weeks.
Islamabad, Pakistan. Pure chemical of isoniazid was obtained from
the Schazoo Laboratories (Pvt.), Lahore.
Recording and interpretation of results
Collection and processing of samples
The Bijoux bottles were inspected weekly for appearance of growth.
A total number of 172 pulmonary and extra-pulmonary tuberculosis When the growth was evident on LJ medium, colony morphology
diagnosed (AFB positive) patients were selected from six different was noted. One culture bottle was exposed to day light for 1 h and
sources. The patients of all age groups were selected, regardless of re-incubated to be examined for pigmentation on the following day.
their age, gender and previous therapeutic profile. The sputum, pus The cultures with no growth were discarded after 8 weeks of
and bronchial washings were collected, labeled and stored, incubation. The presence and amount of growth in term of number
separately. All the samples were centrifuged decontaminated and of colonies on control and drug inoculated medium recorded. The
processed as described by Taha et al. (2010). These concentrated results were interpreted for resistance on the basis of percentage of
residues of sputum, pus and bronchial washings were used for colonies on drug containing media in comparison to the growth on
primary culture. drug free medium. The strains showing susceptibility were again
incubated and examined at 6 weeks before declaring as sensitive.
The growth pattern, number of colonies and contamination were
Preparation and inoculation of Lowenstein Jensen (LJ) checked carefully on weekly basis.
medium

Lowenstein Jensen medium was prepared by the method described RESULTS

by Nazir et al. (Uplekar et al., 2001) and used for primary culturing
of the processed samples. A 15 ml of medium was poured into
sterilized 25 ml McCartney vials and closed with sterilized silver A total number of 172 samples were collected from six
caps. The medium was autoclaved at 115°C under 15 lb/inch2 different local sources; 41 (23.8%) outdoor, Mayo
pressures for 20 min. Then it was solidified in slanting position, Hospital, 110(64%) indoor, Mayo Hospital, 14 (8.1%)
cooled, labeled and stored at 2 to 8°C. The LJ medium slants were Jinnah Hospital, 6 (3.5%) WAPDA Hospital, Lahore,
inoculated with processed samples in class-II safety cabinet Pakistan (Table 1). The specimens comprised of 146
(Telstar, Spain). The inoculated slants were kept in incubator at
37°C for 4 weeks. The growth of M. tuberculosis thus obtained was
(84.9%) sputum, 18 (10.5%) pus and 8 (4.7%) bronchial
identified though acid fast staining and further used for sensitivity washings with 145 (84.30%) pulmonary and 27 (15.69%)
testing. extra-pulmonary specimens. There were 122 (70.9%)
males and 50 (29.1%) females out of total 172 clinical
isolates (Table 2).
Preparation of inoculums of Mycobacterium tuberculosis
Data showed the isoniazid susceptibility profile as 25
The over seeding of a drug containing medium was prevented by (14.5%) resistant and 147 (85.5%) sensitive strains. On
standardization of inoculums that mitigate the possibility of miss basis of number of colonies resistance was found as; 30
judgment of considering the resistance to a susceptible strain. and 50 colonies shown two different strains, 100 colonies
Approximately 1 mg wet weight bacilli/ml was estimated to vary by 21 (84%) strains and 200 colonies by resting 2 (8%)
between 106 and 108 CFU. The representative samples of growth strain out of 25 (14.53%) isoniazid resistant isolates of a
containing minimally 50 colonies were taken from primary culture
and placed into McCartney vials containing 1 ml of distilled water
total 172 M. tuberculosis (Table 3).
and 5 glass beads. The mixtures were homogenized by vigorous Furthermore M. tuberculosis strains were inoculated
stirring by Vortex Mixer (Eyela, Japan) for 1 to 3 min and left in over the LJ medium incorporated with five different
class-II safety cabinet (Telstar, Spain). The opacity of the suspen- isoniazid levels. All of the 25 (100%) strains were
sions was adjusted by the addition of sterile distilled water to that of founded resistant at first (0.2 µg/ml) and second levels (3
a 0.5 Mac Farland turbidity standard. Four serial 10 fold dilutions of
µg/ml). 9 (36%) strains were resistant up to third level (6
inoculums were prepared that is 10 to 1, 10 to 2, 10 to 3 and 10 to 4
in sterilized test tubes and labeled as 1, 2, 3 and 4, respectively. µg/ml), 7 (28%) strains upto forth level (9 µg/ml), 6 (24%)
The tube No. 3 of dilution inoculums of 10 to 3 and tube No. 5 of strains up to fifth level (12 µg/ml) and 3 (12%) strains
 Nazir et al. 4293

Table 1. Source and number of samples of M. tuberculosis strains.

S/N Source Frequency Percentage (%) Valid (%) Cumulative (%)

1 Mayo Hospital outdoor 41 23.8 23.8 95.9
2 Mayo Hospital indoor 110 64.0 64.0 64.0
3 Jinnah Hospital 14 8.1 8.1 72.1
4 DOTS 6 3.5 3.5 99.4
5 WAPDA Hospital 1 0.6 0.6 100.0
Total 172 100.0 100.0

Table 2. Gender wise patient distribution.

Gender Frequency Percent Valid Cumulative percent

Females 50 29.1 29.1 29.1
Males 122 70.9 70.9 100.0
Total 172 100.0 100.0

Table 3. Comparison of resistance percentage over quantity of growth of indigenous Mycobacterium TB in isoniazid incorporated Lowenstein
Jensen media.

Valid Cumulative
Number of colonies Frequency Percentage (%) Percentage (%) Percentage (%)
30 1 4 4 4
50 1 4 4 8
100 21 84 84 92
200 2 8 8 100
Total 25 100 100

higher than fifth level (>12 µg/ml) (Table 4). Russian Federation, 11.3 % resistance in china and
15.4% resistance in India. Study by Canada
Communicable Disease Report (2004), reveals similar
DISCUSSION finding of 9.3% most common type of drug resistance
against isoniazid. The possible reason of difference of
These findings are in conformity with Bitar et al. (2001) results may lie in the facts of the differences in living
reported that majority of cases 89% (n = 607) were of standards, health facilities, over all hygienic condition and
pulmonary TB, 6% (n = 39) presenting extra-pulmonary socio-economic privileges. These findings are in
tuberculosis and 5% (n = 37) cases for whom site of agreement with Herendra and Shah (1998), who reported
disease was unknown. 20 to 30% isoniazid resistance. These findings are in
Gender comparison depicts greater percentage of male consistent with Miah et al. (2000) who reported 15.8%
than females. These findings have been substantiated by isoniazid resistance and 5% MDR in Bangladesh. Our
Uplekar et al. (2001) who reported a 70% excess of finding are in line with Sumathi and Srivastava (2004),
males over females globally each year. The reasons for who reported 11 (14.7%) isolates were seen resistant to
this difference are unclear as yet. These findings are also isoniazid by all the three methods - proportion method by
in consistent with Haq et al. (2002) who reported 68% agar dilution on Middlebrook 7H11 agar, proportion
male and 32% female tuberculosis patients. Our findings method using the conventional Lowenstein-Jensen (L-J)
are in conformity with WHO/IUALTD (2000) that reported medium and E test strip method. Findings of the study
67% of male tuberculosis patients. These findings are in under discussion substantiated by Rizwan et al. (2003),
agreement with Bitar et el. (2001) who have reported who reported the 26% isoniazid resistance and 16%
70% males with a Male: Famale sex ratio of 2:8. MDR. Primary and acquired isoniazid resistance was 20
Our findings are supported the work of WHO/IUALTD and 33% with statistically significant difference.
(2000), who reported the 19.4% isoniazid resistance in The findings regarding the level of isoniazid resistant
4294 Afr. J. Microbiol. Res.

Table 4. Level of resistance (in % age) of isoniazid resistant Mycobacterium TB.

ISN Levels in LJ media Isoniazid ug/ ml MTB strains Resistance (%) Valid (%) Cumulative (%)
1 0.2 25 100 0 0
2 3 25 100 0 0
3 6 9 36 36 36
4 9 7 28 28 64
5 12 6 24 24 88
5+ 12+ 3 12 12 100
Total 25 100 100

clinical isolate of M. tuberculosis are in line with the work al. (2001), who reported 0.2 ug/ml MIC, 300 mg standard
of Jesudason et al. (2003), who reported the resistance dose, maximum plasma concentration in slow acetylation
of M. tuberculosis above than optimum concentration of 5.4 ± 2.0 ug/ml and in fast acetylation 7.1 ± 1.9 ug/ml.
0.2 ug/ml. This work also supported by Victor et al. These findings are in consistent with Holdiness (1984),
(1997), who reported M. tuberculosis resistance against who reported the peak serum concentration 3 to 7 ug/mL
isoniazid more than 0.2 ug/ml. our finding are also in line (21.9 to 51 micromoles/L) after a single 300 mg oral
with Stephen (2002), who studied the quantitative dose. 0.57 to 0.76 L/ kg is the volume of distribution
difference of doses required in individual and combination (Kergueris, 1983). 1 to 2 h peak serum concentration
therapy. He reported that isoniazid resistant tuberculosis time (tmax) and 4 ug/ml peak plasma concentratioin
poses a significant threat to human health and is (Cmax) (Venkatesan, 1989). Findings of study under
important to understand how the resistance emerges if discussion are substantiated by Juan (2006), who
we are to reverse the upward trend. Treatment with reported oral or IM administered of isoniazid with
internationally approved regimens has resulted in a very bactericidal effect. Its daily dosage for children is 5 to 10
high cure rates, without the emergence of resistance. mg/kg and for adult is 5 mg/kg. The maximum daily
These regimens are effective in preventing the administrable regimen is 300 mg.
emergence of resistance because of inhibiting the deve- In conclusion, most seriously ill tubercular patients are
lopment of spontaneous resistance due to mutation. The generally seen in hospitals, the epidemiological data has
concentrations of isoniazid incorporated in LJ medium pointed out five time higher prevalence of pulmonary
exceed the therapeutic range of 4 ug/ml Richard et al. tuberculosis than extra-pulmonary tuberculosis. The final
(2006), therefore can not be maintained in plasma of concentrations incorporated in LJ medium exceeded the
tuberculosis patients in actual clinical practice. The therapeutic index and can’t be used in actual clinical
maximum isoniazid regimen is 400 mg (Leon et al., practice. Therefore; we have to replace, modify or finding
2004). If exceeded, unwanted effects including hypersen- some other effective procedure to stop the mortality and
sitivity, paresthenia, hepatic enzyme elevation, hepatitis morbidity due to isoniazid resistant M. tuberculosis.
and peripheral neuropathy, nausea, vomiting, epigastric
distress, agranulocytosis, thrombocytopenia, fever,
various rashes, dermatological and rheumatoid and ACKNOWLEDGEMENTS
lupoid syndromes may produce serious health hazards.
The adverse effects are related to dosage and duration of The authors are grateful to Ghulab Devi (Memorial)
administration. Paresthenia is the most common adverse Hospital for TB and chest diseases, Lahore, Pakistan;
effect, appears to be due to a relative pyridoxine Pakistan Medical Research Council (PMRC), Mayo
deficiency. Which is corrected by pyridoxine (vitamin B6) Hospital, Lahore, Pakistan; and National Institute of
supplementation and therefore particularly recommended Biotechnology and Genetic Engineering, Faisalabad,
to lactating women to intake vitamin supplementation? Pakistan.
The findings of the study under discussion substantiated
by Richard et al. (2006), who reported the standard MIC
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