Antibiotic Susceptibility Pattern of Mycobacterium tuberculosis

Background: The emergence and spread of DR and MDR-TB threat global TB control. The susceptibility patterns of M. tuberculosis isolates against anti-tuberculosis drugs informs an important aspect of TB controls and surveillance and analysis of local rates of TB drug resistance helps in the detection and monitoring of the extent of DR and MDR strains, indicating the quality of TB control in the country.


INTRODUCTION
Tuberculosis (TB) is a treatable and preventable disease. 1 It is an infectious bacterial disease caused by Mycobacterium an acid fast bacilli. 2 The SAARC region accounts more than 29% of global burden of tuberculosis with 0.6 million deaths every year and 2 million new cases annually. 3Tuberculosis is a socio-medical problem. 4 Ziehl-Neelson Staining or by Fluoro-chrome staining allows highly accurate diagnosis widely available, simple and multi-purpose equipment. 5Culture increases the number of tuberculosis cases found, often by 30-50% and detects cases earlier, often before they become infectious. 6Most of the recent advances in the laboratory diagnosis of TB BY rapid culture, identifi cation, and susceptibility systems. 7ug resistant tuberculosis is a case of tuberculosis (usually pulmonary) excreting bacilli resistant to one or more anti-tuberculosis drugs.MDR-TB is resistant to at least Isoniazide and Rifampicin, the main antituberculosis drugs. 8TS strategy as one of the most cost effective health interaction and recommends that effective TB treatment be a part of the essential clinical service package available in primary health care.In Nepal, DOTS strategy has been implemented since 1996 and has already reduced the number of deaths. 9

METHODS
A cross sectional study conducted to fi nd out antibiotic susceptibility pattern of Mycobacterium tuberculosis in pulmonary tuberculosis patients was at National Tuberculosis Centre from September 2005 to May 2006.A total of 295 clinically suspected patients having sputum positive diagnosed by fl uorescent microscopy were included in the study.The microscopy produced bright yellowish bacteria against a dark background.The sputum samples were decontaminated and cultured on 2% modifi ed Ogawa media and smear examination was done from pellet.The M. tuberculosis bacteria were confi rmed by different biochemical tests.The culture positive isolates were tested for anti-TB drug susceptibility testing by Proportional Method as standard protocol.
Each strain was tested against four antibiotics at the following concentrations: Isoniazid (INH) at 0.25μm/ml and 8μm/ml; Ethambutol (EMB) at 1μm/ml and 2μm/ml; Streptomycin(SM) at 4μm/ml and 8μm/ml and Rifampicin (RMP) at 20μm/ml and 40μm/ml.The LJ medium is used for all the resistance tests.Working drug dilutions was prepared on the day of use; 1 ml of working solution added to 500 ml of LJ medium will yield fi nal drug concentrations equivalent to the different critical concentrations.The medium is distributed in volumes of 6-8 ml in sterile 17 mm x 170 mm screw-capped test tubes, coagulated at 85 °C for 45 minutes, and allowed to cool at room temperature for 24 hours; the screw caps are then tightened and the tubes stored at 4 °C.The control medium without drugs is prepared at the same time as the drug-containing media.The period of validity of the media stored at 4 °C is 2 months.

RESULTS
A total of 295 suspected PTB cases attending at NTC was included in the study.The sputum samples from patients under study were subjected to test for Fluorochrome staining, culture and antibiotic susceptibility testing for culture positive isolates.
Among 295 cultures positive M. tuberculosis isolates studied from drug susceptibility testing, resistance to one or more anti-drugs were exhibited.
Out of 295 isolates, 45 isolates were from untreated patients and 250 isolates were from treated patients.
Among the untreated patients, 35.55 %( n=16) were sensitive to all the 4 drugs whereas the remaining were resistant to two or more drugs.Resistant to only one drug was shown by 20% (n=9) of the isolates.Resistant to two drugs was shown by 17.77% (n=8), three drugs to 11.11% (n=5) and four drugs to 6.66% (n=3) of the isolates (Table 3).
Out of the 250 treated TB patients, 25.20% (n=63) were sensitive to all the four anti-TB drugs and the remaining187 isolates were resistant to one or more drugs.Resistant to one drug was found in 23.6 % (n=59); two drugs to 12.4 % (n=31) and three drugs to 16.4% (n=41).
Multi Drug Resistance (MDR) in untreated TB patients were found in 22.22% (n=10) and in treated TB patients was 37.20 % (n=93).
The primary drug resistance (PDR) to one drug was 20%, to two drugs was 17.77%, to 3 drugs was 11.11% to four drugs was 6.66% and primary MDR was in 22.22% of the isolates (Table 5).Similarly, acquired drug resistance to one drug was 23.60%,to two drugs was 12.40%, to three rugs was 16.40% , to four drugs was 18.80% and acquired MDR was in 37.20% of the isolates.7).The study demonstrated that age and sex were not signifi cantly related to drug resistance (P<0.05).
The study demonstrated that age and sex were not signifi cantly related to drug resistance.Among 79 drug sensitive strains isolated from PTB patients, 40.50% (n=32) were isolated from those patients who had previous cases of TB in his/her family and 59.50% (n=47) had no previous cases of PTB in his/her family.

DISCUSSION
Tuberculosis has become a grave concern in all part of the world because of recent resurgence of TB.Reasons of this resurgence have been identifi ed by WHO as mainly due to HIV pandemic, less health priority given to the disease and signifi cant increase of multidrug resistant tubercle bacilli as a result of inadequate treatment.The causative agent was discovered more than 100 years ago and highly effective drugs and vaccines are available making TB a preventable and curable disease.It remains as the most signifi cant cause of morbidity and mortality due to a single infectious agent in the world.
Tuberculosis is one of the major public health problems in the third world countries with approximately estimated 60% of the adult population being infected with TB.In Nepal, about 45% of the total population is infected with TB.Every year 40,000 people develop active TB of whom 20,000 have infectious pulmonary disease.Nepal, by estimated number of cases, is ranked at 27 globally.Despite the expansion and implementation of a much improved National Tuberculosis Programme (NTP) through DOTS Strategy throughout the country,6000-7000 people still die from TB each year in Nepal. 9e main objective of this study is to know the resistance pattern of the anti-TB drugs in PTB patients  12 Smith reported that as in most countries of the world, in Nepal, the reported incidence of TB is higher in man than women.Rijal reported that the study conducted at NTC, among the 325 cases, 75.69% were male and 24.30% were female. 12-19Thus the incidence of TB was found higher in male than female patients; male patients are affected more than female patients.According to the signifi cance test, the prevalence of TB in male and female was found to be statistically signifi cant.These possible factors explain the gender differences observed, the most commonly accepted being that women are less exposed to infection than men.The second might be the biological difference, such as an increased susceptibility in male.Finally, infected women may progress more frequently to disease and die more rapidly, leaving a cohort with a low prevalence of infection.
In this study, out of 295 culture positive isolates, 15.25% ( n=45) isolates from the untreated TB cases and 84.74% (n=250) isolates from the treated TB cases.The study showed that 26.77 %( n=79) were sensitive to all the four drugs and 68.81 %( n=203) cases were drug resistant to one or more drugs.Monoresistance to INH, SM, RMP and EMB was detected in 41(13.89%),19(6.44%),1(0.33%) and 7(2.37%) strains respectively; and 23.05% to a total single drug resistance.Similarly, resistance to two drugs was detected in 39 strains(13.22%);resistant to three drugs was detected in 46 strains (16.94%).The level of Multi-drug resistant cases were found to be 34.91%(22.22%Primary MDR and 37.20% Acquired MDR).
The highest rates of drug resistance were discovered for isoniazid and streptomycin.In both new and treated cases, 13.33% and 14% of the strains isolated were resistant to isoniazid respectively; whereas 6.66% and 6.40% of the strains isolated from new and previously treated patients were resistant to streptomycin respectively.
The initial drug resistance case was found in 8.47% to one or more of the four anti-TB drugs was found to be 60.33%.In addition, the primary drug resistance(PDR) to one drug was 20%, to two drugs was 17.77%,to 3 drugs was 11.11% and to four drugs was 6.66%.And the acquired drug resistant to one drug was 23.60%, to two drugs was 12.40%, to three drugs was 16.40% and to four drugs was 18.80%.The primary MDR was found to 22.22% of the isolates and the acquired MDR was in 37.20% of isolates.This fi nding of this study is similar with the latest third surveillance report of WHO 2004 in Nepal.The report revealed that 1.3% and 20.5% of the new and old cases had MDR in Nepal.The rate of acquired MDR-TB was higher (19.25%) than the rate of primary MDR-TB (2.63%).
Out of 295 culture positives isolates obtained from the PTB patients,171 isolates were from relapse cases,45 isolates from chronic cases,19 isolates from followup cases,8 isolates from default cases and 7 isolates from treatment failure cases.The highest percentage of MDR was obtained from the chronic cases (64.44%) followed by follow-up cases (47.36%), treatment failure cases (42.85%), relapse cases (27.48%) and default cases (12.5%).
The fi nding of this study is in agreement with other studies conducted at different places.Al Marri in the state of Qatar reported that 85% of the cases were sensitive to anti-TB drugs and 15% cases were resistant to one or more anti-TB drugs among 406 cases of PTB. 14 In West Province of Cameroon, the level of initial drug resistance was found in 14.28% and acquired drug resistance was found in 0.74%,the rate of MDR was found 0.98% (0.49%initial MDR and 1% for acquired MDR); Kuban et al. reported 4.1%MDR case among 566 isolates, 15.2% initial drug resistance and 11.6% acquired drug resistance and 1.06% and acquired MDR cases 3%). 15Indian studies showed that 3.4% of the new cases and 25% of the old cases had MDR-TB (WHO,2000). 23The anti-TB drug sensitivity test conducted in Nepal 1987-1990 revealed that 1.6% and 9.6% of the new and old cases respectively had MDR-TB.GC   [17][18][19][20][21][22][23] The alarming increment in MDR-TB cases may be owing to late identifi cation of suspected MDR-TB cases.Identifi cation of all cases of MDR-TB would require culture and susceptibility testing of tuberculosis suspects, an ideal that is unachievable in developing countries like Nepal.The culture and sensitivity facilities for M. tuberculosis in our country are country are only in National Tuberculosis Centre (NTC) and Germany-Nepal Tuberculosis.Project (GENETUP).Other reasons for increase in MDR-TB may have smear negative TB and hence may even remain undiagnosed with tuberculosis.Even among those with smear positive disease, initial response to treatment may be good, and MDR-TB may not be suspected in some cases.
Our study revealed that there were high rates of initial drug resistance against isoniazid and streptomycin among both new and treated cases .This may be due to low cost and widespread use in the treatment of TB.The most signifi cant fi nding of our study was the low frequency of primary resistance to rifampicin which is a good indicator for success of DOTS.Mono resistance to Rifampicin was not observed at all in new cases.Resistance to Rifampicin predicted resistance to isoniazid and streptomycin and served as marker of MDR.
The high rates of resistance among new cases indicated that drug resistant strains are circulating and are being transmitted from patient to patient in our country Nepal.Transmission of already resistant strains as a serious problem and threat, as it is different to treat patients infected with drug resistance, it is important for a TB control programme to have reliable laboratory facilities for susceptibility testing of M .tuberculosisisolates.our study that neither nor acquired drug resistance were associated with sex or age. 14,18kewise, the family history of TB cases, smoking and alcoholic habit of the patients did not show any signifi cant relation with drug resistance.This study was similar to the study of Leung in Hong Kong; and Toungoussova et al., 2002; Archangels in Russia. 10,20crease cases of MDR-TB are a global problem.MDR-TB can be cured by the effective implementation of DOTS strategy.Regular monitoring of MDR-TB and policy in accordance with the operational research fi nding enables the controls and drug quality assessment is helpful for emerging MDR-TB.

This
In 1997 the World Health Organization (WHO), the International Union Against Tuberculosis and Lung Disease (IUATLD) and partners world-wide released the fi rst report of the global project on anti-tuberculosis drug resistance. 22The data generated in this report were reinforced in a recently published second report.Directly observed treatment short-course (DOTS), the WHO strategy for TB control cures virtually all patients with drug-susceptible TB and some drug resistant TB through the administration of short-course chemotherapy with fi rst-line drugs.
However, patients with multidrug-resistant (MDR) tuberculosis (TB) to at least isoniazid and rifampicin are more likely to fail short-course chemotherapy.In recent years there has been encouraging evidence that patients with MDR TB can be cured with appropriate management based on second-line drugs.Unfortunately, second-line drugs are inherently more toxic and less effective than fi rst-line drugs and reliable assessment of drug resistance is an essential prerequisite for appropriate use.Treatment is prolonged and signifi cantly more expensive.Accurate laboratory drug susceptibility testing (DST) data to second-line drugs will support clinical decision making and help to prevent the emergence of further drug resistance in patients with MDR TB.In order to meet the challenges posed by MDR TB, the WHO established the DOTS-Plus initiative to assess the feasibility and cost-effectiveness of using second-line drugs to manage patients with MDR TB primarily in middle and low-income countries.
DOTS-Plus is needed in areas where MDR-TB has emerged due to previous inadequate TB control programmes.Therefore, DOTS-Plus pilot projects are only recommended in settings where the standard DOTS strategy is fully in place to protect against the creation of further drug resistance.DOTS-Plus is designed to cure MDR-TB using second-line TB drugs.These drugs should be stored and dispensed at specialized health centers with appropriate facilities and well-trained staff.It is vital that DOTS-Plus pilot projects follow WHO recommendations in order to minimize the risk of creating drug resistance to second line TB drugs.DOTS-Plus works as a supplement to standard DOTS-based TB programmes already in place.
If patients failing DOTS are presumed to have MDR-TB, and if drug-susceptibility testing is limited, they might be placed on an empirical treatment regimen consisting of second-line TB drugs.Under DOTS Plus, they must endure an additional two years of daily, observed combination therapy, including injectable antibiotics, which can produce unpleasant side-effects.As of July 2002, the Green Light Committee (GLC) had approved seven pilot projects to implement the DOTS-Plus strategy, and is currently reviewing fi ve further applications.Preliminary results from those programmes already under way show percentages of culture negativization to be between 46 and 79 percent.Continued support for these projects -together with the implementation of new programmes in other countries -will contribute to the building of a sound policy for the control of MDR-TB.
Estonia's country-wide DOTS-Plus programme began in March 2001, and allows for the enrolment of 200 patients over a two-year period.Preliminary results show a sputum negativization of 46 percent of patients after six months of treatment.The pilot project has become a leverage tool to promote the expansion of the DOTS strategy in Estonia.
Latvia began to implement DOTS fully in 1997.However, poor case management in the past and the overcrowded conditions of TB wards still helped to make Latvia the country with the second highest MDR-TB rate in the world.The proportion of MDR-TB among new TB patients in this country is 9.5 percent.On February 2001, the GLC approved a countrywide DOTS-Plus pilot project.
Strengthening MDR-TB control now through DOTS-Plus will help to reduce morbidity, mortality and transmission due to MDR-TB.By directing MDR-TB patients to effective treatment protocols now, we are saving direct costs.And by controlling the primary cycle of MDR-TB transmission now, we are saving future funds and indirect costs that would otherwise have to be diverted into treatment for both sick individuals and those that they infect.

CONCLUSION
MDR-TB was found the highest in chronic cases (64.44%) followed by follow-up case (47.36%), treatment failure cases (42.85%), relapse cases (27.48%) and Default cases (12.5%).A statistical analysis reveals no signifi cant relationship between age and sex with the emergence of drug resistant isolates.
study showed there were no any relation of age and sex, with drug resistance.The result of the present study was in agreement with the study of Al-Marri in Qatar; Warndroff et al in Karongo District, Malavi also supports Antibiotic Susceptibility Pattern of Mycobacterium tuberculosis JNHRC Vol. 7 No. 1 Issue 14 Apr 2009 36 . A total 295 cases were included in this study carried out from September 2005 to May 2006.Out of 295 cases, 250 cases were previously treated cases and 45 were untreated cases.During this study, among the 295 cases of TB, 73.89 %( n=218) males were found higher in number than female 26.10% (n=77) in age group discussion from 11 to above 80 year.The highest number of cases belonged to the age group 21-30 (29.81%).This fi nding was concordant with similar studies in other countries.In a similar study in Italy, Ponticiella et al. (1997) reported 82.2% males and 17.8% of females among 90 active PTB cases; Blumberg et al (1991-1997) in Atlanta, USA reported 74% of the male and 26%of the female TB cases among 1536 cases.Likewise, in Archangeh, Russia, Toungoussova et al in Korea reported 66.49% if the male and 34.31% of the female TB cases among 2486 cases.Kuban et al in Cameroon, Yaunde, reported 65.76% of male and 34.25% of the female TB cases among 111 cases; Riantawan et al in Thailand reported 77% male and 23% of the female cases among 1441 cases. 10-15Tuberculosis Control Programme, Nepal reported 66.77% male and 33.23% female of TB cases among 14,384 newly diagnosed TB cases during 2002/2003.All above fi ndings are consistent with this study.Shrestha et al reported 47% males and 3.05% of female TB cases in histopathological specimens at Tribhuvan University Teaching Hospital.
et al.(2001) found 8.57% of initial MDR cases and 100% of acquired MDR cases; Bhattarai et al. (2003) obtained 4.16% of primary MDR cases and 5% of acquired MDR cases.Similarly, Rijal et al. (2003) found primary MDR in 2.63% of the isolates and acquired MDR in 19.25% of isolates.

Table 1 .
Age and sexwise distribution of PTB patients

Table 3 .
Antibiotic susceptibility pattern in patients with or without past history of treatment

Table 4 .
Antibiotic susceptibility pattern in patients with past treatment

Table 5 .
Comparison of primary and acquired anti-TB drug resistance.

Table 7 . Age wise distribution of resistant M. tuberculosis in female
Toungousssova OS, Caugant DA, Sandven PB, June G.Drug resistance of Mycobacterium tuberculosis strains isolated from patients with pulmonary tuberculosis in Archangels, Russia.The international journal of TB and lung disease 2002;6:5406-14.11.Raintawan P, Punnotok J, Chaisuksuwan R, Prasugarit V (1997) Resistance of Mycobacterium tuberculosis to anti-tuberculosis drugs in the central region of Thailand.The International Journal of Tuberculosis and Lung Disease 1997;1(4): p. 299-301.12. Shrestha B, Nehar A, Breyer U. The pattern of anti-tuberculosis drug resistance in patients treated at an urban TB clinic in Kathmandu Valley.J Nepal Med Assoc TB special 1996;117:36-40 13.Rijal KR, Ghimire P, Bam DS, Rijal B. An epidemiological study of anti-tuberculosis drug resistance pattern in the pulmonary tuberculosis patients visiting National Tuberculosis Centre.In: A Dissertation submitted to the Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal, 2004.14.Al-Marri MRHA.Pattern of mycobacterial resistance to four anti-tuberculosis drugs in pulmonary tuberculosis patients in the state of Qarter after the implementation of DOTS and limited expatriate screening Programme.The international journal of Tuberculosis and Lung Disease 2001;5(12):1116-1121. 15.Kuban C, Bercion R, Noeske J, Cunin P, Nkamsse P, Ngo Niobe S. Anti-tuberculosis drug resistance in the West Province of Cameroon.The International Journal of Tuberculosis and lung Disease 2002;4(4):356-60.