Trends of anti-tuberculosis drug resistance pattern in new cases and previously treated cases of extrapulmonary tuberculosis cases in referral hospitals in northern IndiaAK Maurya1, S Kant1, VL Nag2, RAS Kushwaha1, TN Dhole2
1 Department of Pulmonary Medicine, Chhatrapati Shahuji Maharaj Medical University, (Erstwhile King George Medical College), Lucknow, Uttar Pradesh, India
2 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Correspondence Address: Source of Support: Indian Council of Medical Research, New Delhi (Extramural ICMR Project Sanction No. 5/8/5/4/2007-ECD-I), Conflict of Interest: None DOI: 10.4103/0022-3859.101379
Source of Support: Indian Council of Medical Research, New Delhi (Extramural ICMR Project Sanction No. 5/8/5/4/2007-ECD-I), Conflict of Interest: None
Background: Drug-resistant tuberculosis is one of major current challenges to global public health. The transmission of resistant strains is increasing as a burden of multidrug-resistant tuberculosis (MDR-TB) patients in extra pulmonary tuberculosis (EPTB) cases in India. Aim and Objectives: The aim was to study trends of anti-tuberculosis drug resistance pattern in new cases and previously treated cases of EPTB in referral hospitals in northern India. Study Design and Setting: A prospectively observational study and referral medical institutions in northern India. Materials and Methods: All EPTB specimens were processed for Ziehl Neelsen staining, BACTEC culture and BACTEC NAP test for Mycobacterium tuberculosis complex. All M. tuberculosis complex isolates were performed for radiometric-based drug susceptibility pattern against streptomycin, isoniazid, rifampicin and ethambutol using the 1% proportion method. Results: We found that 165/756 (20.5%) isolates were identified as M. tuberculosis complex by the NAP test. We observed that 39.9% were resistant to first-line antitubercular drugs. The resistance rate was higher in previously treated patients: H (30.3%), R (16.3%), E (15.7%) and S (16.3%). MDR-TB was observed in 13.4%, but, in new cases, this was 11.4% and 19.1% of the previously treated patients (P<0.05). Conclusion: MDR-TB is gradually increased in EPTB cases and predominant resistance to previous treated cases of EPTB. The molecular drug sensitivity test (DST) method can be an early decision for chemotherapy in MDR-TB patients. The International Standards of TB Care need to be used by the RNTCP and professional medical associations as a tool to improve TB care in the country.
Keywords: Anti-tuberculosis drug, extrapulmonary tuberculosis, multidrug-resistant tuberculosis, Mycobacterium tuberculosis complex
Tuberculosis (TB) is one of the major public health problems in India. India has about 1.8 million new cases of TB annually, accounting for one-fifth of new cases in the world - a greater number than in any other country. , Drug-resistant tuberculosis (DR-TB) is a man-made problem, because of inadequate use of drugs, inappropriate prescription or poor adherence to treatment, which permits the multiplication of drug-resistant strains. , The emergence of DR-TB strains is a global problem, which is a threat to the best efforts of prevention and TB control. The World Health Organization (WHO) reported that DR-TB is increasing in various parts of the world as well as in India.  The WHO and The International Union Against Tuberculosis and Lung Diseases has launched a Global Project on Anti-Tuberculosis Drug Resistance Surveillance, which uses standardized methods to measure the prevalence of drug resistance and assess its correlation with display of TB control.  Multidrug-resistant TB (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has become a significant public health dilemma in a number of countries and an obstacle to effective global TB control.  Extrapulmonary TB (EPTB) is a significant health problem in both developing and developed countries. , Diagnosis of EPTB in its different clinical presentations remains a true major challenge.  Resistance with new cases results from direct transmission of DR Mycobacterium tuberculosis; resistance rates generally increase when resistance among previously treated cases is already high and conditions for spread are favorable.  The Baltic region and countries of the former Soviet Union have reported the highest rates of MDR-TB in the world (approximately 10% in new and 25% in previously treated cases).  Recent reports from India state that MDR-TB has been found in 3% of new cases and 12% of previous treated patients.  Various studies have been focused on the pattern of drug resistance in pulmonary TB, but very few data is available for DR-TB in EPTB. , To the best of our knowledge, this is first study to report the patterns of anti-tuberculosis drug-resistance patterns among EPTB cases in northern India. The aim of the study to determine the anti-tuberculosis drug resistance patterns and its trends in new cases and previously treated cases of EPTB cases in referral hospitals in northern India.
Settings: The study was conducted between July 2007 and December 2010 in two tertiary care referral medical institutions in northern India. The study was a prospective observational study. The study protocol was assessed and approved by the respective Institutional Ethics Review Boards.
Data Collection: The entire patient population was attending indoor (IPD) and outdoor (OPD) wards from two tertiary care centers in northern India. A written informed consent was obtained from the patients before enrollment into the study. The clinical history regarding present and past history of anti-tubercular treatment (ATT), family history of TB and any other associated disease was taken in the prescribed performa.
Sample Size: The sample size for the study was calculated by the formula proposed by Lemeshow et al.  , where Z was equal to 1.96 (80% power of the study), the expected population proportional was 0.37, relative precision (%) was 20 and, at 95% confidence interval, the sample size for the study was 165 cases. Therefore, we have taken 165 strains of Mycobacterium tuberculosis complex (MTBC) from EPTB cases.
The formula for sample size calculation is as follows:
where, P: expected proportion, €: relative precision, a: significance level
Criteria for Inclusion: Patients included in the study were consenting new or previously treated EPTB cases patients from any age group in whom TB was confirmed by culture and in whom DST against MTBC strains had been performed. A flow chart of the inclusion criteria of MTBC isolates from EPTB cases in the study is presented in [Figure 1].
Non-consenting patients, those infected with Mycobacterium other than tuberculosis (MOTT) and bacillary patients with an unknown bacteriological profile were not included in the study, as were those patients not willing to participate in the study.
Definitions of Resistance Types: Resistance in new cases was defined as the presence of resistant strains of MTBC in patients who had never received anti-tuberculosis drugs or who had been treated for less than 1 month. Resistance in previously treated cases was defined as the presence of a resistant strain of MTBC in patients who had received anti-tuberculosis drugs in the past or who had been treated for more than 1 month.
Smear examination and culture: Specimens were processed for mycobacterial smear preparation and BACTEC culture. Smears were stained by the Ziehl Neelsen (ZN) method and examined for acid-fast bacilli (AFB).  Specimens from sterile sites were centrifuged and the sediment was inoculated into the BACTEC 12 B vial supplemented with the antimicrobial mixture PANTA (Becton Dickinson, Sparks, MD, USA). BACTEC vials were incubated at 37 ±1°C and interpreted as per Becton Dickinson manual instructions.  All inoculated BACTEC 12 B vials were tested twice a week for the first 3 weeks and then once a week for the remaining 3 weeks. Positive vials were subjected to smear microscopy for further confirmation. No growth after 8 weeks of incubation was treated as negative. Growth of MTBC was typed by niacin production, catalase activity at 68°C and pH 7 and susceptibility to P-nitrobenzoic acid. 
Biochemical identification of MTBC: The final identification of isolates belonging to MTBC was performed by the BACTEC NAP test (Becton Dickinson and Company, Sparks, MD, USA), where NAP (P-nitro-a-acetylamino-b-hydroxypropiophenone) was added to the culture to inhibit the growth of bacteria belonging to the MTBC.  Standard strain M. tuberculosis, H37Rv ATCCδ no. 27294 was used as positive control.
Radiometric-based drug susceptibility test
Radiometric-based drug susceptibility testing of MTBC by the BACTEC 460 TB (Becton Dickinson and Company) system was carried out as per the manufacture's instructions. , First-line drugs were provided in a drug kit (Becton and Dickinson). The following final concentrations were used: streptomycin (S) 6.0 mcg/mL, isoniazid (H) 0.1 mcg/mL, rifampicin (R) 2.0 mcg/mL and ethambutol (E) 7.5 mcg/mL. Briefly, 0.1 mL of the appropriate drug solution was injected into labeled BACTEC 12 B vials, which resulted in the desired concentration of a drug in the medium. This was followed by inoculation of 0.1 mL of bacterial suspension from a positive BACTEC 12B vial with a GI 500-800. For control, the bacterial inoculums were diluted 1:100 before inoculation. The inoculated 12 B vials were incubated and read daily on the instrument till the GI of the control reached >30. Reference strain of M. tuberculosis H37Rv was used as a quality control on a weekly basis.
All statistical calculations were performed using Statistical Package for Social Science (SPSS) Version 15.0. P<0.05 was considered statistically significant.
A total of 756 specimens from 756 patients with a presumptive clinical diagnosis of EPTB were evaluated for the presence of M. tuberculosis infection. Of the 756 specimens, 270 (35.8%) were lymph node aspirate and cold abscesses, 96 (12.7%) pleural fluid, 74 (9.8%) Cerebrospinal fluid, 73 (9.6%) urine, 50 (6.7%) biopsy materials, 47 pus (6.2%), 46 ascitic fluid (6.1%), 29 (3.9%) wound swabs, 19 (2.5%) gastric aspirate, 18 (2.3%) pericardial fluid, 17 (2.2%) synovial fluid and 17 (2.2%) bone marrow aspirates. Of the 756 specimens, 71 (9.3%) were positive for AFB by ZN staining and 227 (30.1%) were positive for mycobacteria by BACTEC culture. One hundred and sixty-five (20.6%) were confirmed as MTBC by the NAP test. Among 165 MTBC patients, the mean age of all patients was 32.12 + 5.23years; 94 (56.9%) were males and 71 (43.1%) were females, with 123 (74.5%) new cases (they had not taken ATT in the past or were less than 1 month on ATT) and 42 (25.5%) were cases as previous treated cases (they had taken ATT in the past or were more than 1 month on ATT) (P<0.05). The history of contact with TB patients was determined in 42 cases (25.4%); 19 (11.5%) were having a history of diabetic mellitus, family history of TB was present in 23 (13.9%) and three (1.8%) cases were HIV positive.
One hundred and sixty-five MTBC isolates were performed for first-line DST. Among the 165 MTBC isolates, 123 (74.5%) strains were seen in new cases and 42 (25.4%) strains were seen in previously treated cases of EPTB. We found that higher presence of resistant strains was seen in previous treated cases in comparison with new cases of EPTB (P<0.05). The resistance pattern of the 123 MTBC strains from the new cases is summarized in [Table 1]. Seventy-six (61.8%) were fully susceptible (SS), 44 (38.2%) were resistant and 14 (11.4%) were MDR-TB. The pattern of MDR-TB was five (4.1%) HR, three (2.4%) HR, three (2.4%) SHR and three (2.4%) HRES. The resistance pattern of 42 MTBC strains from previously treated cases is summarized in [Table 1]; 24 (57.2%) strains were SS and eight (19.1%) were MDR-TB. The pattern of MDR-TB was two (4.7%) HR, three (7.1%) SHR, three (7.1%) HRES and no resistant strains in HRE. Our results showed that 22 (17.8%) in monoresistance and 13 (10.5%) in two drug resistance were noted in the new cases, which was a higher percentage of resistant strains in comparison with the previously treated cases of EPTB (P>0.05). But, three drug resistance and four drug resistance of MTBC strains were higher presence of resistant strains in previously treated cases in comparison with the new cases of EPTB (P<0.05).
The resistance rate (RR) to first-line agent H, R, E and S in new cases, previously treated cases and overall RR was seen in 27.6%, 38.1% and 30.3%, 14.6%; 21.4% and 16.3%, 14.6%; 19.1% and 15.7% and 13.8%; 23.8% and 16.3%. Results are summarized in [Table 2]. Among the 23 MDR-TB strains with HIV association, three cases were HIV positive (13.1%) and 15 (65.2%) were negative, and five (21.7%) cases of HIV test were not performed due to unaccepted HIV testing (P<0.05). In the HIV and MDR-TB-positive cases, they had on-treatment of ART and ATT.
Our study confirmed that the high rate of drug resistance included MDR-TB among new cases and previously treated cases of EPTB at tertiary care hospitals in northern India. Overall, 39.9% of the EPTB cases had resistance to anti-tubercular drugs (38.2% of new cases and 42.8% of previously treated cases).
Gurang et al.  and Sachdeva et al.  have reported 10.5% from 513 EPTB patients in Nepal and 9.14% from 350 EPTB patients in India for LJ-based culture positivity of M. tuberculosis. In comparison, we found a 30.1% culture positivity of M. tuberculosis from 756 EPTB patients in northern India. We found a higher positive rate of M. tuberculosis due to use of liquid media for culture of mycobacteria in this study. Previous studies  revealed that the BACTEC system was found to be more advantageous in paucibacillary specimens, especially in case of extrapulmonary cases like cerebrospinal fluid, body fluids and fine needle cytology, where LJ media yielded very scanty growth or no growth.
Previous studies , have reported 88.9% of MTBC from Nepal and 62.5% presence of MTBC in EPTB specimens from India, but we found 72.6% of MTBC in EPTB specimens at tertiary care hospitals from northern India. We found that the maximum number of isolates in this study were isolates from lymph node aspiration. The findings are concordant with similar studies in other countries. ,,
Earlier studies reported that the prevalence of MDR tuberculosis has been shown to vary widely over different regions, with the higher rates being found in Nepal (48%), Gujarat, India (34%), New York city (USA) (30%), Brolivia (15%) and South Korea (15%).  As these results relate to pulmonary TB, comparisons are not strictly valid. Previous studies reported , a high rate of MDR-TB in EPTB cases (12.5%) from Nepal and from Delhi (10%), India, but we found that the prevalence of MDR-TB was 13.5% in EPTB cases at our tertiary care hospitals in northern India. The reason for this high rate of MDR-TB may be that the highly suspected drug-resistant cases were considered for DST in our tertiary care referral center. The high frequency of chronic cases may explain the high resistance rate in previously treated cases. Patient who had received anti-tuberculous drugs in the past were significantly more likely to be drug resistant than those who had never received them (P<0.05). Overall, 39.9% strains were seen to be resistant in our study, although the percentage of new cases to monodrug resistance and two-drug resistance was seen to be higher in comparison with previous treated cases. Our results were similar to other studies reported from other regions,  which showed that the resistance rate to isoniazid was highest. There is rare and contracts with finding that have been published by other studies. ,, It remains to be seen whether this pattern will persist in the near future. A spurious result due to methodology error seems unlikely, as all the laboratory methods have remained unchanged and effective quality control has been maintained.
We found that rate of TB-HIV co-infection was high (13.1%) in MDR-TB patients. This supports the results of the study done by Gandhi et al.  and the WHO/IUATLD 2002-2007 (31.6%) Study.  The response to TB treatment is usually similar in patients with and without HIV, except that HIV patients have a greater risk of drug toxicity and of mortality during treatment. 
The limitations of the current study are that it contains potential biases that could limit the validity of the results, such as the methods of patient selection and data collection, including data related to treatment history, which was used to differentiate between new and previously treated cases. Other details about other predisposing factors for development of drug resistance may be missed, and clinical outcome of drug-resistant cases in each and every patient is not available. However, despite the possible limitations, the current study strongly suggests problems in the control of MDR-TB among EPTB in the tertiary care hospitals in northern India.
Radiometric-based drug susceptibility testing of MTBC by the BACTEC 460 TB system is based on modified proportional method and is FDA approved as first-line anti-TB drug.  This method is standardized to increases the accuracy of the results. We found that susceptibility results are available within 6-8 days compare with 2-4 weeks by LJ. This showed that we could report complete results of isolation, identification and drug susceptibility testing of isolate culture with an average of 4 weeks time, as recommended by the Center of Disease and Prevention Control.  Surveillance of MTBC drug susceptibility testing can guide verdict makers in important standardization procedures for chemotherapy and routine evaluation of the quality of the TB control programme.
Further molecular-based rapid MDR-TB screening tests that achieve a substantial reduction in diagnostic delay and early availability of DST results would benefit patients harboring MDR-TB with appropriate regimens. The impact of rapid assays on patient outcomes is highly dependent on the quality of the rest of the TB control program.
In conclusion, the prevalence of MDR-TB among EPTB has been increasing in northern India. The trend of MDR-TB in previously treated cases of EPTB cases has reached an alarming level. Circulating of MDR-TB strains within society is a serious problem. This fact should be kept in mind that one MDR-TB patient if left untreated or not treated properly will create another 10-15 new cases of MDR-TB within 1 year. There is an urgent need of surveillance programmes, and newer molecular DST tests can aid in the rapid detection of Rifampicin and Isoniazid, which can be used as molecular markers for MDR-TB. The early availability of DST results would be beneficial to patients harboring MDR-TB strains to enable them receive effective treatment with appropriate regimens.
This work was supported by a grant from the Indian Council of Medical Research, New Delhi (Extramural ICMR Project Sanction No. 5/8/5/4/2007-ECD-I). The authors would like to thank the Technical Members of the Mycobacteriology Laboratory, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Science, Lucknow, India for their technical support during the research work.
[Table 1], [Table 2]