Tuberculosis is a major public health problem and causes 2 million deaths each year [1,2]. After the AIDS epidemic, however, its re-emergence in several countries has become a serious concern, whereas in endemic countries the development of drug resistance is an additional burden on the population at risk. The World Health Organization (WHO) estimates that up to 50 million individuals worldwide may be infected with drug-resistant strains of tuberculosis. According to one study , approximately 4.3% of all new and previously treated cases had multidrug-resistant (MDR) tuberculosis only in 2004. These cases can be treated successfully with second-line drugs, but more recently M. tuberculosis has also shown resistance to the available second-line drugs. These cases are almost untreatable and carry high fatality rates. This prompted scientists to coin the phrase ‘extensively drug-resistant’ (XDR) to define the state of tuberculosis in which the infecting M. tuberculosis strain develops resistance to at least two main first-line drugs (isoniazid and rifampicin) and is also resistant to any fluoroquinolone, and at least one of three injectable second-line drugs (capreomycin, kanamycin, and amikacin) . Data reported by Gandhi et al. from South Africa showed that 221 of 536 patients with HIV–tuberculosis co-infection had MDR strains. Of these, 53 were found to have XDR tuberculosis; 52 of them died. As the incidence of MDR tuberculosis in India is comparatively greater [3,6] than the global rate except for China, the possibility of the occurrence of XDR tuberculosis in India is expected to be high. Therefore, we carried out a hospital-based study to find the number of XDR tuberculosis strains in Indian patients co-infected with HIV attending the All India Institute of Medical Sciences, New Delhi, a tertiary care hospital.
The All India Institute of Medical Sciences handles approximately 1 600 000–1 700 000 patients annually hailing from Delhi and the adjoining states. In the preceding year this number was 16 92 832, of which 13 008 patients were suspected to have tuberculosis and were referred to us for a confirmation of tuberculosis. This prospective study was carried out from March to December 2006. Besides the clinically suspected tuberculosis patients, all HIV-positive symptomatic patients were also included in the study. After counseling and informed consent their HIV status was confirmed in both previously HIV tested and untested patients as per WHO guidelines (3 R/E/S) and appropriate clinical samples [sputum, cerebrospinal fluid (CSF), lymph node aspirate (LNA)] were collected from these patients. Whenever possible more than one sample was collected from each patient (except CSF and LNA). The specimens from the non-sterile sites were subjected to decontamination by the NALC-sodium hydroxide method. The decontaminated samples were inoculated into BACTEC MGIT 960 culture tubes (BD, USA) according to the manufacturer's instructions. Mycobacterial growth in the flashed positive tubes was confirmed by acid-fast staining and biochemical assays including nitrate reduction, the niacin test and aryl sulphatase test. Identification was also performed with the AccuProbe MTB complex culture identification test (Gen-Probe Incorp., San Diego, California, USA) and an in-house polymerase chain reaction protocol . The confirmed M. tuberculosis isolates were subjected to first-line (streptomycin, isoniazid, rifampicin, and ethambutol) antimycobacterial drug susceptibility testing (DST), and those having multidrug resistance were further subjected to second-line DST against ciprofloxacin, ofloxacin, kanamycin, p-amino salicylic acid using the BACTEC MGIT 960 system . As a quality control, DST was also performed by a proportional method for isoniazid, rifampicin, streptomycin, and ethambutol on Lowenstein–Jensen medium at concentrations of 0.2, 40, 4.0, and 2.0 mg/ml, respectively. DST against second-line drugs (kanamycin, ciprofloxacin, ofloxacin and p-amino salicylic acid) was performed using two critical proportions of 1% and 10% only [9,10]. The internal quality control tests were carried out for each batch of the prepared medium with a reference strain, M. tuberculosis H37Rv (kind gift from Dr V.M. Katoch, National JALMA Institute of Leprosy and other Mycobacterial Diseases, Agra, India), sensitive to all drugs. Drugs were serially diluted to appropriate concentrations to determine variations in minimal inhibitory concentrations following the WHO recommendations for second-line drug susceptibility testing .
During the study period, 54 patients were found to be HIV–tuberculosis co-infected. From these, a total of 139 clinical specimens (sputum, 131; LNA, three; CSF, three; blood, two) were collected. Sixty-four (46.04%) of 139 samples representing 24 (44.4%) of 54 patients were found to be culture positive for M. tuberculosis. One isolate of M. tuberculosis from each patient (24) was subjected to in-vitro DST and 12 (50.0%) of 24 were found to have MDR. All second-line DST results were evaluated in accordance with the definition of the Centers for Disease Control and Prevention and WHO . Accordingly, out of 12 MDR isolates, four (33.33%) isolates fulfilled the definition of XDR tuberculosis. The site of mycobacterial isolation and DST profile are shown in Table 1. Of the four patients (all adults with a mean age of 32 years; 21–49) with XDR tuberculosis, one was female and three were men. The mean CD4 and CD8 cell counts in these patients were 102 (51–246) and 425 (314–612) cells/μl, respectively. All XDR tuberculosis patients died within 2.6 months of diagnosis.
Our preliminary findings suggest that XDR tuberculosis is a reality in India. Even though XDR tuberculosis in four (16.7%) out of 24 cases may not truly reflect the prevalence rate, it clearly shows the existence of such strains and that these patients carry an extremely high and rapid mortality rate. This study also has significance in that from 54 HIV-infected patients, M. tuberculosis was isolated during a study period of 9 months only. We are continuing our work on XDR tuberculosis evaluation in HIV-positive and negative patients from both hospital and community settings. Earlier findings from a survey conducted by the WHO and Centers for Disease Control and Prevention  found that XDR tuberculosis is prevalent in 4% of MDR tuberculosis cases in the United States and 19% of MDR tuberculosis cases from Latvia. A study from KwaZulu Natal, South Africa  showed the prevalence of MDR tuberculosis as 39% and XDR tuberculosis in 23.45% of MDR cases . From Asian countries, one report from South Korea showed XDR in 15% of MDR cases and in 10.9% cases from Iran [11,12]. Therefore, to the best of our knowledge ours is the first documentation of XDR tuberculosis from India.
Sponsorship: Financial assistance of the Department of Biotechnology and the Indian Council of Medical Research, Governmentt of India, to S.S. is greatly appreciated.
1. Espinal MA, Laserson K, Camacho M, Fusheng Z, Kim SJ, Tlali RE, et al
. Determinants of drug-resistant tuberculosis: analysis of 11 countries. Int J Tuberc Lung Dis 2001; 5:887–893.
2. Horsburgh CR Jr. The global problem of multidrug-resistant tuberculosis: the genie is out of the bottle. JAMA 2000; 283:2575–2576.
3. Aziz MA, Wright A, Laszlo A, De Muynck A, Portaels F, Van Deun A, et al
. Epidemiology of antituberculosis drug resistance (the Global Project on Antituberculosis Drug Resistance Surveillance): an updated analysis. Lancet 2006; 368:2142–2154.
4. Centers for Disease Control and Prevention. Revised definition of extensively drug-resistant tuberculosis
. MMWR Morb Mortal Wkly Rep
5. Gandhi NR, Moll A, Sturm AW, Pawinski R, Govender T, Lalloo U, et al
. Extensively drug resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 2006; 368:1575–1580.
6. Gopinath K, Mani Sankar M, Kumar S, Singh S. Controlling multi drug resistant tuberculosis in India. Lancet 2007; 369:741–742.
7. Singh S, Gopinath K, Shahdad S, Singh S. Nontuberculous mycobacterial infections in Indian AIDS patients detected by a novel set of ESAT-6 polymerase chain reaction primers. Jpn J Infect Dis 2007; 60:14–18.
8. Rusch-Gerdes S, Pfyffer GE, Casal M, Chadwick M, Siddiqi S. Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuberculosis
to classical second-line drugs and newer antimicrobials. J Clin Microbiol 2006; 44:688–692.
9. World Health Organization. Guidelines for drug susceptibility testing for second line antituberculosis drugs for DOTS-plus. WHO/CDC/TB/2001.288. Geneva, Switzerland: WHO; 2001.
10. Kent PT, Kubica GP. Public health mycobacteriology: a guide for level III laboratory. Atlanta, GA: Public Health Services, US Department of Health and Human Services, Centers for Diseases Control; 1985.
11. Centers for Disease Control and Prevention. Emergence ofMycobacterium tuberculosiswith extensive resistance to second-line drugs – worldwide, 2000–2004
. MMWR Morb Mortal Wkly Rep
12. Masjedi MR, Farnia P, Sorooch S, Pooramiri MV, Mansoori SD, Zarifi AZ, et al
. Extensively drug resistant tuberculosis: 2 years of surveillance in Iran. Clin Infect Dis 2006; 43:841–847.