Current status of multidrug resistant tuberculosis in a tertiary care hospital of East DelhiT Sagar, NP Singh, B Kashyap, IR Kaur
Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.118031
Source of Support: None, Conflict of Interest: None
Background and Objective: Multidrug resistant tuberculosis (MDR-TB) is caused by infection due to Mycobacterium tuberculosis which is resistant to both isoniazid (INH) and rifampicin (RIF). It is caused by selection of resistant mutant strains due to inadequate treatment and poor compliance. MDR-TB is a major public health problem as the treatment is complicated, cure rates are well below those for drug susceptible tuberculosis and patient remains infectious for months despite receiving the best available therapy. The drug susceptibility pattern of M. tuberculosis is essential for proper control of MDR-TB in every health care setting, hence the study was initiated with the aim of studying the prevalence of MDR-TB in patients attending a tertiary care hospital in east Delhi. Materials and Methods: Five hundred and forty-three pulmonary and extrapulmonary samples from suspected cases of tuberculosis received in the mycobacteriology laboratory from November 2009 through October 2010 were investigated for M. tuberculosis. All the samples were subjected to direct microscopic examination for demonstration of acid fast bacilli followed by culture on Lowenstein-Jensen (LJ) medium to isolate M. tuberculosis. Identification was done by conventional biochemical methods. Drug susceptibility of isolated M. tuberculosis strains was done by conventional 1% proportion method followed by sequencing of RIF resistant isolates to detect mutations to confirm resistance. Results and Conclusions: M. tuberculosis was isolated from 75 out of 543 suspected cases of pulmonary/extrapulmonary TB. Three of the total 75 M. tuberculosis isolates (4%) showed resistance to any one of the first line drugs. Prevalence of MDR-TB was 1.3%. The sequencing of single MDR strain showed mutations at codons 516, 517, and 518. Amplification of rpoB and sequential analysis of the amplicon is a better way of detection of mutation and the evidence of new mutation in this study indicate that mutations continue to arise, probably due to the ability of M. tuberculosis to adapt to drug exposure.
Keywords: Antituberculosis drugs resistance, drug resistance, multidrug resistant tuberculosis, mutation, rpoB gene, tuberculosis
Tuberculosis continues to be a major health problem worldwide despite the fact that the causative agent was discovered more than 100 years ago. Though the disease primarily effects lungs and causes pulmonary tuberculosis, it can also affect intestine, meninges, bones and joints, lymph glands, skin, and other tissues of the body. The disease is more prevalent in developing countries with poor economy, limited infrastructure, overcrowding, and undernourishment. In India, the disease is estimated to kill 500,000 individuals per year; about 1,000 everyday; and one per minute. Multidrug-resistant tuberculosis (MDR-TB) has been a topic of growing interest in the last decade. The exact magnitude of the problem of resistance to antituberculosis drugs worldwide was not known till 1994-1997 when the global project on antituberculosis drugs resistance surveillance was initiated by World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUAT-LD). MDR TB is defined as the disease due to Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampicin (RIF) with or without resistance to other drugs. Prevalence of MDR-TB mirrors the functional state and efficacy of tuberculosis control programs and realistic attitude of the community towards implementation of such programs.  Treating MDR-TB can be difficult because of inability to use the two most potent antituberculosis drugs (i.e., INH and RIF) that implies less effective first line therapy and more toxic and less efficacious second line therapy. In all countries and especially those where the number of cases of tuberculosis is rising rapidly because of association with HIV, the development and spread of resistant strains of tuberculosis is a serious concern. In the present study, we determined the prevalence of MDR-TB in a tertiary care hospital of east Delhi along with the mutations in rpoB gene of an isolated MDR strain.
A prospective study was carried out from November 2009 through October 2010 in the Department of Microbiology at University College of Medical Sciences (UCMS) and Guru Teg Bahadur (GTB) Hospital. Five hundred and forty-three samples from clinically suspected pulmonary and extrapulmonary tuberculosis patients received in the mycobacteriology laboratory were investigated for M. tuberculosis. A total of 75 M. tuberculosis isolates from 543 samples of pulmonary and extrapulmonary tuberculosis cases were further analyzed for drug resistance by conventional 1% proportion method.
Smears were prepared from all the 543 samples and stained by Ziehl-Neelsen (ZN) staining for demonstration of acid fast bacilli after which they were examined under light microscope and graded according to Revised National Tuberculosis Program (RNTCP) protocol. 
Isolation and identification of M. tuberculosis
All the 543 specimens were processed and cultured on Lowenstein Jensen (LJ) medium as per the standard protocol. The sediments were inoculated onto LJ medium slopes and incubated for 8 weeks. The growth of M. tuberculosis was identified by colony morphology, Ziehl-Neelsen staining, and the conventional biochemical tests as per the standard protocol followed in our laboratory.  The niacin test was carried out using commercially prepared BBL Taxo TB niacin test strips (from BD Diagnostic) according to manufactures' protocol.
Drug susceptibility testing for M. tuberculosis
The DST was carried out on the M. isolates by conventional 1% proportion method against INH (0.2 μg/ml), ethambutol (2 μg/ml), streptomycin (4 μg/ml), and RIF (40 μg/ml).  H37Rv was used as the reference strain.
The mycobacterial DNA was extracted from the MDR isolate (Bioneer, USA) according to the manufacturer's protocol. The extracted DNA was stored at −70°C till further processing.
Polymerase chain reaction of MDR strain targeting rpoB gene
The 255 base pair (bp) region of rpoB gene was amplified using the following primers.
Backward primer: 5'ATGACCACCCAGGACGTG3'.
Forward primer: 5'GGTTTCGATCGGGCACAT3'.
Polymerase chain reaction (PCR)  was carried out in 50 mL of a reaction mixture containing 5 μl of × 10 Taq buffer (Fermentas), 200 mM of deoxynucleoside triphosphates (Fermentas), 1 U of Taq polymerase (Fermentas),10 pmoles of each set of primers (Genetix), and 10 ng of chromosomal DNA followed by addition of nuclease free water to make up to final volume (50 μl). The thermal cycle conditions were: 95°C for 5 min; 35 cycles of 95°C × 1 min, 50°C × 1 min, 72°C × 1 min, and final extension at 72°C × 10 min. The PCR products were analyzed on 1% agarose gel and the amplicon size was compared with appropriate DNA ladder (HiMedia; Thermal cycler Master cycle personel Eppendorf AG 22331 Hamburg, Germany).
Sequencing of amplicon
The amplicon of the MDR strain was outsourced for sequencing (Invitrogen, Gurgaon). The sequence of the rpoB amplicon of the test RIF resistance strain of M. tuberculosis was matched with the known sequence of rpoB gene of the sensitive H37RV strain for mutations.
Among the total of 543 clinically suspected cases of pulmonary and extrapulmonary TB that were recruited during the study period, only 75 were diagnosed to be positive and M. tuberculosis was isolated from them. Out of the total of 75 isolates of M. tuberculosis, 36 and 39 samples were from pulmonary and extrapulmonary tuberculosis cases, respectively. Three of the 75 (4%) M. tuberculosis isolates showed resistance to one or two drugs and the interpretation could be done by 28 days in one (33%) and by 42 days in two (67%) isolates [Table 1]. Two of these isolates (one MDR and the INH monoresistant strain) were from cases of pulmonary tuberculosis and one isolate (resistant to INH and streptomycin) was from a case of infertility. All the three isolates showed resistance to INH out of which only one isolate was monoresistant. Among the remaining two isolates, one was resistant to RIF (MDR) (1.3%) and the other was resistant to streptomycin (1.3%) in addition to INH [Table 1]. Rest of the 72/75 (96%) isolates were sensitive to all the drugs.
Deletion of codons 516, 517, and 518 was detected in RIF resistance determining region (RRDR) of rpoB gene in the single MDR strain sequenced.
Tuberculosis is one of the oldest diseases known to affect humans. It is a major cause of death worldwide. If properly treated tuberculosis caused by drug susceptible strain is curable virtually in all cases. In recent years the treatment of tuberculosis has been threatened by the increasing number of patients with MDR tuberculosis. Both INH and RIF are potent bactericidal antituberculosis drugs used in tuberculosis control programs. The outcome of treatment of patients harboring MDR M. tuberculosis strains has been poor with high mortality rate. Their chance of being cured is very low and they require significant expenditure of healthcare resources. Moreover, these patients remain infectious for a prolonged period and may therefore be more likely to infect others. The level of drug resistance is said to provide an epidemiological indicator to assess the amount of resistant bacterial transmission in the community as well as the success of tuberculosis control programs. Further, this influences the therapeutic regimens and policy decision also.
The prevalence of MDR-TB in our study was 1.3% which is reasonably in agreement with various other studies shown in [Table 2]. ,,,,,,, The variation in studies could be due to different selection criteria of patients, extent of misuse of drugs, quality of questionnaire used for eliciting history of previous treatment, and inadequate laboratory support and reporting system. In the single MDR case that was found, there was no history of intake of antituberculosis treatment in the past, neither was their family history of TB. According to the directly observed treatment shortcourse (DOTS) records, the patient belongs to low socioeconomic status but was compliant with the therapy. MDR-TB largely occurs as a result of pressure selection during disease treatment. Drug resistance in M. tuberculosis is caused by mutation in relatively restricted region of the genome. Mutation associated with drug resistance occur in rpoB gene for RIF, katG gene for INH, gene embB for ethambutol, pncA gene for pyrazinamide, and rpsl and rrs genes for streptomycin. RIF is one of the most important chemotherapeutic agents used to combat infections by M. tuberculosis and can be assumed to be a surrogate marker for MDR-TB.  Moreover, more than 90% of RIF-resistant isolates are also resistant to INH; therefore, detection of RIF resistance could also identify MDR strains.  The above observations have led to the recent development of several genotypic methods for rapidly detecting RIF-resistance conferring mutations, including DNA sequencing, line probe assay, single strand conformation polymorphism, DNA microarrays, RNA/RNA mismatch, and molecular beacons.
The development of resistance to RIF is due to mutation in a well-defined 81 bp (27 codon) central region of the gene that includes the β-subunit of RNA polymerase (rpoB). More than 96% of the RIF-resistant strains contain a mutation in this 81 bp region of rpoB gene which is referred to as the rifampicin resistance determining region (RRDR), thus facilitating a straight forward approach to detecting RIF resistance and/or MDR, rapidly.  Different groups of workers from diverse regions of the world ,, have reported substitutions, deletions, and insertions in the RRDR of the rpoB gene. The highest frequencies of mutation are seen in codon 531 (83%), followed by codon 526 (7%), codon 516 (5%), codon 533 (4%), and codon 513 (2%).  Various workers worldwide have shown different frequencies of mutation at codon 531 (29-74%), 526 (0-43%), and 516 (0-38%). , In our study, MDR-TB strain was amplified for 255 bp region of rpoB gene and the amplicon was sequenced to know the mutation. Deletion mutation was found in codon 516, 517, and 518. The deletion mutation of codons 516, 517, and 518 together has been rarely reported. The deletion of both codon 517 and 518 or codon 516 and 517 or single deletion of codon 517 or 518 has been reported.  Only one case has been reported from south India where deletion of codon 517 was detected.  In our study there was deletion of codon 516, 517, and 518 and such a mutation has not been reported earlier from this part of the country to the best of our knowledge.
Despite the large number of mutations already reported in other studies, the evidence of new mutation in this study indicates that mutations continue to arise, probably due to the ability of M. tuberculosis to adapt to drug exposure. Newer techniques based on the principle of hybridization probes rapidly identify clinical isolates as members of the M. tuberculosis along with determining the presence of point mutation within the RRDR of the rpoB gene. Such tests are designed according to the expected mutations in an area. Amplification of rpoB gene followed by sequential analysis of the amplicon is a better way of detection of mutation.
[Table 1], [Table 2]