Isolation of Diverse Mycobacterium Tuberculosis Strains Employing Automated and Conventional Culture from Lymphadenitis in a Tertiary Care Center, Pondicherry, India

Kandhakumari Gandhi¹, Stephen Selvaraj^2* and Kavitha Kannaiyan³

^*Correspondence: Stephen Selvaraj, Department of Microbiology, Mahatma Gandhi Medical College & Research Institute, Sri Balaji Vidyapeeth (Deemed-to-be-University), Pondicherry, India, Email:

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Introduction

In developing countries like India where the incidence of Tuberculosis (TB) is high, TB of the lymph node (Tuberculous lymphadenitis) remains the most common form infecting 30%-52% among extrapulmonary tuberculosis cases [1]. Lymph node tuberculosis occurs due to lymphatic dissemination of Mycobacterium tuberculosis. The infected lymph nodes in the later stage may break open discharging pus and ultimately the wound will not heal for several years. Lymphadenitis is very common in childhood and commonly reported from females when compared to the male. But today lymphadenitis has been reported from people between the age group of 20 years to 40 years [2]. Various conditions like viral or bacterial adenitis, fungal disease, toxoplasmosis, cat scratch fever, carcinoma and other conditions present the same cytology or histopathology that mimics granulomatous lymphadenopathy. These require the differential diagnosis to distinguish them from Tuberculous lymphadenitis. Presence of acid fast bacilli (AFB) in smear and isolation of M. tuberculosis from lymphadenitis pus is the standard diagnostic method [3]. Due to limitations with conventional diagnostic techniques and extensive differential diagnosis, conditions like Tuberculous lymphadenitis need a diagnostic tool which is highly sensitive and specific. Smear microscopy is less sensitive and in developing countries like India, Lowenstein-Jensen (LJ) culture remains the gold standard, although it is time consuming. However automated liquid culture systems like Mycobacterium Growth Indicator Tube (MGIT 960) have been reported to have better sensitivity with short turnaround time compared to solid culture. Spacer oligonucleotide typing (Spoligotyping) is a polymerase chain reaction (PCR)-based genotyping method which detects and differentiates strains of Mycobacterium tuberculosis complex based on DNA polymorphism present at the "Direct Repeat" (DR) region. The DR region is made up of one or more IS6110 elements and consists of direct repeat sequences which are interspersed by non-repetitive DNA spacers. Presence or absences of DNA spacers in the DR region helps to characterize the diverse strains and to understand the epidemiology. So far, studies on M. tuberculosis strains causing Tuberculous lymphadenitis are not available. Hence in this study, we aimed to isolate M. tuberculosis from lymphadenitis sample employing LJ and MGIT and to study the genotypic diversity by spoliotyping.

Materials and Methods

This work has been carried out in a tertiary care super specialty teaching hospital at Pondicherry. Written informed consent was obtained from patients and the work was approved by Institutional Human Ethical Committee. Patients with pulmonary tuberculosis, sputum positive cases and those treated with Antituberculous treatment drugs were excluded from the study. Forty nine single specimens collected from patients who presented with enlarged superficial lymph nodes for more than 1 month duration with or without other constitutional symptoms like fever and weight loss were included in this study. All 49 samples were processed for smear microscopy and LJ culture. However, only 45 samples were processed in MGIT due to insufficient quantity. With sterile precautions, specimens were processed for direct Ziehl-Neelsen (ZN) staining without decontamination. For culture, the specimens were digested and decontaminated by NaOH-NALC (Sodium hydroxide-N-acetyl-L-cysteine) procedure before inoculation. The inoculated LJ and MGIT tubes were monitored for eight and six weeks respectively. Growth of M. tuberculosis complex (MTBC) recovered in LJ and MGIT was identified and confirmed following standard protocols [4]. Drug susceptibility testing was carried out for five first line drugs (Streptomycin, Isoniazid, Rifampin, Ethambutol and Pyrazinamide) in MGIT following the recommended procedure of the manufacturer. Reference strain of M. tuberculosis H37Rv was used as control. The different strains of M. tuberculosis isolated from lymphadenitis cases were spoligotyped at National Institute of Research in Tuberculosis, Chennai following the procedure of Kamerbeek et al. [5].

Results

Among the 49 samples included 16 samples were positive by culture. Out of these 16 patients 13 (81.3%) were male and 3 (18.7%) were female. The median age of the patient was 26 years (range 1 year to 68 years). ZN microscopy was positive for AFB in 28.6% (14/49) cases, LJ positivity among 20.4% (10/49), and MGIT positivity in 26.7% (12/45) cases. Presence of AFB growth on LJ and/or MGIT was considered as culture positive. Culture grew M. tuberculosis complex (15 strains of M. tuberculosis and one strain of M. bovis BCG) in 32.7% (16/49) of the samples processed. All the isolates which have grown on LJ were positive by smear. But four isolates which were positive by smear has not grown in LJ, but isolated using MGIT. In our study two smear and LJ culture negative specimens has grown exclusively in MGIT. In this study six isolates which has not grown in LJ has been isolated using MGIT. But on the contrary two smear and LJ positive isolates has not grown in MGIT.

The mean turnaround time for LJ culture positivity from lymphadenitis pus was 31.8 days and the median for BACTEC MGIT 960 TB culture was 14.5 days. Growth on LJ and in MGIT tubes by smear positive and negative cases are presented in Table 1. All sixteen isolates were identified as MTBC by using SD Bioline immunochromatographic rapid kit for production of MPT64Ag. All isolates were sensitive to Para nitro benzoic acid (PNB) susceptibility assay in MGIT960. Conventional tests like Acid fastness, Cord factor, Niacin production and Nitrate reduction carried out identified 15 isolates as Mycobacterium tuberculosis and one isolate as Mycobacterium bovis. Spoligotyping characterized M. bovis as a BCG vaccine strain. Spoligotyping technique, besides serving as an epidemiological tool for genotyping study, also serves as an identification tool in the differentiation of species within the M. tuberculosis Complex, which further confirmed the identification by conventional biochemical tests.

Table 1: Different laboratory parameters of M. tuberculosis and M. bovis BCG strains isolated from lymphadenitis

Among 16 isolates subjected to anti-mycobacterial drug susceptibility testing 8 (50%) were sensitive to all first line drugs tested. Two isolates showed dual resistance to streptomycin and Isoniazid, whereas two isolates showed mono-resistance to Isoniazid. Isoniazid resistance with or without combination with other drugs was seen in four isolates. One strain of M. bovis BCG showed natural resistance to pyrazinamide. Mono-resistance to Rifampicin, Ethambutol and Pyrazinamide each was seen in one isolate respectively. The different spoligotypes from lymphadenitis pus is given in Table 1 [6].

Discussion

Tuberculous lymphadenitis is caused mainly by M. tuberculosis and non tuberculous mycobacteria in developing countries like India and other developed countries. In our study we have isolated 15 strains of M. tuberculosis and one M. bovis BCG vaccine strain, which was isolated from a case of lymphadenitis. This child was vaccinated with BCG. BCG vaccines are given as a protective measure against severe forms of EPTB such as TB meningitis and hence infants are vaccinated with single dose of Bacille Calmette-Guérin (BCG) vaccine after birth according to childhood immunization programmes in high TB burden countries. Conventional identification techniques are capable of recovering the organism as only M. bovis. But spoligotyping identified it as a vaccine strain of M. bovis (BOVIS1_BCG). Hence spoligotyping differentiated the animal strain M. bovis and the vaccine strain, and hence epidemiological tools like spoligotyping are useful for the proper characterization of strains.

In India, Ziehl-Neelsen microscopy is widely used for diagnostic purpose. The sensitivity of this microscopy varies depending upon the source of sample. Unlike sputum specimen, extrapulmonary specimens like pus, and body fluids like Cerebrospinal fluid, pleural fluid, synovial fluid etc., are paucibacillary. Ghariani et al. [7] reported only scanty bacilli from 75.6% of the smear positive extrapulmonary specimens. Pus collected from lymph nodes yield increased positivity by smear microscopy compared to body fluids. Handa et al. [2] reported increase in AFB positivity smears prepared from purulent aspirates. AFB smear positivity in lymphadenitis sample ranges from 15% to 47% depending on the presence of necrosis [1,8]. Mittal et al. [9] reported the sensitivity and specificity of AFB smear microscopy from lymph node aspirates were 76.47% and 100% respectively. Studies have reported that concentration techniques increase the sensitivity. Patwardhan et al. [10] reported 30.7% from lymphadenitis samples while using direct microscopy and 41.5% using concentrated ZN technique. Mirza et al. [11] reported 42% smear positivity from cytology positive lymphadenitis patients. Reddy et al. [12] reported 12% smear positivity from tuberculous lymphadenitis and 0% from granulomatous lymphadenitis. Another study by Sharma et al. [13] reported 30% and 0% positivity from confirmed and suspected case of lymphadenitis respectively. The present research recorded 28.6% using smear microscopy.

Patwardhan et al. [10] reported LJ positivity from lymphadenitis samples ranging from 14% to 57%. Presence of necrosis increases the culture positivity ranging from 35% to 65% [1]. The highest isolation rate of 50.7%, followed by 45% and 32.6% positivity using LJ was reported by few authors [10,12,14]. In the present study, 20.4% LJ positivity was reported among the 28.6% AFB smear positive cases. All the isolates which have grown on LJ were smear positive and nearly 28.6% (4/14) of the smear positive cases failed to grow on LJ. Among the total culture positive samples only 62.5% isolates has grown in LJ and others were isolated using MGIT system.

Gautam et al. [15] reported 18% positivity using MGIT from lymphadenitis specimens. Four smear positive isolates which has not grown in LJ was isolated using only MGIT system. Also two smear and LJ culture negative specimens have grown exclusively in MGIT. Thus, six isolates which has not grown in LJ has been isolated using MGIT. On the contrary, two smear positive and LJ positive isolates has not grown in MGIT and has grown only in LJ which would have been missed if LJ is not included.

The mean TAT (Turn around Time) on LJ was 26 days and 30 days and in MGIT it was 13 days and 20 days in case of smear positive and negative specimens respectively [10]. In our study the mean TAT for LJ and was reported as 31.8 days and the median TAT for MGIT was 14.5 days respectively. So MGIT is far superior to LJ in terms of higher yield and short turnaround time. Hence from purulent pus materials, the positivity of AFB in ZN smear and liquid culture is quite high. Thus automated liquid culture system can be very helpful for optimal isolation of Mycobacterium from pus sample unlike body fluids.

Drug resistance is very common in M. tuberculosis isolated from pulmonary specimens than extrapulmonary. DST for M. tuberculosis isolates from TB lymphadenitis show different levels of resistance. Thus Zewdie et al. [16], from Ethiopia observed that 8.3% of their isolates from TB lymphadenitis were MDR TB and 1.7% showed mono-resistance to any one of the four first line drugs. According to an earlier report by Biadglegne et al. [17], 8.1% of resistance was recorded comprising 1.4% MDR-TB and 6.7% showing mono-resistance. Recently Sharma et al. [18], reported that among their 63 isolates of MTB from TB lymphadenitis, six were MDR TB and two were INH mono-resistant, with a percentage resistance of 8.7%. A supranational reference laboratory from India by Dusthackeer et al. [6], has reported a very high resistance of 57.0% based on a large number of isolates from Lymph nodes. They recorded MDR TB of 19.0% and 38.0% isolates showing mono-resistance. This report which is made from a Tuberculosis Research Laboratory at Chennai, India, which is about 160 Km away from Pondicherry, is very much similar to our present study.

Salvador et al. [19] has discussed about the M. tuberculosis strains and its association with specific sites. Sankar et al. [20] from North India reported the association of Manu strains with extrapulmonary tuberculosis. In our study majority, around 47% of M. tuberculosis strains were profiles described as orphans, which may indicate their association with lymphadenitis cases. No specific spoligotypes were seen in specific age or sex groups.

Conclusion

In TB endemic countries, TB lymphadenitis remains the most common extrapulmonary manifestation of tuberculosis. High index of clinical suspicion and diagnostic tests with high sensitivity and specificity helps in the definitive diagnosis of lymphadenitis. Combination of conventional and automated tests increases the percentage positivity. Automated systems like MGIT960 can differentiate the live and dead bacilli, helps to detect drug resistance and serves as a right tool for proper management of patient by avoiding empirical treatment. In our study majority, around 47% of M. tuberculosis strains were profiles described as orphans, which may indicate their association with lymphadenitis cases.

Acknowledgments

The authors express their sincere thanks to Dr. S. Sivakumar and Dr. S. Narayanan, Department of Immunology, National Institute for Research in Tuberculosis (NIRT-ICMR), Chennai, Tamil Nadu, India, for their immense help to carry out spoligotyping work. Authors also thank the Chairman, Vice-Chancellor, Dean- Academic, Dean of Faculty and Dean-Research of Mahatma Gandhi Medical College and Research Institute, Sri Balaji Vidyapeeth (Deemed-to-be-University), Pondicherry, for the facilities provided.

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this manuscript.

References

1. Singh KK, Muralidhar M, Kumar A, et al. Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy. J Clin Pathol 2000; 53:355-61.
2. Handa U, Mundi I, Mohan S. Nodal tuberculosis revisited: A review. J Infect Dev Ctries 2012; 6:6-12.
3. Kandola P, Meena LS. Extra-pulmonary tuberculosis: Overview, manifestations, diagnostic and treatment techniques. Adv Mater Rev 2014; 1:13-9.
4. Kandhakumari G, Stephen S. Extra pulmonary tuberculosis: Rapid identification of Mycobacterium tuberculosis grown in Mycobacterium growth indicator tube 960 and Lowenstein-Jensen media, employing Standard diagnostics Bioline Mycobacterium tuberculosis protein 64 antigen detection kit. Indian J Med Microbiol 2015; 33:S122-125.
5. Kamerbeek J, Schouls L, Kolk A, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997; 35:907-14.
6. Dusthackeer A, Sekar G, Chidambaram S, et al. Drug resistance among extrapulmonary TB patients: Six years experience from a supranational reference laboratory. Indian J Med Res 2015; 142:568.
7. Ghariani A, Jaouadi T, Smaoui S, et al. Diagnosis of lymph node tuberculosis using the GeneXpert MTB/RIF in Tunisia. Int J Mycobacteriol 2015; 4:270-5.
8. Kandhakumari G, Stephen S. Evaluation of a new rapid kit, BD MGIT TBc identifi cation test for confirmation of Mycobacterium tuberculosis complex. Indian J Pathol Microbiol 2017; 60:243-6.
9. Mittal P, Handa U, Mohan H, et al. Comparative evaluation of fine needle aspiration cytology, culture, and PCR in diagnosis of tuberculous lymphadenitis. Diagn Cytopathol 2011; 39:822-6.
10. Patwardhan SA, Bhargava P, Bhide VM, et al. A study of tubercular lymphadenitis: A comparison of various laboratory diagnostic modalities with a special reference to tubercular polymerase chain reaction. Indian J Med Microbiol 2011; 29:389-94.
11. Mirza S, Restrepo BI, McCormick JB, et al. Diagnosis of tuberculosis lymphadenitis using a polymerase chain reaction on peripheral blood mononuclear cells. Am J Trop Med Hyg 2003; 69:461-5.
12. Reddy VK, Aparna S, Prasad CE, et al. Mycobacterial culture of fine needle aspirate-A useful tool in diagnosing tuberculous lymphadenitis. Indian J Med Microbiol 2008; 26:259.
13. Sharma K, Gupta N, Sharma A, et al. Multiplex polymerase chain reaction using insertion sequence 6110 (IS6110) and mycobacterial protein fraction from BCG of Rm 0.64 in electrophoresis target genes for diagnosis of tuberculous lymphadenitis. Indian J Med Microbiol 2013; 31:24-8.
14. Verma JS, Dhavan I, Nair D, et al. Rapid culture diagnosis of tuberculous lymphadenitis from a tertiary care centre in an endemic nation: Potential and pitfalls. Indian J Med Microbiol 2012; 30:342-5.
15. Gautam H, Agrawal SK, Verma SK, et al. Cervical tuberculous lymphadenitis: Clinical profile and diagnostic modalities. Int J Mycobacteriol 2018; 7:212-6
16. Zewdie O, Mihret A, Abebe T, et al. Genotyping and molecular detection of multidrug-resistant Mycobacterium tuberculosis among tuberculosis lymphadenitis cases in Addis Ababa, Ethiopia. New Microbes New Infect 2018; 21:36-41.
17. Biadglegne F, Tessema B, Sack U, et al. Drug resistance of Mycobacterium tuberculosis isolates from tuberculosis lymphadenitis patients in Ethiopia. Indian J Med Res 2014;140:116-22.
18. Sharma S, Hanif M, Chopra KK, et al. Detection of multidrug resistance and extensively drug resistance among smear-negative extrapulmonary tuberculosis cases in a reference laboratory. Biomed Biotechnol Res J 2018; 2:132-5.
19. Salvador F, Los-Arcos I, Sánchez-Montalvá A, et al. Epidemiology and diagnosis of tuberculous lymphadenitis in a tuberculosis low-burden country. Medicine (Baltimore) 2015; 94:e509.
20. Sankar MM, Singh J, Diana SC, et al. Molecular characterization of Mycobacterium tuberculosis isolates from North Indian patients with extrapulmonary tuberculosis. Tuberculosis (Edinb) 2013; 93:75-83.