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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 2  |  Issue : 2  |  Page : 132-135

Detection of multidrug resistance and extensively drug resistance among smear-negative extrapulmonary tuberculosis cases in a reference laboratory


1 Department of Microbiology, Maharaja Vinayak Global University, Jaipur, Rajasthan, India
2 New Delhi Tuberculosis Centre, New Delhi, India

Date of Web Publication14-Jun-2018

Correspondence Address:
Dr. M Hanif
New Delhi Tuberculosis Centre, Jawaharlal Nehru Marg, Delhi Gate, New Delhi - 110 002
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_48_18

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  Abstract 


Background: Extrapulmonary tuberculosis (EPTB) constitutes about 15%–20% of all cases of TB. Patients with EPTB are more likely to have negative sputum smear results. It is often hard to diagnose because of difficulty of sampling and paucibacillary nature of samples. Methods: Smear negative extra pulmonary (EP) specimens were included in the study. An attempt was made to recover M. tuberculosis (MTB) from such EP specimens using rapid liquid culture (MGIT 960). Molecular Line probe assay (LPA) was used to determine the resistant for first line drugs, and second line drug resistant was determined using liquid culture. Results: Culture positivity was found in 21.3% (133/623) of specimens; of these 95.48% (127/133) were found to be MTB and 4.51% (6/133) of specimens were found to be non tubercular mycobacteria. Among MTB detected 18.9% of specimens were multi drug resistant, 3.90% were Rifampicin mono-resistant and 13.30% were Isoniazid resistant. Second line DST (N=29) was performed for Kanamycin and Ofloxacin; of which 3.4 % was found to be resistant to both drugs, 3.4 % was found to be resistant to Ofloxacin and 93.1% were sensitive to both drugs. Conclusion: Large percentage of drug resistance was observed in the study. Early recovery of MTB and determination of its drug resistance helped in early initiation of treatment and controlled further transmission of drug-resistant TB in the population.

Keywords: Culture positive, extrapulmonary tuberculosis, multidrug resistance, smear negative, extensively drug resistance


How to cite this article:
Sharma S, Hanif M, Chopra KK, Sharma M, Dwivedi KK, Sidiq Z, Vashistha H, Ahmed V, Dubey M. 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

How to cite this URL:
Sharma S, Hanif M, Chopra KK, Sharma M, Dwivedi KK, Sidiq Z, Vashistha H, Ahmed V, Dubey M. Detection of multidrug resistance and extensively drug resistance among smear-negative extrapulmonary tuberculosis cases in a reference laboratory. Biomed Biotechnol Res J [serial online] 2018 [cited 2019 May 19];2:132-5. Available from: http://www.bmbtrj.org/text.asp?2018/2/2/132/234460




  Introduction Top


Tuberculosis (TB) is a disease of global importance. As per the recent World Health Organization estimates, one-third of the world population is estimated to be infected with Mycobacterium tuberculosis (MTB), and 10.6 million new cases of TB arise each year. It has been estimated that globally, there are 8.6 million incident cases of TB, of which 80% are in 22 countries, with India being ranked as one of the highest-burden countries.[1]

Extra-pulmonary tuberculosis (EPTB) constitutes about 15 to 20 % of all cases of Tuberculosis (TB). Patients with EPTB are more likely to have negative sputum smear results, and many EPTB cases do not have direct lung involvement. EPTB diagnosis is much more lacking in rural health facilities that are used by about 70% of the population in developing countries.[2]

EPTB is often hard to diagnose with conventional methods, due to the longer time required, and also the difficulty of sampling and the paucibacillary nature of samples.[3] EPTB in patient with active MTBinfection is often initially misdiagnosed as cancer.[4] Hence, there is a need for rapid, sensitive, and accurate detection system. In mycobacteriology, several new rapid technologies for the growth and detection of mycobacteria have been developed to reduce the time to detect and identify mycobacteria in clinical specimens; among these, the Mycobacterium Growth Indicator Tube (MGIT 960 system), Becton Dickinson Microbiology System, Sparks, NV, USA, is one of the better culture systems to provide early and reliable results.

Numbers of studies have been published on evaluating efficiency of MGIT 960 system on pulmonary samples [5],[6],[7],[8] but there a few reports on extrapulmonary samples. The aim of the present study was to determine the recovery of M. tuberculosis in smear negative extrapulmonary samples through liquid culture using MGIT 960 and also to determine their susceptibility against first line drugs using Line Probe assay (LPA) and second line drugs using MGIT 960 respectively from MGIT positive culture.


  Methods Top


Study population

A total of 623 extrapulmonary specimens received during March 2015 to April 2016 at Intermediate Reference Laboratory, New Delhi Tuberculosis Centre were included in the study have been shown in the [Figure 1]. Of these specimens, Cerebrospinal fluid (CSF) constitute large number (35.7%) followed by pus (29.5%) and lymph node aspirate (10.1%). abscess (3%), and knee aspirates (6%).
Figure 1: Diagrammatic representation of types of extrapulmonary specimens included in the study

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Acid-fast bacilli staining and microscopy

Smears were prepared directly from all specimens, stained with Auramine stain,[9] and examined under fluorescent microscope as per the Revised National Tuberculosis Control Programme (RNTCP) guideline.

Mycobacterium Growth Indicator Tube liquid culture

All specimens were decontaminated by N-acetyl-L-cysteine–sodium hydroxide (NALC-NaOH) method.[10] Briefly, equal amount of NALC-NaOH mixture was added in the sample and allowed to hold for 15 min at room temperature. Sediment was then neutralized with phosphate buffer (pH 6.8) and centrifuged at 3000 rcf for 15 min. After centrifugation, supernatant was discarded and pellet was resuspended in 1.5 ml of sterile phosphate buffer. 0.5 ml of sediment was inoculated in MGIT 960 7 ml BBL tube (Becton Dickinson).

Identification ofMycobacterium tuberculosis complex

Positive culture were identified by: (a) examining smears for microscopic cords of acid-fast bacilli and (b) an immunochromatographic test that use mouse monoclonal anti–MPT64 from SD Bioline MPT64, as per the manufacturer instructions.[11] Briefly; 100 μl of the growth suspension was added in the cassette and incubated for 15 minutes at room temperature. The presence of only control band (pink color) in the absence of test band was considered as negative for MPT64 antigen (NTM). Presence of both control and test band indicated a positive result and interpreted as presence of MPT64 antigen (MTB).

Line probe assay (genotypic drug susceptibility testing)

This assay was performed using MTBDRplus version 2 test kit for the detection of drug sensitivity by two drugs Rif and INH as per the manufacturer's instruction.[12] This test comprises three main steps: (a) DNA extraction, (b) preparation of master mix and addition of DNA in to the master mix and its amplification, and (c) development of blot by reverse hybridization technique. All three steps were carried out in separate rooms with restricted access and unidirectional work flow.

DNA extraction

One thousand microliters of positive culture from each MGIT tube was centrifuged at 10000 g for 15 min, the supernatant discarded, and the pellet was resuspended in 100 μl of lysis buffer. This solution was incubated at 95°C in a hot air oven for 5 min. After that, 100 μl of neutralizing buffer was added to each sample followed by centrifugation at 13000 g for 5 min, the supernatant was transferred in a separate 1.5 ml Eppendorf tube, and the pellet was discarded.

Master mix and amplification

Master mix was prepared by adding 45 μl amplification mix consisting of 10 μl AM-A and 35 μl AM-B provided with the kit. 5 μl DNA template was added in each tube in a separate room, and amplification was performed with final volume of 50 μl. Polymerase chain reaction was performed using a thermocycler from Applied Biosystems 3500 using the manufacturer-designed protocol.[12]

Hybridization and detection

Reverse hybridization was performed using GT Blot 48 (Hain Life Sciences, Germany). After hybridization and washing, the strips were removed, allowed to air dry, and fixed on paper.

Interpretation of results

The MTBDRplus assay strip contains 27 reaction zones; 21 of them are probes for mutations and six are control probes for verification of the test procedures. The six control probes include a conjugate control and amplification control (AC), an MTB complex-specific control (TUB), an rpoB locus AC, a katG locus AC, and an inhA locus AC. For the detection of RIF resistance, the probes cover the rpoB gene, while for the INH resistance, specific probes cover positions in katG and inhA. The absence of at least one of the wild-type bands or the presence of bands indicating a mutation in each drug resistance-related gene implies that the sample tested is resistant to the respective antibiotic. When all the wild-type probes of a gene stain positive and there is no detectable mutation within the region examined, the sample tested is susceptible to the respective antibiotic.

Phenotypic drug susceptibility testing

Using MGIT 960 system, DST for the second-line anti-TB drugs, namely kanamycin and ofloxacin, was performed on the culture isolates. Working solution of each drug was prepared at the concentration of 2.0 and 2.5, respectively, for kanamycin and ofloxacin according to the manufacturer's recommendations.[13] All liquid MGIT-positive MTB culture from 1 to 5 days was used for DST.


  Results Top


Of 623 participants from whom EP specimens were collected, both males and females were found in the equal proportion (male: 315 and females: 308). Average age of the study population was 20 years ranging from 3 to 68 years.

The culture positivity was found in (133/623) 21.3% specimens, of which (127/133) 95.48% were found to be, MTB and in 4.51% (6/133) were found to be NTM . Culture contamination was observed in 4.96% (31/623) of the specimens and the remaining 73.6% (459/623) of specimens were found to be culture negative.

Of variety of specimens tested, nearly 50% (12/24) of biopsy specimens received were found to be positive for MTB, followed by abscess 27.7% (5/18), CSF 22.4% (50/223), and lymph node aspirate 22.2% (14/63). Among all six (4.51%) NTMs detected in the study, three were found in the CSF, two in the pleural fluid, and one in pus.

Among cultures positive, 36.20% (46/127) of specimens were found to be resistant for either first-line monodrug or multidrug; of these, 18.9% (24/127) of specimens were multidrug resistances (MDRs), 3.90% (5/127) were Rif monoresistant, and 13.30% (17/127) were INH resistant. A total of 29 isolates were subjected to DST for the second-line drugs – kanamycin and ofloxacin, of which (3.4%) 1 was found to be resistant to both drugs, (3.4%) 1 was found to be resistant to ofloxacin, and (93.1%) 27 were sensitive to both drugs [Table 1].
Table 1: Details of specimens, culture positivity, and pattern of drug resistance

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  Discussion Top


In most developing countries, bacteriological confirmation of extrapulmonary TB cases relies on smear microscopy which lacks sensitivity. Isolation of mycobacteria from clinical specimens is considered to be the best method for the diagnosis of EPTB. Culture isolation followed by reliable and accurate DST is essential to determine an effective treatment regimen and to avoid further development of drug resistance.[14],[15] Under the RNTCP program, all the newer diagnostic techniques namely MGIT, LPA, and CBNAAT that are being used are adopted.

Therefore, in this study, an attempt was made to detect TB and drug resistant among cases who are smear negative but still have viable mycobacterium, which is a probable cause of spreading the infection in the community. Even though molecular methods are preferred over other phenotypic methods, Xpert MTB/RIF assay (Cepheid, CA, USA) was not available in the facility and LPA which was the only available method could not be applied to EP specimen as per the manufacturer's instruction. Therefore, MGIT system was used to isolate mycobacterium from these specimens. However, once the culture growth was obtained, they were subjected to LPA so that to reduce the turnaround time.

The frequency of extrapulmonary tuberculosis in our study was 21.2% which correlates with the study of Rishi et al. (21%).[16] However different recovery rate have also been reported earlier, Smaoui etal have reported (15.4%),[3] Bunger et al. have reported (12%).[17] Butt et al. have reported (25%)[18] in their study. Possible reason for the difference in inclusion criteria, we have included non respiratory samples only where as other studies have included both respiratory & non respiratory samples. Moreover, lower recovery rate was also observed by Hillemann et al. (8%),[19] because they have used Lowenstein Jensen media for recovery of mycobacteria.

Apart from microbiological reason, TB is also linked to lower socioeconomic strata leading to malnutrition, poor immune status, and thus acquisition of tubercular infection. Studies done in Pakistan (Butt et al., 2003),[18] Tunisia (Smaoui et al., 2015),[3] and other parts of the world where study participants belonged to poor socioeconomic status showed higher positivity rate. Higher culture positivity in our study further supported this theory.

In this study, higher culture positivity was observed in biopsy specimens 12/24 (50%) from different sites, but it cannot be considered significantly due to lesser number of denominator. Among all culture-positive specimens, abscess yields 27% isolates followed by CSF (22.4%), lymph node (22.2%), pleural fluid (21.4%), pus (16.3%), and knee aspirates (13.5%). Other studies have reported a higher rate of culture positivity in pus (44.9%).[18] Culture contamination rate observed is within the recommended range of RNTCP.

Culture yield was very good in this study considering the fact that EP specimens are paucibacillary in nature. Our laboratory is certified as IRL by the CTD, Ministry of Health and Family Welfare, and GOI. Moreover, this laboratory regularly participates in EQA program conducted by National Reference Laboratory as per the guidelines of the RNTCP.

We observed that the high level of MDR (22.8%) in our isolates and single-drug resistant was 3.1%. Previous studies have reported 12.5% MDR in EPTB in Nepal,[20] 10% in Delhi,[21] and 21.4% in Pakistan.[18] The possible reason of higher observation of drug resistant in our study can be the inclusion of new cases as well as retreatment cases and previously treated cases.


  Conclusion Top


Early recovery of MTB and determination of its drug resistance helped in early initiation of treatment and controlled further transmission of drug-resistant TB in population with EPTB.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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WHO. Updates 2017. Available from: http://www.who.int/features/factfiles/tuberculosis/en/. [Last accessed on 2017 Apr 27].  Back to cited text no. 1
    
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Purohit M, Mustafa T. Laboratory diagnosis of extra-pulmonary tuberculosis (EPTB) in resource-constrained setting: State of the art, challenges and the need. J Clin Diagn Res 2015;9:EE01-6.  Back to cited text no. 2
    
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Smaoui S, Kammoun S, Marouane C, Slim L, Messadi-Akrout F. Evaluation of the BACTEC MGIT 960 TB with solid media for recovery of mycobacteria from extrapulmonary specimens in South Tunisia. J Med Diagn Methods 2015;4:1-5.  Back to cited text no. 3
    
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Aisenberg GM, Jacobson K, Chemaly RF, Rolston KV, Raad II, Safdar A, et al. Extrapulmonary tuberculosis active infection misdiagnosed as cancer: Mycobacterium tuberculosis disease in patients at a comprehensive cancer center (2001-2005). Cancer 2005;104:2882-7.  Back to cited text no. 4
    
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Hanna BA, Ebrahimzadeh A, Elliott LB, Morgan MA, Novak SM, Rusch-Gerdes S, et al. Multicenter evaluation of the BACTEC MGIT 960 system for recovery of mycobacteria. J Clin Microbiol 1999;37:748-52.  Back to cited text no. 5
    
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Somoskövi A, Ködmön C, Lantos A, Bártfai Z, Tamási L, Füzy J, et al. Comparison of recoveries of Mycobacterium tuberculosis using the automated BACTEC MGIT 960 system, the BACTEC 460 TB system, and Löwenstein-Jensen medium. J Clin Microbiol 2000;38:2395-7.  Back to cited text no. 6
    
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Koh WJ, Ko Y, Kim CK, Park KS, Lee NY. Rapid diagnosis of tuberculosis and multidrug resistance using a MGIT 960 system. Ann Lab Med 2012;32:264-9.  Back to cited text no. 7
    
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Hasan M, Munshi SK, Banu Momi MS, Rahman F, Noor R. Evaluation of the effectiveness of BACTEC MGIT 960 for the detection of mycobacteria in Bangladesh. Int J Mycobacteriol 2013;2:214-9.  Back to cited text no. 8
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Manual for Sputum Smear Fluorescence Microscopy – Central TB Division, Ministry of Health & Family Welfare, Government of India. Available from: http://www.tbcindia.nic.in.  Back to cited text no. 9
    
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Kent PT, Kubica GP. Public Health Microbiology, a Guide for the Level III Laboratory. Atlanta, GA: Centers for Disease Control, Division of Laboratory Training and Consultation; 1985.  Back to cited text no. 10
    
11.
Gaillard T, Fabre M, Martinaud C, Vong R, Brisou P, Soler C. Assessment of the SD Bioline Ag MPT64 RapidTM and the MGITTM TBc identification tests for the diagnosis of tuberculosis. Diagn Microbiol Infect Dis 2011;70:154-6.  Back to cited text no. 11
    
12.
Genotype MTBDRplusTM, version 2.0 [product insert]. Nehren, Germany: Hain Lifescience, GmbH. Hain Life Science. Available from: https://www.ghdonline.org/uploads/MTBDRplusV2_0212_304A-02-02.pdf. [Last accessed on 2015 Mar 04].  Back to cited text no. 12
    
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Siddiqi SH. MGIT procedure manual. Bactec MGIT 960 TB system, Culture and Drug Susceptibility. Becton Dickinson, Sparks, MD. 2006.  Back to cited text no. 13
    
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World Health Organization. Guidelines for drug susceptibility testing for second-line anti-tuberculosis drugs for DOTS-Plus. WHO/CDS/TB/ 2001.288. World Health Organization, Geneva, Switzerland. 2001.  Back to cited text no. 14
    
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Rishi S, Sinha P, Malhotra B, Pal N. A comparative study for the detection of mycobacteria by BACTEC MGIT 960, Lowenstein Jensen media and direct AFB smear examination. Indian J Med Microbiol 2007;25:383-6.  Back to cited text no. 16
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Bunger R, Singh VA, Avneet, Mehta S, Pathania D. Evaluation of BACTEC micro MGIT with Lowenstein Jensen media for detection of mycobacteria in clinically suspected patients of extra pulmonary tuberculosis in a tertiary care hospital at Mullana (Ambala). J Med Microb Diagn 2013;2:1-3.  Back to cited text no. 17
    
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Butt T, Kazmi SY, Ahmad RN, Mahmood A, Karamat KA, Anwar M, et al. Frequency and antibiotic susceptibility pattern of mycobacterial isolates from extra-pulmonary tuberculosis cases. J Pak Med Assoc 2003;53:328-32.  Back to cited text no. 18
    
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Hillemann D, Richter E, Rüsch-Gerdes S. Use of the BACTEC mycobacteria growth indicator tube 960 automated system for recovery of mycobacteria from 9,558 extrapulmonary specimens, including urine samples. J Clin Microbiol 2006;44:4014-7.  Back to cited text no. 19
    
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Maurya AK, Kant S, Nag VL, Kushwaha R, Dhole TN. Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis. Indian J Med Microbiol 2012;30:182-6.  Back to cited text no. 20
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