Abstract
Background
Very few studies report resistance pattern exclusively in musculoskeletal tuberculosis (MSK-TB).
Methods
This study of 100 pus samples from patients of MSK-TB with active disease in whom Mycobacterium tuberculosis (MTB) was detected by cartridge-based nucleic acid amplification test (CBNAAT), revealed the pattern of resistance among newly diagnosed and previously treated cases. Liquid culture and drug susceptibility testing (DST) using MGIT 960 was done for 11 anti-tubercular drugs.
Results
Among these 100 cases; 22% were AFB positive; MGIT 960 detected MTB in 58.33% (35/60) new cases and 30.0% (12/40) previously treated cases. Five new and 10 previously treated cases had drug resistance and 12 were detected rifampicin resistance (Rif-R) by CBNAAT. Among new cases MGIT-DST detected mono-INH resistant in 2.86% (1/35), mono-STR resistant in 2.86% (1/35), MDR-TB in 5.7% (2/35) and pre-XDR in 2.9%(1/35).Among previously treated cases Rif-R was found in 10% (4/40) where MTB was not detected by MGIT and MGIT-DST detected mono-INH resistant in 8.33% (1/12); MDR-TB in 8.33% (1/12) and pre-XDR in 33.3%. There were no cases of XDR-TB.
Conclusion
High disease burden of various type drug resistance were seen more commonly in previously treated cases and was not uncommon in new cases of MSK-TB. Both CBNAAT and DST are essential for detecting resistance pattern in MSK-TB.
Keywords: EPTB, MSK-TB, Pre-XDR, Drug-resistant tuberculosis, GeneXpert MTB/RIF
Introduction
World Health Organization (WHO) reported10.0 million (range 9.9–11.1 million) people diagnosed with tuberculosis (TB) worldwide in the year 2018, with India sharing 27.0% of the total burden. Among these new TB cases, 15% were reported to be extra-pulmonary TB (EPTB) [1]. Approximately 10–11% of extra pulmonary TB involves the musculoskeletal system [2].
WHO reported 3.4% of new and 18% of previously treated cases had multidrug-resistant TB or MDR-TB defined as resistance to at least isoniazid [INH] and rifampicin [RIF] and 9.5% of these cases eventually had extensive drug-resistant TB or XDR-TB defined as MDR-TB plus resistance to any fluoroquinolone [FQ] and at least one of the three second-line anti-tuberculosis injectable drugs (capreomycin, kanamycin or amikacin) among pulmonary TB cases [1]. Pre-XDR-TB is defined as TB with resistance to isoniazid and rifampicin and either a FQ or a second line injectable drug but not both [3].
The aim of the present study was to profile the drug resistance pattern in newly diagnosed cases and previously treated musculoskeletal tuberculosis (MSK-TB) cases by genotypic (CBNAAT) as well as phenotypic methods (MGIT-DST).
Methods
This prospective study was done in cases of MSK-TB cases who reported for the advanced investigations or treatment at Lok Nayak Hospital, a tertiary care teaching hospital in New Delhi between October 2017 and April 2019. The study was conducted after a written informed consent of all patients and approval of the institutional ethics committee.
The sample size was based on one of the very few exclusive studies of musculoskeletal TB reported by Chen et al. [4] of 113 positive culture cases of bone and joint TB among 206 patients where the samples were collected from bone, tissue and pus during skeletal operations. Our study included all new cases of musculoskeletal tuberculosis as well as those on antitubercular therapy (ATT) of any duration, where a sample could be taken from a cold abscess or joint fluid. Only those samples which detected MTB by CBNAAT were finally included in the study. All patients of MSK TB where the samples were inaccessible for aspiration and did not need a therapeutic surgical intervention were excluded from the study. Though our study had a smaller sample size of 100 cases, the strength of our study was the case selection being limited specifically to patients of MSK-TB having active disease where MTB could be detected by CBNAAT (GeneXpert) at the accredited laboratory of the national tuberculosis elimination program (NTEP) of India at the TB chest clinic, Lok Nayak Hospital, New Delhi.
In all these 100 samples where MTB was detected by CBNAAT, auramine staining for acid fast bacillus under the polarizing microscope; liquid culture and drug susceptibility testing (DST) using MGIT 960 for 11 anti-tubercular drugs namely isoniazid (INH), rifampicin (RIF), streptomycin (STR), ethambutol (EMB), kanamycin (KAM), capreomycin (CAP), amikacin (AMK), PAS, ethionamide (ETH), ofloxacin (OFL), levofloxacin (LVX) were done at the New Delhi Tuberculosis Centre, having laboratories accredited by the national accreditation board for testing and calibration laboratories (NABL) and NTEP Intermediate reference laboratory.
Results
The samples included pus from cold abscess (71/100), joint aspiration (14/100), surgically drained sites (11/100) and discharging sinuses (4/100). Samples of cold abscess were all from spinal TB except one case of sternal TB, while the joint commonly affected were knee, ankle and wrist (Table 1). Majority of the cases were of spinal tuberculosis (77/100) which was also the commonest site of drug resistance.
Table 1.
Sites of musculoskeletal tuberculosis
| Site | Total cases | Resistant cases |
|---|---|---|
| Spine | 77 | 12 |
| Knee | 4 | 1 |
| Ankle | 4 | 1 |
| Wrist | 4 | 0 |
| Hip | 3 | 0 |
| Shoulder | 2 | 0 |
| Elbow | 2 | 1 |
| Sternum | 2 | 0 |
| Ribs | 2 | 0 |
| Total | 100 | 15 |
Out of the 100 samples, 60 were from newly diagnosed cases who had not received antitubercular therapy and 40 were from previously treated cases (who received 1 month or more of anti-TB drugs in the past). Acid fast bacillus was detected on fluorescent microscopy in only 22% cases of which 28.33% were in new cases (17/60) and 12.5% in previously treated cases (5/40). Our results were classified as Rif-R by CBNAAT and on the basis of MGIT-DST either as mono drug resistance or MDR (INH + Rifampicin resistance), pre-XDR or XDR.
There were 15 cases of drug-resistant TB; 5 in new and 10 in previously treated cases. CBNAAT detected rifampicin resistance (Rif-R) in 12% (12/100), three in new and nine in previously treated cases referred to as genotypic resistance. Phenotypic resistance to various anti-tubercular drugs was detected by DST only in the isolates in which MTB could be detected by MGIT (Table 2). There were two samples from newly diagnosed cases and one from previously treated cases which were contaminated on culture and discarded for DST. DST could be done in 35/60 new cases and 12/40 previously treated cases.
Table 2.
Drug resistance profile
| Resistance pattern | Drug resistance combinations | New cases | Previously treated | No. of patients |
|---|---|---|---|---|
| Mono-drug resistant | INH | 1 | 1 | 2 |
| STR | 1 | 0 | 1 | |
| Multidrug-resistant MDR | INH, RIF, | 1 | 1 | 2 |
| INH, RIF, EMB | 1 | 0 | 1 | |
| Pre-XDR | INH, RIF, STR, EMB, OFL, LVX | 0 | 3 | 3 |
| INH, RIF, STR, OFL, LVX | 1 | 1 | 2 | |
| Rif-R by CBNAAT but MTB undetected by MGIT | 0 | 4 | 4 | |
| Total | 5 | 10 | 15 | |
INH: Isoniazid: RIF: Rifampicin: STR: Streptomycin; EMB: Ethambutol; OFL: Ofloxacin; LVX: Levofloxacin
Among the 60 new cases, genotypic resistance (Rif-R) by CBNAAT was detected in 3 patients. The phenotypic drug resistance could only be ascertained from 35/60 samples (58.33%) which detected MTB by MGIT-960 after ignoring the 85.7% (30/35) isolates which were fully susceptible to all tested drugs. The remaining 5/35 (14.3%) isolates showed variable drug resistance pattern including INH-mono resistant in 1/35 (2.8%); STR-mono resistance in 1/35 (2.8%); MDR in 2/35 (5.7%) and pre-XDR in 1/35 (2.8%).
Out of the 40 previously treated cases, genotypic resistance (Rif-R) by CBNAAT was detected in 9 patients. The phenotypic resistance by DST in these nine patients could only be done in five samples which flagged positive as MTB detected by MGIT and the DST revealed one case of MDR and four cases of Pre-XDR. Thus four samples of RIF-R did not detect MTB by MGIT and could not be subject to DST and these patients were clinically treated as MDR. Among the remaining 31/40 samples which were not Rif-R by CBNAAT, seven samples detected MTB by MGIT-DST of which 1 was mono-INH resistance. Since MTB was detected by MGIT-960 in 12/40 samples of the previously treated cases INH mono resistance was calculated as 1/12 (8.33%). Similar basis of calculation revealed MDR-TB in 1/12 (8.33%) and pre-XDR in 4/12 (33.3%).
Neither Rifampicin mono-resistance nor XDR-TB were detected by DST in any sample (Fig. 1).
Fig. 1.
Systematic description of drug resistance among patients
Among the previously treated cases, 13 patients had taken anti-tubercular therapy (ATT) in the past with a median duration of 18 months (standard deviation 10.9) and 8/13 (61.53%) were found to be drug resistant. Among the remaining 27/40 patients who had taken an inadequate course of ATT, with a mean duration of 4.07 months (standard deviation-1.69) 2/27 (7.4%) were detected as pre-XDR.
The mean duration of symptoms among the drug sensitive cases (85/100) was 7.79 months ± 5.25 ranging from 1–24 months while among the resistant cases the mean was 6.30 ± 3.55 months, ranging from 15 days to 12 months.
Discussion
In our study knee, ankle, wrist cases were more than hip TB (Table 1). Although over the past decades even standard books have mentioned hip as the second commonest site of MSK-TB after spine [5], however recently some studies have reported that knee and other sites are more commonly involved than hip [6, 7].
Out of the 100 samples of MSK-TB in our study only 22%, detected MTB on fluorescent microscopy, more among new cases (28.33%) than the previously treated cases (12.5%). Azger et al. found 33.6% AFB positivity rate among EPTB samples [8].
In our study, resistance to one or more anti tubercular drugs was observed in 8.33% (5/60) of new cases and 25% (10/40) previously treated patients. Arockiaraj et al. reported resistance to at least one drug in 10.2%, out of which there were 3.6% MDR; 0.4% XDR; 4.6% mono-drug resistance and 1.4% poly-drug resistance detected by culture in 898 MSK-TB cases irrespective of their duration of treatment [9].
A study among spinal tuberculosis patients by Mohan et al. [10] found 16.6% drug resistance to at least one anti tubercular drug. Among these, 78.3% cases were multidrug resistant and 2.7% were XDR. Prevalence of resistance to isoniazid, rifampicin and streptomycin was 15.0%, 13.5% and 11.2%, respectively.
An important observation in our study was the varying pattern of drug resistance to one or more drugs in both new as well as previously treated cases. Some of the resistance pattern could not detected by one method alone, either CBNAAT or DST. Hence it is important for the orthopaedic surgeons to be aware that all attempts should be made to collect maximum quantity of sample and subject it to both CBNAAT and DST by MGIT.
The strength of this study was the selection of CBNAAT proven cases of MSK-TB and not just clinic-radiologically suspected cases of MSK-TB. The limitation of this study, was exclusion of some cases of spinal TB in whom samples could not be taken since there was no therapeutic indication for a surgical decompression, we are not aware of the resistance pattern of these cases. Another observation in our study, was a lower than expected detection rate of MTB by MGIT (47/100) making it difficult to assess the exact drug resistance pattern. This skewed observation in our accredited laboratories could be attributed to the fact that approximately 105–106 colony forming units (CFU) of MTB are required per ml of medium to effectively produce results [11]. Thus, the 4 previously treated cases in which Rif-R was detected on CBNAAT, but not detected by MGIT-DST created an uncertainty in the final identification of mono-drug, MDR, pre-XDR and XDR-TB pattern in the 100 cases of our study.
Conclusion
Varying pattern of drug resistance was seen more commonly in previously treated cases but was not uncommon in new cases of MSK-TB. Though MGIT culture and DST are considered to be the gold standard for detecting drug resistance in TB but less than half (47%) of the cases in our study could be detected by this method, hence Rif-R by CBNAAT can be used as a surrogate marker for detection of MDR-TB in cases these samples do not detect MTB by MGIT. Detection of mono drug resistance pattern which are not Rif-R as well as multi drug resistance, emphasizes the need for both CBNAAT and MGIT-DST to complement each other to detect the entire drug resistance profile for appropriate treatment of MSK-TB.
Acknowledgements
The authors appreciate the support of technical staff of Orthopaedics and TB Chest Clinic department of Lok Nayak Hospital and also of the New Delhi Tuberculosis Centre, New Delhi for this study.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
Ethical Standard
This article does not contain any studies with human or animal subjects performed by the any of the authors.
Informed Consent
For this type of study informed consent is not required.
Footnotes
Publisher's Note
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