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 ~  Abstract
 ~ Introduction
 ~ Methods
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
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BRIEF COMMUNICATION
Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 590-593
 

Improved case detection using Xpert Mycobacterium tuberculosis/rifampicin assay in skeletal tuberculosis


Department of Microbiology, Seth G. S. Medical College and KEM Hospital, Mumbai, Maharashtra, India

Date of Web Publication18-Mar-2019

Correspondence Address:
Dr. Gita Nataraj
Department of Microbiology, 7th Floor, Multi-Storey Building, Seth G. S. Medical College and KEM Hospital, Parel, Mumbai - 400 012, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_10

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 ~ Abstract 

Background: In India, musculoskeletal tuberculosis (TB) accounts for 10%–25% of extrapulmonary TB. Data on drug-resistant skeletal TB are lacking. At present, the diagnosis is based mainly on radiological techniques. Laboratory confirmation of skeletal TB is delayed as 6–8 weeks are required for culture results. Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay is a fully automated test which simultaneously detects MTB and RIF resistance within 3 h. Hence, this study was done to compare the yield of case detection using Xpert assay in comparison with culture in specimens received from clinically suspected skeletal TB cases. Methods: Retrospective analysis of microscopy, culture and Xpert assay results was carried out on specimens received in laboratory from skeletal TB cases from January 2016 to December 2017. Results: Of the 201 patients analysed, majority of the specimens were obtained from the spine (55.72%). MTB was detected in 48.68% of tissue and 24% of pus specimens. Xpert assay was detected MTB in 67 (33.33%) specimens of which 53 (47.32%) were from the spine. Culture was detected MTB in 66 (32.83%) specimens. Xpert assay was detected two specimens more than culture. One specimen was positive by only culture. RIF-resistant MTB was detected in 10 (14.92%) specimens by Xpert assay. Conclusion: The spine is the most common site involved. Tissue specimen is better for early diagnosis. High RIF resistance in skeletal TB is an alarming situation. Ability of Xpert MTB/RIF assay for rapid and simultaneous detection of MTB and RIF resistance in comparison with culture makes it a useful diagnostic tool in skeletal TB.


Keywords: Skeletal tuberculosis, culture, Xpert Mycobacterium tuberculosis/rifampicin assay


How to cite this article:
Kanade SR, Nataraj G, Mehta PR. Improved case detection using Xpert Mycobacterium tuberculosis/rifampicin assay in skeletal tuberculosis. Indian J Med Microbiol 2018;36:590-3

How to cite this URL:
Kanade SR, Nataraj G, Mehta PR. Improved case detection using Xpert Mycobacterium tuberculosis/rifampicin assay in skeletal tuberculosis. Indian J Med Microbiol [serial online] 2018 [cited 2019 Jul 18];36:590-3. Available from: http://www.ijmm.org/text.asp?2018/36/4/590/254406



 ~ Introduction Top


Tuberculosis (TB) is an ancient disease. Skeletal TB is one of the oldest diseases reported in human. Evidence of osteoarticular TB was detected in Egyptian mummies dating back to 3400 BC.[1] It is one of the common manifestations of extrapulmonary TB (EPTB). Bone and joint TB currently account for 2.2%–4.7% of all TB cases in Europe and the USA and around 10%–15% of EPTB cases.[2] In India, EPTB accounts for 10%–42% of TB cases, 10%–25% of which are musculoskeletal TB.[3],[4]

Clinical presentation of skeletal TB is not specific and includes localised pain, swelling and restriction of movement. If not diagnosed early, it may lead to joint destruction and deformity. In spinal TB, it may lead to neurological deficit resulting in long-term morbidity and disability.[5],[6] Laboratory diagnosis of skeletal TB is a challenge due to difficulty in collecting specimens from deep-seated infection and paucibacillary nature of the disease. At present, the diagnosis is based mainly on radiological findings. Conventional laboratory tests give poor yield (microscopy) or are time consuming (culture). Improving accuracy and reducing delay in the diagnosis are of paramount importance in skeletal TB to prevent permanent complications. In patients with delayed diagnosis, surgical interventions are required in as high as 98% patients.[7]

Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay ® (Xpert assay) is a cartridge-based fully automated nucleic acid amplification test, endorsed by the WHO in 2013 for initial diagnosis of EPTB.[8] Both the International Standards for TB Care and the Standards for TB Care in India, 2014, recommend Xpert assay for EPTB.[9],[10] It simultaneously detects MTB and RIF resistance within 3 h. As the limit of detection of the assay is 131 CFU/ml of the specimen, it is expected to have better accuracy in paucibacillary diseases such as skeletal TB. As treatment of skeletal TB is comparatively lengthy, early and confirmed laboratory diagnosis with knowledge of drug resistance is the need of the hour. Data on drug-resistant skeletal TB are lacking in India. Hence, this retrospective study was undertaken to compare the yield of case detection using microscopy, culture and Xpert assay in specimens received from clinically suspected cases of skeletal TB and to determine proportion of RIF resistance in skeletal TB.


 ~ Methods Top


Pus and tissue biopsy specimens collected in a sterile container during surgical debridement from suspected skeletal TB cases are routinely submitted to the laboratory for the detection of MTB. Tissue specimens are submitted in sterile normal saline. Microscopy by Ziehl–Neelsen staining was performed as per the RNTCP guidelines.[11] Culture was done on Lowenstein–Jensen medium after decontamination by NALC-NaOH method.[12] Xpert MTB/RIF assay was performed as per the guidelines issued by the WHO for processing extrapulmonary specimens.[13] This assay provided the result as 'MTB detected' or 'MTB not detected'. When MTB is detected in the sample, it provides a semi-quantitative estimation of bacterial load as very low, low, medium and high as well as RIF sensitive/resistant. All the results were entered in the laboratory register.

Ethics committee permission was obtained to conduct a retrospective analysis of the results of microscopy, culture and Xpert assay performed on specimens from suspected cases of skeletal TB over 2 years (January 2016–December 2017). Percentage positivity and incremental yield using Xpert MTB/RIF assay were determined. Chi-square test was applied to determine significance of difference in number of cases detected. P < 0.05 was considered statistically significant.


 ~ Results Top


Two hundred and ten consecutive cases of skeletal TB referred to this laboratory over 2 years were included in the study. An equal number of specimens were processed. Nine (4.28%) specimens were excluded from analysis due to inconclusive results in either culture or Xpert assay. Of the 201 cases included for analysis, 119 were male and 82 were female. About 48% of patients were >60 years of age, 49% were between 30 and 60 years and 3% were <14 years old. One hundred and twenty-five (63.19%) specimens were pus and 76 (37.81%) were biopsy from tissue. Anatomically, majority of the specimens were obtained from the spine (55.72%) followed by knee joint (21.39%). Of the 201 specimens processed, MTB was detected in 67 (33.33%) of specimens by any of the three methods. About 48.68% (37/76) of tissue biopsy and 24% (30/125) of pus specimens were positive for MTB.

Microscopy by fluorescent staining was detected acid-fast bacilli only in 7 (3.48%) specimens. All of them were pus specimens. Xpert assay was detected MTB in 67 (33.33%) specimens of which 53 (47.32%) were specimens from the spine [Table 1]. 38.81%, 55.22% and 5.97% specimen had very low, low and medium load of bacilli, respectively [Table 2]. High load of bacilli was not detected in any specimen. Ten (14.92%) cases were detected as RIF-resistant MTB by Xpert assay.
Table 1: Positivity by microscopy, culture and Xpert assay in various specimens from skeletal tuberculosis

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Table 2: Rifampicin status and load of bacilli by Xpert assay

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Culture on Lowenstein–Jensen medium was detected MTB in 66 (32.83%) specimens. When the results of Xpert assay and culture were compared, three discordant results were observed. Xpert assay was detected two specimens more than culture. One specimen was positive by culture and negative by Xpert assay.


 ~ Discussion Top


Skeletal TB is the third most common type of EPTB after pleural and lymphatic disease. Spine- and weight-bearing joints are the most vulnerable sites of infection.[14] Skeletal TB is a chronic infection, which if not diagnosed and treated properly, leads to lifetime morbidity in the form of deformity or neurological deficit.[14] The development of Xpert assay for the diagnosis of TB was a breakthrough due to its rapid turnaround time of 3 h and better accuracy.[15] Very few studies are available to determine the contribution of Xpert assay in providing a definitive diagnosis of skeletal TB.

In the present study, common site affected by TB in clinically suspected skeletal TB cases referred to the laboratory had was spine (47.32%) followed by large joints. Skeletal TB generally arises from reactivation of bacilli lodged in bone during mycobacteraemia of primary infection. Rarely TB bacilli travel from the lung to the spine along the venous plexus or by lymphatic drainage to the paraortic lymph node.[16] The predilection of the TB bacilli for the spine and large joints is attributed to the rich vascular supply of vertebra and growth plates of long bones, respectively.[15] Spinal TB usually involves more than one vertebra because the segmental artery bifurcates to supply two adjacent vertebrae.[17],[18] This causes major problem with weight-bearing capacity of the spine, and the patient may become bedridden or results in and major deformity in posture.

Very low sensitivity of 10.77% was observed by microscopy using Ziehl–Neelsen staining in comparison with culture. All the smear-positive specimens were pus indicating that microscopy does not play a major role in TB diagnosis in biopsy of bone tissue. Very low smear positivity in skeletal TB may be attributed to the paucibacillary nature of the disease. This finding is supported by the results of Xpert assay wherein the bacillary load in MTB-positive specimens was either very low (38.81%) or low (55.23%). Another reason may be difficulty in breaking a very small piece of the bone tissue obtained during biopsy.

Of the 201 specimens, 67 (33.33%) were positive by Xpert assay and 66 (32.83%) were detected by culture. Three discordant results were obtained and all three specimens were bone tissue. Xpert assay was detected two cases more than culture. These two cases would have been missed without Xpert assay. These two specimens were very small pieces of vertebral tissue obtained during surgery. Culture is considered as gold standard for the diagnosis of active TB. However, decontamination done for any type of culture is a very critical step which affects the growth of bacilli. Considering the paucibacillary nature of skeletal TB, extended decontamination even for few minutes may kill the TB bacilli giving negative results in culture. In the present study, decontamination process did not exceed more than 20 min. Negative results in culture may also be due to uneven distribution of the bacilli in a very hard bone tissue. Furthermore, bacilli have a tendency to form clumps. Tissue specimens received in sterile container with approximately 2 ml of sterile normal saline were homogenised using a sterile tissue homogeniser and processed for Xpert assay. Proper care was taken to avoid transferring any clumps of tissue which have not been properly homogenised in the cartridge. Better homogenisation and liquefaction of the sample are probably obtained using sample reagent of Xpert assay than culture. In the present study, Xpert assay may have received a larger portion of tissue as compared to culture due to the more rapid turnaround time and simultaneous detection of RIF resistance. Xpert assay had sensitivity of 97.01%, specificity of 99.25%, positive predictive value of 98.48% and negative predictive value of 98.52% in comparison with culture. It is higher than pooled sensitivity and specificity of 81% and 83%, respectively, in a meta-analysis.[19] Variable results were documented in other studies done on Xpert assay and other PCR techniques.[20],[21],[22]

One specimen was positive by culture and negative by Xpert assay. This specimen was tissue biopsy from cervical vertebra. Xpert assay result was valid as sample processing control was amplified and probe check control also worked. False-negative Xpert assay result may be attributed to the presence of lesser number of bacilli compared to limit of detection of the assay. It may also be due to uneven distribution of mycobacteria in tissue samples, presence of extensive necrosis in the tissues and presence of inhibitors which inhibited the amplification of the MTB genome without any effect on the internal control.

One of the important advantages of Xpert assay is that RIF status of the MTB detected in the specimen is available at the same time as detection. Of the 67 specimens positive for MTB, 10 (14.92%) were RIF resistant. These patients were put on treatment for RIF-resistant TB within 24 h of submitting the sample to the laboratory due to the rapidity of Xpert assay. Two specimens showed RIF indeterminate result with very low load of bacilli. On retesting, they were reported as RIF sensitive. Data on drug resistance in skeletal TB are lacking in literature.

Major problem with extrapulmonary specimens is the presence of blood in the specimen. As skeletal specimens are collected by either imaging-guided needle biopsy or surgical biopsy from the affected site, they usually contain significant amount of blood which interferes with Xpert assay giving 'ERROR' result. In the present study, 'ERROR' was obtained in 20 (9.52%) specimens of which 11 could be retested. MTB was detected in two of these specimens, both being RIF sensitive.

Of the total 201 specimens, 125 (62.19%) were pus and 76 (37.81%) were tissue specimens. Positivity was better in tissue specimens (48.68%) as compared to pus specimens (24%). Tissue specimens giving positive results were pieces of vertebral tissue from different locations. RIF resistance was detected in 20% and 10.81% of pus and biopsy specimens, respectively.


 ~ Conclusion Top


The spine is the most common site involved in skeletal TB. Tissue specimen is better than pus or fluid to obtain early diagnosis. High RIF resistance in skeletal TB, especially spine TB, is an alarming situation. Xpert MTB/RIF assay is a point of care test and its ability of rapid and simultaneous detection of MTB and RIF resistance in comparison with culture as a gold standard makes it a useful diagnostic tool in skeletal TB.

Acknowledgement

Dr. Daksha Shah from Mumbai District Tuberculosis Control Society provided the consumables required for Xpert MTB/RIF assay under Revised National Tuberculosis Control Program.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Chastel C. When the Egyptian mummies are speaking about the infections that have made them ill. Hist Sci Med 2004;38:147-55.  Back to cited text no. 1
    
2.
Pigrau-Serrallach C, Rodríguez-Pardo D. Bone and joint tuberculosis. Eur Spine J 2013;22 Suppl 4:556-66.  Back to cited text no. 2
    
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World Health Organization. Global Tuberculosis Report. World Health Organization; 2014.  Back to cited text no. 3
    
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Trecarichi EM, Di Meco E, Mazzotta V, Fantoni M. Tuberculous spondylodiscitis: Epidemiology, clinical features, treatment, and outcome. Eur Rev Med Pharmacol Sci 2012;16 Suppl 2:58-72.  Back to cited text no. 4
    
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Rajasekaran S, Shanmugasundaram TK. Prediction of the angle of gibbus deformity in tuberculosis of the spine. J Bone Joint Surg Am 1987;69:503-9.  Back to cited text no. 5
    
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Watts HG, Lifeso RM. Tuberculosis of bones and joints. J Bone Joint Surg Am 1996;78:288-98.  Back to cited text no. 6
    
7.
Turgut M. Spinal tuberculosis (Pott's disease): Its clinical presentation, surgical management, and outcome. A survey study on 694 patients. Neurosurg Rev 2001;24:8-13.  Back to cited text no. 7
    
8.
World Health Organization. Policy Update. Automated Real-Time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifampicin Resistance: Xpert MTB/RIF Assay for the Diagnosis of Pulmonary and Extrapulmonary TB in Adults and Children. WHO/HTM/TB/2013.16. Geneva, Switzerland: World Health Organization; 2013.  Back to cited text no. 8
    
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TB CARE. International Standard for Tuberculosis Care. 3rd ed. 2014.  Back to cited text no. 9
    
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World Health Organization. Standard for TB care in India. World Health Organization; 2014.  Back to cited text no. 10
    
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Central TB Division, Ministry of Health and Family Welfare. Revised National Tuberculosis Control Programme, Module for Laboratory Technicians; March, 2006.  Back to cited text no. 11
    
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Global Tuberculosis Programme. World Health Organization Laboratory Services in Tuberculosis Control Part III Culture. WHO/TB/98.258. World Health Organization; 1998.  Back to cited text no. 12
    
13.
World Health Organization. Standard Operating Procedure (SOP): Specimen Processing of CSF, Lymph Node and other Tissues for Xpert MTB/RIF. World Health Organization; 2014.  Back to cited text no. 13
    
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Leonard MK, Blumberg HM. Musculoskeletal tuberculosis. Microbiol Spectr 2017;5:371-92.  Back to cited text no. 14
    
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Oommen S, Banaji N. Laboratory diagnosis of tuberculosis: Advances in technology and drug susceptibility testing. Indian J Med Microbiol 2017;35:323-31.  Back to cited text no. 15
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Gardam M, Lim S. Mycobacterial osteomyelitis and arthritis. Infect Dis Clin North Am 2005;19:819-30.  Back to cited text no. 16
    
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Jain AK. Tuberculosis of the spine: A fresh look at an old disease. J Bone Joint Surg Br 2010;92:905-13.  Back to cited text no. 17
    
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Jain AK, Dhammi IK. Tuberculosis of the spine: A review. Clin Orthop Relat Res 2007;460:39-49.  Back to cited text no. 18
    
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Wen H, Li P, Ma H, Lv G. Diagnostic accuracy of xpert MTB/RIF assay for musculoskeletal tuberculosis: A meta-analysis. Infect Drug Resist 2017;10:299-305.  Back to cited text no. 19
    
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Held M, Laubscher M, Zar HJ, Dunn RN. GeneXpert polymerase chain reaction for spinal tuberculosis: An accurate and rapid diagnostic test. Bone Joint J 2014;96-B: 1366-9.  Back to cited text no. 20
    
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Gu Y, Wang G, Dong W, Li Y, Ma Y, Shang Y, et al. Xpert MTB/RIF and genoType MTBDRplus assays for the rapid diagnosis of bone and joint tuberculosis. Int J Infect Dis 2015;36:27-30.  Back to cited text no. 21
    
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Negi SS, Gupta S, Khare S, Lal S. Comparison of various microbiological tests including polymerase chain reaction for the diagnosis of osteoarticular tuberculosis. Indian J Med Microbiol 2005;23:245-8.  Back to cited text no. 22
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