CB-NAAT MTB/RIF assay and histopathology correlation in diagnosis of osteoarticular tuberculosis using culture as reference standard

1. Introduction

Tuberculosis (TB) remains one of the world's deadliest communicable diseases. World Health Organization (WHO) fact sheet of the year 2015 shows a mixed picture of global tuberculosis (TB); on one hand, there is a decline in the incidence and the mortality rate attributed to TB, while on the other hand, the decrease in rate is very slow and TB is still the greatest killer worldwide due to a single infectious agent.^1,2.

As TB is a curable disease, the role of accurate diagnosis and treatment is vital for improving the global picture. Between the years 2000 and 2013, approximately, 37 million lives were saved through TB diagnosis and treatment.^1,2 Apart from the poor detection rate, increase from the incidence of multidrug-resistant (MDR) TB and extensively drug resistant (XDR) TB is also critical. In the same year, 3.5% of new and 20.5% of previously treated TB cases were diagnosed to have MDR-TB and 9% of these had XDR-TB.³

Extrapulmonary tuberculosis (EPTB) affects 15–20% patients with tuberculosis. Skeletal tuberculosis constitutes 10% of EPTB, of which spinal tuberculosis accounts for nearly 50%.⁴ Extrapulmonary infection with members of the Mycobacterium tuberculosis complex (MTBC) remains a diagnosis that is often difficult to establish, since the number of bacteria in extrapulmonary specimens is often lower than the number in pulmonary specimens. Furthermore, collection of extrapulmonary material often requires invasive procedures, and it is not easy to obtain additional samples.⁵ Delayed diagnosis is typical; and this worsens the prognosis and increases morbidity. Early diagnosis and treatment can prevent deformities and other dangerous sequelae like neurologic deficit, commonly seen in cases of spinal tuberculosis.

A history of tuberculosis, a positive skin test (its value declines in endemic areas), and an elevated erythrocyte sedimentation rate (ESR) are used for the diagnosis of osteoarticular TB. Biopsy plays a valuable role in the diagnosis. Culturing the organism is tedious and slow. In a small number of cases with imaging and clinical findings suggestive of tubercular infection, no organism can be cultured despite multiple attempts.⁶ Numerous molecular tests are described and validated in pulmonary tuberculosis however there is little or no validation of the same in osteoarticular tuberculosis.⁷

Now, major thrust is being laid on the accessibility of economically feasible accurate diagnostic assays.⁸ In early 2011, WHO endorsed a novel, rapid, automated, molecular diagnostic test, the CB-NAAT MTB/RIF assay that can simultaneously detect TB and rifampicin resistance.⁹ CB-NAAT MTB/RIF assay provides result from 2 h, thus the patients can be started with proper treatment even on the same day. Furthermore, it requires minimal hands-on technical time, training, and can be installed in unconventional laboratories.^10,11

2. Material and methodology

This cross sectional study included on 210 patients out of which 45.07% female and 54.93% male were present. Mean of age of distribution was 30.31 ± 15.13 with range (3–70 years) of age. A provisional diagnosis of osteoarticular tuberculosis was made based on the clinical and radiological symptoms and signs. All patients with clinical and radiological suspicion of osteoarticular tuberculosis included who followed inclusion criteria (Patients with clinical and radiological suspicion of osteoarticular tuberculosis) and exclusion criteria (patient's presently on anti-tubercular treatment, patients with past history of taking anti tubercular treatment and patients with an inadequate sample yield) were included in the study. Total 210 patients were screened from outpatient clinic on the basis of constitutional symptoms of TB (Joint pain, Fever, Malaise, Weight loss, Night sweating, Anorexia, Spinal deformity, sometimes associated with numbness, paraesthesia, or muscle weakness of the legs, difficulty in standing, kyphotic deformity etc.), haematological investigations like Complete blood counts with ESR, Quantitative CRP, and Manteaux test and radiological investigation including Plane radiograph of the respective part, CT scan of the respective part, MRI scan of the respective part. If associated abscess was present, ultrasound of the respective part was done. Chest radiographs PA view was done to rule out associated pulmonary Koch's if patient has with pulmonary symptoms. Out of this 210 patients 140 were suspected cases of osteoarticular out of which 69 was excluded from study (35 patients – were taking ATT currently, 12 patients had a history of taking ATT, 20 patients did not yield representative material for study).

In this study patient of suspected osteoarticular TB with of different joints involvement (Table 1) of which maximum was diagnosed with spine tuberculosis. (66.20%)

Table 1.

Frequency of joint involvement in TB.

	Frequency	Percentage
Right 2nd MTP Joint TB	1	1.41%
Left ankle TB	2	2.82%
Right distal Tibia TB	1	1.41%
Left Elbow TB	2	2.82%
Right Knee TB	3	4.23%
Left Knee TB	2	2.82%
Left Shoulder TB	1	1.41%
Pott's Spine	47	66.20%
Right Proximal Tibia TB	1	1.41%
Right Elbow TB	3	4.23%
Right Ankle TB	3	4.23%
Right Hip TB	1	1.41%
Right Index Finger TB	1	1.41%
Right Shoulder TB	1	1.41%
Right Sternoclavicular Joint TB	1	1.41%
Right Wrist TB	1	1.41%
Total	71	100.00%

Under aseptic precautions biopsy from representative tissue, fluid or disease from predetermined areas of disease was taken and sent for histopathological examination, AFB stain, and culture for AFB and CB-NAAT MTB/RIF assay evaluation. Formalin fixed paraffin embedded sections were prepared histopathological and AFB stain was done for evidence of tuberculosis. The CBNAAT assay evaluation done by purification, concentration, amplification and identified targeted TB nuclei acid it sequences in the TB genome (rpo b), and provided results. AFB culture was used as gold standard for the study.(see Table 2, Table 3, Table 4)

Table 2.

Showing cross tabulation (results) of Gene Xpert and culture for AFB(gold standard).

		AFB Culture		Total	P value	Kappa
		negative	Positive
Gene Xpert	Negative	23 (32.39%)	4 (5.63%)	27 (38.03%)	<.0001	0.599
	Positive	10 (14.08%)	34 (47.89%)	44 (61.97%)
Total		33 (46.48%)	38 (53.52%)	71 (100.00%)

Table 3.

Showing cross tabulation of histopathological examination and culture for AFB culture (gold standard).

		AFB Culture		Total	P value	Kappa
		negative	Positive
HISTOPATHOLOGICAL EXAMINATION	Negative	24 (33.80%)	11 (15.49%)	35 (49.30%)	0.001	0.409
	positive	9 (12.68%)	27 (38.03%)	36 (50.70%)
Total		33 (46.48%)	38 (53.52%)	71 (100.00%)

Table 4.

Showing comparison of sensitivity of Gene Xpert and histopathological examination.

		Histopathological examination		Total	P value
		Negative	Positive
GENE XPERT	Negative	2 (5.26%)	2 (5.26%)	4 (10.53%)	0.039
	Positive	10 (26.32%)	24 (63.16%)	34 (89.47%)
Total		12 (31.58%)	26 (68.42%)	38 (100.00%)

3. Results

In our study 140 were suspected cases of osteoarticular out of which 71 patients met the inclusion criteria of study in which maximum number (66.20%) were diagnosed with pott's spine. Mean ESR found to be 44.75 ± 19.82 and 83.10% patients CRP was positive which more than 6 mg/l was and Mantoux test was positive which was more than 15 mm in 84.51% patients.

AFB stain found to be positive about only 14.08% patients despite of radiological suspicion osteoarticular TB as diagnosis. Sensitivity of AFB stain was 23.68% and specificity was 96.97%. AFB culture was positive about only 53.52% patients of suspected osteoarticular tuberculosis. Histopathological examination was positive in 50.7% patients of suspected osteoarticular tuberculosis with sensitivity was 71.05% and specificity was 72.73%. Positive predictable value of histopathological examination was 75.00% and negative predictive value was 68.57%.

Out of 71 patients of suspected osteoarticular TB 61.9% patient were positive about gene xpert on sensitivity was 89.47% and specificity was 69.71%. Positive predictable value of gene xpert was 77.27% and negative predictive value was 85.19%. Rifampicin resistance was seen in 8.45% patients of suspected osteoarticular TB only.

CB-NAAT MTB/RIF assay total positive in 30.3% patients out of which 15.15% patients were also positive for histopathological examination and 15.15% were only positive for CB-NAAT MTB/RIF assay but negative for histopathological examination while comparing hisopathological examination and CB-NAAT MTB/RIF assay.

4. Discussion

Establishing the diagnosis of TB beyond doubt is very important when considering the cost and prolonged duration of treatment and the effects of delayed treatment including psychosocial implications.¹² Even in disease endemic countries, the diagnostic modalities available are not sufficient for accurate diagnosis the diseased and most of the diagnosis are based on only clinical suspicion and imaging results¹³ so the question arise why is it so difficult to diagnose osteoarticular tuberculosis. Firstly, the AFB are fastidious and slow-growing organism. Secondly AFB osteoarticular tuberculosis is a paucibacillary disease and being a deep-seated lesion it is difficult to procure the tissue.² Thereby sensitivity of most tests is very low in articular TB, as there is dilution of tubercle bacilli in synovial fluid.⁵ Despite a wide array of the available tests like AFB culture sensitivity, AFB staining, histopathology, or even polymerase chain reaction (PCR), no single test claims to be of 100% diagnostic accuracy. In the absence of a single specific, sensitive, and rapid diagnostic method, especially in cases of early tubercular arthritis, an important priority in TB research remains to persistently find such a method for diagnosis.^14,15

The diagnosis of osteoarticular tuberculosis in endemic areas is clinico-radiological. It is justified to treat the patient's clinico-radiologically in classical lesions of the bone. The clinical and radiological response can be observed in 8–12 weeks. The priority areas to achieve these surmountable targets include administration of highly effective treatment to prevent MDR-TB and second, expansion of rapid diagnostic assays for MDR-TB.¹¹ However, diagnosis with the bacterial culture still remains the gold standard in many developing countries and the 125-year-old sputum microscopic examination is still the most widely used method for the detection of TB.^10,16 The role of PCR in extrapulmonary have been evaluated in multiple studied which have reported the sensitivity variably ranging from 32 to 78%^6,7 but no study till now has evaluated the diagnostic role of CB-NAAT MTB/RIF assay in osteoarticular TB and also correlation with histopathological examination.⁵ A single CB-NAAT MTB/RIF assay run can provide both detection of TB and detection of rifampicin resistance within 2 h.¹⁰ It is a quick assay with minimum hands-on time and negligible safety concerns for persons handling the samples. WHO recommends the use of CB-NAAT MTB/RIF assay worldwide.¹⁶

In this study out of 71 patients of suspected osteoarticular TB, Culture for AFB were positive in 53.52% patients 61.9% patient were positive for CB-NAAT MTB/RIF assay, with sensitivity of 89.47%and specificity of 69.7%. In 47.89% patients CB-NAAT MTB/RIF assay as well AFB culture was positive whereas histopathological examination turned to be positive in 50.70% patient. The strength of agreement between CB-NAAT MTB/RIF assay and AFB culture was found to be statistically insignificant with p value of 0.0001 and kappa value of 0.599.

The new CB-NAAT MTB/RIF assay tested in our study targets the rifampin resistance-associated rpoB gene region by heminested PCR with three specific primers and combines the sensitive detection of M. tuberculosis DNA and determination of RMP resistance. Furthermore, the hands-on time is short due to automation of bacterial lysis, DNA extraction, real-time PCR amplification, and amplicon detection in a single system.¹⁷ These features of simplicity and safety of use could allow for cost-effective and highly sensitive detection of tuberculosis and drug resistance outside reference centres, which would increase access to testing and decrease delays in diagnosis, without the need to build large numbers of laboratories equipped for advanced biosafety.¹⁸ Numerous studies have assessed the yield of PCR techniques for the diagnosis of extrapulmonary tuberculosis.^3,10

This is the first study to verify the usefulness of application of the CB-NAAT MTB/RIF assay for the rapid diagnosis of osteoarticular tuberculosis. Overall, the sensitivity and specificity of the Xpert assay were very high, with the assay correctly identifying 61.9% of all culture-positive specimens. The high sensitivity is not self-evident, since only small amounts of DNA are expected in any osteoarticular clinical sample.¹⁹

To eliminate TB as a public health problem (as defined by a TB incidence of less than one per million population worldwide) by 2050, TB incidence rates will need to decrease by an average of 16% per year for the next 40 years.²⁰ To achieve this huge target no single intervention will be helpful, so new diagnostic tests with their more sensitivity, specificity and ease to use could make a substantial impact.

Implementation of a nucleic acid amplification test globally was estimated to reduce TB incidence by 28% overall by 2050.²¹ We concluded that CB-NAAT MTB/RIF assay has great potential not only to diagnose clinically suspected osteoarticular TB but also rapidly and very effective in detection of multidrug resistance strain. This will ensure early treatment for patients thus prevent in further transmission of the disease. CB-NAAT MTB/RIF assay is not a substitute for culture. Histopathological examination also make the diagnosis strong in TB with high sensitivity and specificity but it is not a rapid method. So TB programs must adapt their systems to use this technology, increasing access to patients while also focusing on the basics of curative TB treatment so that increased case-finding will translate into more patients cured. Moreover, there is a critical need for increased capacity to diagnose MDR-TB to be matched by increased capacity to effectively treat and cure diagnosed cases. Patient advocates and activists should hold everyone accountable and drive demand for better TB programmes that include use of new tools such as the CB-NAAT MTB/RIF assay. The WHO index- TB guideline 2016–2017 recommended, as CB-NAAT MTB/RIF assay should be used as an additional test to conventional smear microscopy, culture and cytology in FNAC specimens.