View of Line Probe Assay Is Better Than CBNAAT for Diagnosis of Ileocolonic Tuberculosis: A Prospective Study

Line Probe Assay Is Better Than CBNAAT for Diagnosis of Ileocolonic Tuberculosis: A Prospective Study

Dr Anil Kumar G. MD¹, Dr Deepak Suvarna MD, DNB ², Dr Aradya H V, MD, DNB ³ Mr Ravindran kumar ⁴ , Dr Nandeesh H P. MD, DNB , DM ⁵, Dr Vijay Kumar T

R. MD, DM ³, Dr Rithesh Reddy G. MBBS, MD¹

Author Affiliation: Department of Medical Gastroenterology and Department of

Microbiology, JSS Medical College and Hospital, JSS Academy of Higher Education &

Research, Mysore, Karnataka, India

1. Senior resident 2. Associate Professor 3. Assistant Professor 4. Research assistant 5.Professor

Corresponding Author: Dr. Deepak Suvarna, Associate Professor, Department of Medical

Gastroenterology, JSS Hospital, Mysore. Phone number: +91-9964380249,

EMAIL: [email protected]

ABSTRACT

Introduction:

Diagnosis of Ileocolonic Tuberculosis remains a challenge due to its nonspecific clinical and laboratory characteristics. Role of Line probe assay in Ileocolonic ulcers for the diagnosis of intestinal tuberculosis and Non Tubercular Mycobacterium is not well defined. We report the utility of Line Probe Assay (LPA) testing in patients with Ileocolonic ulcers and its comparison with Cartridge based nucleic acid amplification test (CBNAAT) and histopathology. Methodology: A prospective study of 35 patients with Ileocolonic ulcers detected during colonoscopy. Ileocolonic ulcer tissue was subjected to Line probe assay (GenoType Mycobacterium CM kit) for detection of Mycobacterium tuberculosis (MT) and Non tubercular Mycobacterium (NTM), CBNAAT (Gene Xpert), and Histopathology. Patients were followed up for six months and repeat colonoscopy was done for response of therapy and to assess diagnostic accuracy. Results: Out of 35 patients, 31 patients were analysed. 16 patients diagnosed to have TB were started on anti-tubercular treatment based on clinical presentation, LPA, CBNAAT and histopathology. 15 patients had other diagnosis. Sensitivity and specificity of LPA for diagnosis of ITB was 28.57% and 50%whereas The sensitivity of CBNAAT was 7.14% and the specificity was 100%.Histopathology was positive in 12 patients (38.7%). Positive predictive value of both LPA and CBNAAT were comparable. Conclusion: LPA provides an additional investigation in the armamentarium for the diagnosis of Ileocolonic tuberculosis .Although CBNAAT is generally recommended in the diagnosis of tuberculosis in extra-pulmonary specimens, utility of the same in the setting of Ileocolonic ulcers appears limited as demonstrated in this study.

Keywords: Intestinal tuberculosis, Line probe assay, Polymerase chain reaction, Cartridge based Nucleic acid amplification, Gene Xpert, Crohn’s disease, Ileocolonic Ulcers

Introduction:

Intestinal Ulcers (Colonic and Ileal) are common finding during colonoscopy. The aetiology of these ulcers includes Tuberculosis, Crohn’s disease, Nonsteroidal anti-inflammatory drugs, infectious causes like Yersinia, Amoebiasis, Ischemic and Idiopathic ulcers. When histopathological examination reveals granulomas in these ulcers, differential diagnosis of Tuberculosis vs. Crohn’s disease is usually considered. Although there are few differentiating points to diagnose either of these

conditions, in reality it is difficult and many patients end up taking empirical anti tubercular treatment.

Hence we need better diagnostic tools for diagnosis of intestinal tuberculosis.

Literature review:

Tuberculosis is one of the major infections worldwide. Extrapulmonary forms of tuberculosis are increasingly reported. The aetiology of Crohn’s disease is also not clear. Infectious causes including Non tubercular mycobacterium (NTM) has been postulated as etiological factor for Crohn’s disease.^1,2,3.There are, however, many diverse opinions^4-5.To date, more than 160 NTM species have been recognized, while many others await classification. Approximately one-third have been associated with diseases in humans⁶.The most frequent forms of NTM include Mycobacterium avium complex (MAC, 61%), Mycobacterium fortuitum (19%) and Mycobacterium kansasii (10%), with smaller percentages of Mycobacterium gordonae and Mycobacterium chelonae⁷. Mycobacterial involvement of the bowel either by tuberculosis or MAC may lead to diarrhoea. Antony et al⁸ reported that MAC is the most commonly identified organism in patients with chronic diarrhoea and low CD4 lymphocyte counts.

There is a possibility that some patients thought to have Crohn's disease or tuberculosis are indeed cases of atypical mycobacteriosis of the intestine. Correct species identification of NTM isolates is important clinically since NTM species differ in their potential to cause clinical disease in humans and in their response to specific antibiotics⁹

Over recent years clinical laboratory methods of speciation have moved from biochemical to molecular techniques. Matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry has shown good promise in providing rapid speciation of NTM ¹⁰

Of the molecular techniques available, line probe assay (eg, Hain CM and AS) is widely used allowing reasonable identification of common species of NTM^11,12 These molecular techniques are applied on respiratory samples for detection of NTM.

Clinical presentation of Extra-pulmonary tuberculosis (EPTB) is diverse and it may lead to delayed diagnosis due to paucibacillary nature of the samples. Sensitivity of acid-fast bacillus detection by microscopy is poor (0-40%) and smear positive samples requires 10⁴ bacilli/ml of the sample^13,14.BACTEC-MGIT cultures established from colonoscopic biopsy specimens were positive for M. tuberculosis only in 20.29% GITB patients¹⁵ Long incubation period is required to grow Mycobacterium Tuberculosis, limit the usefulness of culture methods for diagnosis of TB. Recently, nucleic acid amplification tests, which can be used for direct detection of TB from samples, have emerged as potentially useful tools for rapid diagnosis of TB¹⁶.

World Health Organization has recommended a cartridge based Nucleic acid amplification test (CBNAAT), Xpert MTB/RIF (by Cepheid, CA, USA) for extra pulmonary samples. But it has limitation of detecting RIF resistance only¹⁷. Line probe assay (LPA) detects resistance to both Isoniazid (INH) and Rifampicin (RIF). LPA Studies have been performed in EPTB diagnosis, but there is limited evidence of LPA use in Ileocolonic ulcers. This study was designed to evaluate the use of LPA for detection of MTB and NTM directly from Colonic and Ileal ulcers, and to compare with Histopathological examination (HPE) and CBNAAT.

Methods:

Methodology: The study was conducted at a tertiary referral centre in India. Ethical committee

to the study. Patients with Ileal / colonic ulcers more than 1 x 1 cm in size were included in the study.

Malignant ulcers and ulcers less than 1 x 1 cm were excluded from the study. Biopsies were obtained from the colonic and Ileal ulcers and transferred into three separate containers one for Histopathology, two other containers containing saline for CBNAAT and Line probe assay.

The Line probe assay was done using GenoType Mycobacterium CM kit which is a qualitative in vitro test for the identification of the Mycobacterium tuberculosis complex as well as the following nontuberculous mycobacterial species: M. avium, M. chelonae, M. abscessus, M. fortuitum, M.

gordonae, M. intracellulare,M. scrofulaceum, M. interjectum, M. kansasii, M. malmoense, M.

marinum/M. ulcerans, M. peregrinum, and M. xenopi.

Genotype Mycobacterium CM test is based on the DNA Strip technology. The whole procedure is divided into three steps:

1. DNA extraction from tissue material

2. A multiplex amplification with biotinylated primers 3. A reverse hybridization.

The CB-NAAT system detects DNA sequences, specific for Mycobacterium tuberculosis. It purifies and concentrates Mycobacterium tuberculosis bacilli from tissue samples, isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR. The process identifies clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons.

Patients were monitored until the final definite diagnosis was made. If the symptoms improved, Colonoscopy was performed after six months to look for resolution of the ulcers. If the symptoms persisted for two months after starting treatment or worsened after initiating treatment, repeat colonoscopy was done at two months. The patients’ clinical, endoscopic, laboratory and histological features were stored in a database.

Data analysis: Data was entered into Microsoft excel data sheet and was analyzed using SPSS 22 version software. Categorical data was represented in the form of frequencies and proportions.

Continuous data was represented as mean and standard deviation. Chi square was used to check for association of qualitative data. Sensitivity , Specificity , Positive and Negative Predictive Value and Diagnostic Accuracy of the test was done in comparison with gold standard test .p value (Probability that the result is true) of <0.05 was considered as statistically significant after assuming all the rules of statistical tests.

Results:

A total of 16 patients were diagnosed to have tuberculosis based on clinical presentation, lab investigations and histopathology whereas 15 patients had other diagnoses (Crohn’s disease, Amoebiasis, nonspecific Ileitis). In patients treated with anti-tubercular therapy (16), Abdominal pain was present in 11 (68.7%) patients, diarrhoea in 9 (56.2%) Weight loss in 6 (37.5%), fever in 2 (12.5%) and bleeding per Rectum in 1(6.2%). Two patients were HIV positive. Ascites was present in 2(6.4%) and 11(35.4%) patients had anaemia. Summary of clinical and laboratory parameters are mentioned in table 1

Table 1. Clinical parameters of the patients studied.

Patients started on ATT (n=16)

Others (n=15) P Value

Clinical features Pain abdomen 11 (68.7%) 7 (46.6%) 0.213

Diarrhoea 9 (56.2%) 10 (66.6%) 0.552

Weight Loss 6 (37.5%) 2 (13.3%) 0.124

Fever 2 (12.5%) 2 (13.3%) 0.945

Bleeding per Rectum 1 (6.2%) 3 (20%) 0.254 Investigations Haemoglobin 11.1 +2.2 11.42 + 2.22 0.701 Total Leucocyte count 6701.7 +3019.6 6643 +3051.9 0.957

ESR 38.89+28.08 37.13+ 27.5 0.861

Colonoscopy Ileum 6 (37.5%) 8(53.3%) 0.376

Caecum 2 (12.5%) 1 ( 6.6%) 0.583

Ileo-caecal 9 (56.2%) 1 (6.6%) 0.003*

Other Part of Colon 4 (25%) 7 (46.6%) 0.208

Histology Granuloma 11 (68.7%) 0 (0%) 0.001*

Caseation necrosis 1 (6.2%) 0 (0%) 0.325

Inflammation 4 (25%) 4 (26.6%) 0.916

USG Findings Lymphadenopathy 2(12.5%) 1 (6.6%) 0.583

Wall Thickening 6 (37.5%) 5 (33.3%) 0.809

Ascites 3 (18.7%) 1 (6.6%) 0.316

CT Findings Lymphadenopathy 4 (25%) 3 (20%) 0.739

Intestinal Wall Thickening

5 (31.2%) 4 (26.6%) 0.779

Ascites 1 (6.2%) 1 (6.6%) 0.962

USG-Ultrasonography, CT-Computerized tomography

LPA was positive in 9(29.03%). MTB Species was positive in 5(16.12%) and NTM was positive in 4 (12.1%). 12(38.7%) Patients had features of tuberculosis on biopsy. Out of the 31 patients, only one was positive for CBNAAT (3.3%).

Diagnostic Ability of Line probe assay for diagnosis on Intestinal Tuberculosis in mentioned in table two.

Table 2

Patients treated with Anti Tubercular Therapy(16) Improved (14) Not improved (02)

Frequency % Frequency %

LINE PROBE ASSAY for MTB

Positive 04 28.57% 01 50%

Negative 10 71.42% 01 50 %

SN- 28.57%

(95%CI-

SP- 50.00%

(95% CI- 1.26% to

PLR-0.57 (95%CI- 0.11 to

NLR- 1.43 (95%CI 0.34 to

PPV-80.00%

(95% CI

44.32% to

NPV- 9.09%(95%

CI 2.35%

Accuracy- 31.25%(95%CI-

11.02% to

58.10%)

( SN- Sensitivity, SP- Specificity, PLR- Positive Likelihood Ratio, NLR- Negative Likelihood Ratio, PPV- Positive Predictive Value, NPV -Negative Predictive Value, MTB-Mycobacterium Tuberculosis )

The diagnostic ability of CBNAAT for ITB is mentioned in the Table 3 TABLE 3

Patients treated with Anti Tubercular Therapy(16) Improved (14) Not improved (02)

Frequency % Frequency %

CBNAAT Positive(1) 01 7.14% 0 0%

Negative(15) 13 92.85% 02 100%

SN- 7.14% CI 95% (0.18%- 33.87%)

SP- 100% CI 95%(15.81%- 100.00%)

PLR- --

NLR- 0.93(

95% CI

0.80-1.07)

PPV-100%

NPV-

13.33%(95%CI 11.74 to 15.10

%)

Accuracy- 18.75% ( 95%CI 4.05- 45.65%)-

( SN- Sensitivity, SP- Specificity, PLR- Positive Likelihood Ratio, NLR- Negative Likelihood Ratio, PPV- Positive Predictive Value, NPV -Negative Predictive Value CBNAAT – Catridge based Nucliec acid amplification test)

Diagnostic Ability of Line robe Assay in Diagnosis ITB when compared to CBNAAT is mentioned in table 4

CBNAAT

Positive (1) Negative (30)

Frequency % Frequency %

LINE PROBE ASSAY

Positive 1 100.0% 8 26.6 %

Negative 0 0.0% 22 73.4 %

SN- 100.00%

(95% CI- 2.50

% to 100.00%) SP-

73.33%(95%CI

54.11% to

87.72%)

PLR- 3.75 (95% CI- 2.07 to 6.79)

NLR-0.00

PPV-11.11%

(95%CI- 6.46% to 18.45%)

NPV- 100.00%

Accuracy- 74.19%

(95% CI- 55.39% to 88.14%)

( SN- Sensitivity, SP- Specificity, PLR- Positive Likelihood Ratio, NLR- Negative Likelihood Ratio, PPV- Positive Predictive Value, NPV -Negative Predictive Value CBNAAT- Catridge based nucleic acid amplification)

Diagnostic Ability of Line robe Assay in Diagnosis TB when compared to Histo pathology is mentioned in table 5

Table 5

( Total 31 cases) Histopathology

Positive(12cases / 38.7%)

Histopathology

Negative(19cases / 61.3%)

Frequency % Frequency %

LINE PROBE ASSAY(

only MTB )

Positive 4 33.3% 1 5.2%

Negative 8 66.6% 18 94.7%

SN- 33.33%

(95%CI- 9.92%

to 65.11%)

SP- 94.74%

(95% CI-

73.97% to 99.87%)

PLR-6.33 ( 95% CI 0.80 to 50.13)

NLR- 0.70 (95% CI - 0.47 to 1.06)

PPV- 80.00%

(95%

33.57% to 96.94%)

NPV- 69.23%

(95% CI 59.80 to 77.29 %)

Accuracy- 70.97%

(95% CI- 51.96% to 85.78%)

( SN- Sensitivity, SP- Specificity, PLR- Positive Likelihood Ratio, NLR- Negative Likelihood Ratio, PPV- Positive Predictive Value, NPV -Negative Predictive Value, MTB-Mycobacterium Tuberculosis)

Discussion

Our study included total of 35 patients with Ileocolonic ulcers. 4 patients were excluded from the final analysis as one developed road traffic accident and died. Three patients stopped the recommended treatment midway and were lost for follow up. 31 patients were considered for final analysis. Of the 31 patients 17 (54.8%) were males. Mean age was 41.58 ± 17.31years. 16 patients were started on ATT after considering their clinical presentation, laboratory investigations, imaging studies, Monteux test, intestinal biopsy histopathology, LPA and CBNAAT.

Among the patients who had been diagnosed with Tuberculosis, abdominal pain was present in 11 (68.7%) patients, Chronic diarrhoea in 9 (56.2%), weight loss in 6 (37.5%), fever in 2 (12.5%) and bleeding per rectum in 1 patients (6.2%). In a study done by Makharia et al¹⁸on intestinal tuberculosis, chronic diarrhoea was present in 37%, blood in stools in 16%, abdomen pain in 90%, fever in 41%

and weight loss in 83% which is almost similar to our study.

On colonoscopy, ulcers were noted most commonly at the ileocaecal area in 9 (56.2%), followed by terminal Ileum in 6 (37.5%), Caecum in 2 (12.5%) and rest of the colon in 4 (25%). These findings were almost similar to the findings Mukewar et al ¹⁹

On Computer tomography findings in 16 of 31 cases started on ATT included bowel thickening in 5 (31.2%) patients, lymphadenopathy in 4 (25%) and ascites in 1 (6.2%) patients which is in contrast to study done by Tariq Sinan et al ²⁰where ascites was seen in more than half the cases (55.2%). Other findings in this study included lymphadenopathy (46.9%), bowel wall thickening (38%) and solid organ involvement (20.4%).

Histological features suggesting ITB include confluent granulomas, multiple granulomas, large granuloma size, bands of epithelioid histiocytes lining ulcers and disproportionate submucosal inflammation that significantly exceeds mucosal inflammation. In our study on intestinal biopsy, in

addition to other findings, granuloma was present in 11 (68.7%).In a study done by Makharia et al¹⁸, granulomas were seen in 62.5% of the patients which was similar to our study.

Of total 31 colonic ulcers patients, 12 cases (38.7%) were suggestive of TB on histopathological examination. Out of these 12 cases, 5 were positive on Line Probe Assay (16.1 %). Of 19 cases which were nonspecific on histopathological examination, 4 were positive for Mycobacterial species (NTM ) on Line Probe assay (12.9 %).

The sensitivity of LPA compared to HPE for diagnosis of ITB was 33.33% (95% confidence interval:

9.92% -65.11%) while the specificity, negative predictive value, the positive predictive value, and accuracy were 94.74% (73.97- 99.87%), 69.23% (59.80-77.29%), 80.00% (33.57- 96.94%) and 70.97

% (51.96- 85.78%), respectively

Out of the 16 patients started on ATT, only one patient was CBNAAT positive. This patient was positive for MTB by LPA also. When LPA was compared to CBNAAT for diagnosis of ITB, Sensitivity was 100% (95% confidence interval: 2.50 to 100%), specificity 73.33% (54.11 to 87.72%).These were similar to observations of Syed et al ²¹

The sensitivity of CBNAAT in our study for diagnosis of ITB was 7.14% (95% confidence interval:

0.18% - 33.87%) while the specificity, negative predictive value, the positive predictive value, and accuracy were 100% (15.81- 100%), 13.33% (11.74%- 15.10%), 100.00% and 18.75 % (4.05%- 45.65%), respectively. However Balaji et al ²² had demonstrated higher sensitivity (32%) of CB NAAT compared to our study.

Multiple studies have been performed on the role of CBNAAT in extra-pulmonary tuberculosis and the sensitivity for detection of tuberculosis has varied from 39 to 82 % and the specificity has varied from 26 to 98%^{23, 24,25}

The sensitivity of LPA for diagnosis of in our study for ITB was 28.57% (95% confidence interval:

8.39% - 58.10%) while the specificity, negative predictive value, the positive predictive value, and accuracy were 50.00% (1.26- 98.74%), 9.09% (2.35%- 29.37%), 80.00% (44.32% - 95.26%) and 31.25 % (11.02%- 58.66%), respectively.

Other studies of LPA on extra-pulmonary samples have obtained a sensitivity ranging from 45-71%

and specificity ranging from 92-94% ^{26, 27}. This is first study of LPA on intestinal ulcers. The sensitivity and specificity of LPA appears to be less as compared to respiratory and other extra- pulmonary samples probably due to the paucibacillary nature of the specimen.

Out of remaining 15 patients who were not started on ATT, 3 were diagnosed as Crohn’s disease, 2 as amoebic colitis and 10 showed nonspecific ulceration.

Of total 31 cases, 9(29.03%) were LPA positive. Out of the 9 LPA positive, 5 (16.12%) were of mycobacterium TB and 4(12.9%) were mycobacterial species positive (NTM). Species differentiation was not possible probably due to low bacterial load in the biopsy specimen. All these patients 9 patients were started on ATT. Four improved among mycobacterial TB positive group and three improved among mycobacterial species positive group. The patient who did not improve in MTB group improved after starting treatment for multidrug resistant TB and the patient who did not improve in the NTM group improved after addition of macrolide.

Of total 31 cases, after considering the clinical presentation, colonoscopy findings, histopathology, imaging, LPA and CBNAAT, 16 Patients were started on ATT. Among the 16 patients who were

started on ATT, overall response to therapy was positive in 14 patients. Among the improved cases, 7 patients were positive for LPA. Among two cases who did not improve on ATT, one was LPA positive for TB and one positive for NTM..

LPA is a better investigation tool compared to CBNAAT for diagnosis of Ileocolonic TB. It can be added as a routine tool in diagnosis of Ileocolonic TB along with histopathology and CBNAAT till better tests arrive.

Conclusion: LPA provides an additional investigation in the armamentarium for the diagnosis in intestinal ulcers. Although CBNAAT is recommended in the diagnosis of tuberculosis in extra pulmonary specimens, utility of CBNAAT in the setting of Ileocolonic ulcers appear limited as demonstrated in this study.

Treatment of intestinal ulcers involves application of multiple clinical and laboratory imaging parameters to reach to a diagnosis. The sensitivity and specificity of all the available investigative modalities are poor and we need better tools.

Limitation of the study : Sample size was small, more studies need to be done with large samples.

Further speciation of NTM was not possible probably due to the paucibacillary nature of the intestinal ulcers

ACKNOWLEDGEMENTS

The authors are thankful to Dr. Sumana Bhat, Head department of microbiology, JSS Hospital, Mysore, India, for her cooperation and support for this study.

Funding: No funding sources Conflict of interest: None declared

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