Lipoarabinomannan (LAM)—a potential biomarker for the diagnosis of tuberculosis from the urine of infected elephants

The spread of tuberculosis (TB) in Asian countries is mainly due to co-existence and close association of humans with elephants and other domestic livestock. Infected animals tend to shed the organism in the preclinical period which risks the transmission of the infection from animals to humans and vice versa. Since tuberculosis infection is chronic and lack specific clinical signs, diagnosis remains challenging. The present study focuses on the utilization of lipoarabinomannan (LAM), a glycolipid for the detection of TB infection in elephants. Usage of urine as the biological sample for the diagnosis makes it more advantageous. Seroprevalence of tuberculosis (TB) in elephants in Kerala were found to be 37.2% (n = 86) using Chembio DPP VetTB assay. Nine (10.46%) out of 86 elephants were positive for AFB and 29 (33.7%) out of 86 elephants were positive for LAM antigen. On comparison of efficiency of LF-LAM assay with that of DPP VetTB assay, LF-LAM assay had a sensitivity of 90.63%, specificity 100%, positive predictive value 100%, negative predictive value 94.7%, accuracy 95.51%, and kappa statistic value 0.924 (p value < 0.001). On comparison of LF-LAM with traditional acid fast staining method, LF-LAM assay had the sensitivity of 100%, specificity 74.03%, positive predictive value 31%, negative predictive value 100%, accuracy 76.74%, and kappa statistic value 0.374 (p value < 0.001). The presence of Mycobacterium tuberculosis was confirmed in trunk wash sample using PCR targeting gene IS6110, at 245 bp amplicon size and 25 seropositive elephants (78.2%) were confirmed positive. Custom sequencing and phylogenetic analysis revealed that the isolate obtained was Mycobacterium tuberculosis. This study in elephants prove that TB LAM Ag (LF-LAM) can be used as potent biomarker for diagnosis of tuberculosis in elephants.


Introduction
Elephants are the largest terrestrial animals on Earth. These majestic pachyderms still form a vital part of the temple and religious festivities. However, there is a decline in the number of Asian elephants in India. Asian elephants are listed as endangered species as per International Union for Conservation of Nature (IUCN). The major causes of decline are due to habitat loss, diseases, and poaching (Baskaran et al. 2011). Tuberculosis (TB) had been reported worldwide in captive elephants. The prognosis of the disease in elephants is grave if not diagnosed earlier. In Asian countries, the occurrence of TB is high among captive wild elephants due to the co-existence and close association with humans and other domestic livestock (Michalak et al. 1998). Infected animals tend to shed the organism in the preclinical period which risks the transmission of the infection to elephants, humans, and other mammals (Lewerin et al. 2005). A definitive antemortem diagnosis of TB in elephants can be challenging and has serious limitations. For the past two decades, the diagnosis of TB predominantly relied on trunk wash culture and acid-fast staining techniques. Trunk wash culture is considered one of the gold standard tests for active TB 13 Page 2 of 7 infection in elephants however, the test has numerous limitations (Lyashchenko et al. 2012).
The present study highlights the exploration of an alternative assay for the early diagnosis of TB and assessment of the reliability and adaptability of the test in the study of wildlife medicine. Urine was selected as the primary biological sample due to its least invasive and low labour-intensive nature. The ease of availability and procurement of the sample highlights the usefulness and future prospects of the study. However, the focal point of the study revolves around the assessment and comparison of the sensitivity, specificity, and reliability of the test in the study with that of the existing tests currently used. Such an investigation and exploration in the wildlife context is the first of its kind.

Selection and collection of samples
The study was conducted in the Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, Pookode, Wayanad, India, during the period from September 2019 to December 2020. A total of 86 captive Asian elephants from Kerala were selected using convenience sampling, and they formed the subjects of the study. All the study subjects were apparently healthy adult animals chosen irrespective of sex, body weight, and parity. Neonates, pregnant animals, and animals with clinical illnesses were excluded from the study.
The clinical samples collected for the study included urine, blood, and trunk wash. For blood collection, animals were placed in their lateral recumbency and blood was drawn from a superficial marginal vein located at the external aspect of the ear using a sterile 16-gauge disposable hypodermic needle ( Fig. 1). Trunk wash samples were collected as per the guidelines for control of tuberculosis in elephants by USDA. Sterile saline (60 ml) was flushed into one nostril of the elephant's trunk using a catheter-tipped syringe. The elephant was then instructed to raise the trunk and leave it elevated for 30-60 s, after which it lowered the trunk and exhaled. Trunk wash contents were collected in a sterile plastic bag and then aseptically transferred into a sterile, leakproof screw-top container. Urine samples were collected from elephants at dawn in the presence of mahout. Mid-stream urine was collected in a sterile urine container. A quantity of 10 ml urine was transferred into centrifugation vials.

Procedures
All 86 elephants included in the study were screened for tuberculosis using USDA-approved DPP VetTB assay, Alere Determine ™ TB LAM (Lipoarabinomannan) Ag assay, and acid-fast staining technique. The samples used for the screening included serum, urine, and trunk wash sediment, respectively. The sera samples were screened using rapid lateral-flow immunochromatographic test-DPP ® (Dual Path Platform) VetTB-Elephant assay kits by Chembio Diagnostic Systems, INC, USA. The test was manually performed, and the results were read after 20 min as per the guidelines of the test kit. The urine samples were screened using a rapid lateral-flow immunochromatographic test-Alere Determine ™ TB LAM (Lipoarabinomannan) Ag assay by Abbott, USA. The test was manually performed, and the results were read after 20 min as per the guidelines of the test kit. The trunk wash sediment was made to a thick smear and screened for acid-fast bacilli.
Further, for confirmation, the trunk wash samples of all seropositive elephants were subjected to a polymerase chain reaction of Mycobacterium tuberculosis targeting bacterial genome IS6110 expression at 245 bp. The forward and reverse primers used were INS1-CGT GAG GGC ATC GAG GTG GC and INS2-GCG TAG GCG TCG GTG ACA AA, respectively (Kulkarni et al. 2012).

Statistical analysis
The statistical analysis was performed using IBM SPSS version 24. The performance of LF-LAM, acid-fast staining tests were evaluated on the basis of indices such as sensitivity, specificity, positive predictive value, and negative predictive value by taking DPP VetTB assay as standard. Cohen's kappa statistic (κ) was estimated to find out the agreement between the tests.

Results
Seroprevalence of tuberculosis in elephants was evaluated in 86 captive elephants from Kerala using Chembio DPP VetTB, which revealed 37.2% seroprevalence (Table 1, Figs. 2 and 3). All 86 elephants were screened for LAM antigen and acidfast bacilli using urine and trunk wash smear, respectively. Twenty-nine (33.7%) out of 86 elephants were positive for LAM antigen (Figs. 4 and 5). Nine (10.46%) out of 86 elephants were positive for AFB. In comparison with the DPP VetTB assay, the LF-LAM Ag test showed a sensitivity of 90.63%, specificity of 100%, the positive predictive value of 100%, the negative predictive value of 94.7%, accuracy of 95.51%, and kappa statistic value of 0.92 (p value < 0.001) ( Table 2). Comparing LF-LAM with the traditional acid-fast staining method, LF-LAM assay had a sensitivity of 100%, specificity 74.03%, positive predictive value 31%, negative predictive value of 100%, accuracy of 76.74%, and kappa statistic value 0.374 (p value < 0.001) (Table 3), whereas, in comparison with the DPP VetTB assay, acid-fast staining showed a sensitivity of 28.13%, specificity of 100%, positive predictive value of 100%, negative predictive value of 70.1%, accuracy of 73.26%, and kappa statistics value of 0.329 (p value < 0.001) ( Table 4).
Trunk wash samples of 32 seropositive elephants were evaluated using PCR targeting gene IS6110, at 245 bp size amplicon to confirm the presence of Mycobacterium tuberculosis (Fig. 6). Out of 32 seropositive elephants, 25 (78.2%) elephants were confirmed positive for Mycobacterium tuberculosis. After custom sequencing and phylogenetic analysis, it was revealed that the isolate obtained was Mycobacterium tuberculosis (Fig. 7).

Discussion
The seroprevalence of tuberculosis in elephants was found to be 37.2% in the study. Previously published studies by Greenwald et al. (2009), Maslow (2011), andKerr et al. (2019) point to the rise in seroprevalence of tuberculosis among elephants touching 15%; nonetheless, the present study demonstrates a higher seroprevalence. This hike in seroprevalence rate may be attributable to the lack of  LF-LAM assay demonstrated sensitivity of 90.63%, specificity of 100%, the positive predictive value of 100%, the negative predictive value of 94.7%, accuracy of 95.51%, and the kappa statistic value of 0.924 (p value < 0.001). These values indicated that the LF-LAM assay could be as efficient as the DPP VetTB assay and could be used as an alternative for the DPP VetTB assay. In comparison with AFB smear microscopy, the LF-LAM assay had a sensitivity of 100%, specificity of 74.03%, positive predictive value of 31.0, a negative predictive value of 100.0, accuracy of 76.74, and kappa statistics value of 0.374 (p value < 0.001). These values indicated that the LAM assay has higher sensitivity than traditional smear microscopy. These findings were in accordance with a previously published report by Elsawy and Redwan (2012). The LAM detection assay by Alere Determine LF-LAM assay was found to have increased sensitivity compared to other LAM detection assays by Chemogen. Although both assays use the same polyclonal antibodies, the differences in the manufacturing process viz matrix composition, pH, and viscosity had a considerable impact on the performance of the test (Minion et al. 2011). The positivity in LAM assay is directly proportional to the bacillary burden in the body (Minion et al. 2011;Agha et al. 2013).
However, a study conducted by Minion et al. (2011) in their systemic review and meta-analysis of LAM in humans found that the sensitivity of the LAM assays in microbiologically confirmed cases was variable ranging from 13 to 93% and specificity 87 to 99%. Nakiyingi et al. (2014) found a sensitivity of 38% and specificity of 100%. The variations in the sensitivity in the present study and previously published studies could be attributed to the variable LAM detection methods, disease severity, patient population, and specimen handling.
AFB smear microscopy demonstrated only 10.46% of positivity for acid-fast bacilli. Comparison with the DPP VetTB acid-fast staining shows a sensitivity of 28.13%, specificity of 100%, positive predictive value of 100%, negative predictive value of 70.1%, accuracy of 73.26%, and the kappa statistics value of 0.329 (p value < 0.001). The sensitivity of smear microscopy obtained in the study was comparatively less when compared to the previously published studies by (Cummings and Schluger 2018;Qureshi et al. 2019) in human sputum samples. The low sensitivity of smear microscopy might be due to the difference in the severity of infection among elephants or might be due to the intermittent shedding of the organisms. In order to demonstrate a positive result in smear microscopy, approximately 5000 to 10,000 bacilli would be required (Qureshi et al. 2019).
Trunk wash samples from 32 seropositive elephants were subjected to PCR targeting gene IS6110, at 245 bp size amplicon to confirm the presence of Mycobacterium tuberculosis (Kulkarni et al. 2012). Out of 32 seropositive elephants, 25 (78.2%) elephants were confirmed positive for Mycobacterium tuberculosis. This finding supports the evidence of active and   latent TB in elephants, which was in agreement with the study conducted by Ong et al. (2013) who observed that only 30% of seropositive samples were positive for trunk wash PCR.
On blasting the acquired trimmed sequence in the National Center for Biotechnology Information (NCBI) showed that the attained sequence shows 98% query cover and 100% identity with other published Mycobacterium tuberculosis sequences in the database. Phylogenetic analysis revealed that the isolates obtained were Mycobacterium tuberculosis. The sequence showed homology with the Mycobacterium tuberculosis strain 1-0013P6C4 chromosome. Among the biological samples, urine is the most abundant biological fluid and least-invasive sample that can be collected makes urine an ideal candidate for biomarker discovery (Siebert et al. 2017;Brondani et al. 2020). Bathla et al. (2015) described urine as an ideal sample for the identification of specific biomolecules as it is a dynamic biological fluid, the composition changes according to the metabolism of the animal at that point of time. This aids in acquiring a true reflection of the metabolic process in animals.

Conclusion
The dire need for alternative methods for the diagnosis of tuberculosis in elephants forms the focus of the research. The study could conclude that the seroprevalence of the disease tuberculosis is increasing in elephants compared to previous reports. Alere Determine TB LAM Ag (LF-LAM) was found to be a reliable diagnostic assay with a sensitivity of 90.63%, specificity of 100%, positive predictive value of 100%, negative predictive value of 94.7%, accuracy of 95.51%, and kappa statistic value of 0.924 (p value < 0.001) in comparison with that of standard DPP VetTB assay. TB LAM Ag (LF-LAM) is a lateral flow assay that has been used in the human field to date; the present study was the first study to demonstrate its adaptability to the veterinary field.