All the patients with diarrhea, who were admitted to Sri Ramaswamy Memorial Medical College Hospital and Research Centre (SRM MCH & RC), Kattankulathur, Tamilnadu, between January 2018 and December 2019, were recruited in the study. All patients ≥18 years were made to understand the purpose of the test, assured of the confidentiality of the results, before obtaining the signed consent. Patients who did not have symptoms of spondyloarthritis post-diarrhea; and patients who weren’t available for follow up, were excluded from the study. All patients with diarrhea and symptoms of spondyloarthritides were diagnosed by the rheumatologist (Dr. C Panchapakesa Rajendran) and sent to the laboratory for further investigations. Stool specimens of all the 1108 patients were collected and transported in glucose phosphate broth and processed using culture methods as per CLSI guidelines, by the microbiologist (1^st author, Jayaprakash T.).

Within 2 h of collection, the samples were processed by laboratory technicians. Stool samples were macroscopically observed for the presence of blood and/or mucus, followedby Gram staining using the modified Hucker’s method to easily detect the morphology of any bacteria present [Humphries RM.¹⁹]. Direct wet-mount preparation was carried out with the stool samples, by adding 2-3 drops of 0.85% saline and examined under a 22-22mm coverslip to detect motile trophozoites. The iodine-mount method was also used to provide a better contrast to detect the presence of bile-stained eggs. Fecal concentration methods were also used, as mentioned in Garcia LS et al. 2018; the sedimentation technique was performed and observed as wet preparation under the microscope using 10x and 40x objectives, with and without the presence of iodine [Garcia L.S.²⁰].

As routine specimen processing, stool samples were inoculated in Selenite F broth, to enrich the salmonella species present. Samples were plated on MacConkey (MAC) media – to differentiate between lactose fermenting and non-lactose fermenting enteropathogens. Selective media such as Xylose Lysine Deoxycholate agar (XLD), and Deoxycholate Citrate agar (DCA) were used – to detect and differentiate the recovery of Salmonella, Shigella species, 5% Sheep blood agar (SBA) was used – to detect the presence of Vibrio spp., Plesiomonas spp., and Aeromonas spp., [Sanchez-Romero MI (21), S.A.²²].

Phenotypic characteristics of the colonies were noted down and compared their consistency with that of enteric pathogens. Furthermore, the selective colonies were screened using biochemical tests such as, Indole test –to aid in differentiate various Enterobacteriaceae members and between P. mirabilis and P. vulgaris, Mannitol Motility Medium (MMM) – to determine the ability of the bacteria to ferment mannitol, and detect motility of the organisms, Triple Sugar Iron (TSI) test – to detect the production of hydrogen sulfide, gas, and the ability to ferment glucose, lactose and/or sucrose; Christenson’s urea agar test – to distinguish Proteus spp. from other enteric bacteria; and Simmons’ citrate medium test; to determine the ability of the organisms to use citrate as its sole carbon source, thus differentiating members of Enterobacteriaceae. All the isolates identified were further subjected to antimicrobial susceptibility tests to detect any resistance to the battery of antimicrobials used. All the culture techniques were performed with the quality control strains obtained from American Type Culture Collection (ATCC).

Among the total(1108), 205 patients were excluded as they were not available for follow-up, while the remaining 903 patients with diarrhea were followed up for 3-4 weeks, to check whether they had articular complaints of axial(unilateral or bilateral sacroiliitis, neck pain) and peripheral joints(shoulder, elbow, wrist, finger, thigh, knee, foot, heel and dorsum of foot pain), followed by extra-articular symptoms such as uveitis, and psoriasis. Laboratory evaluation including Human Leukocyte antigen (HLA-B27), C-reactive protein (CRP) and Erythrocyte Sedimentation Rate (ESR) were done in 29 patients who fulfilled the classification criteria for spondyloarthritis by Assessment of Spondyloarthritis International Society(ASAS). There are no universally accepted classification criteria specific for reactive arthritis and may vary with studies.

Blood samples of patients suspected with reactive arthritis²⁹ were collected by a phlebotomist under aseptic conditions, 2ml in ethylenediamine tetraacetic acid(EDTA) tubes and 2ml in plain blood vacutainers. The EDTA samples were tested for ESR and for the detection of HLA-B27, while the blood collected in plain vacutainers were used to test for CRP using latex agglutination methods.

HLA-B27 (genetic marker)detection was performed by a microbiologist (Jayaprakash T.) using a Chip based Real-Time PCR (RT-PCR) method, based on the principle of TaqMan assay. DNA extraction from whole blood samples collected from patients in EDTA tubes was performed with Trueprep® AUTO/AUTO v2 Universal Cartridge based Sample Prep Device and Trueprep® AUTO/AUTO v2 Universal Cartridge based Sample Prep kit, which uses a proprietary matrix enclosed in a cartridge to purify nucleic acid from clinical samples.

The Truenat™ HLA-B27 chip labelled with patient ID is placed on the chip tray, away from the analyzer, without touching the reaction well. The cap of the microtube containing freeze-dried PCR reagents is removed, and using the filter barrier tip, 6µL of purified DNA is transferred into the microtube. The tube was allowed to stand for 30-60s to get a clear solution. The clear solution (6 µL) was dispensed into the reaction well of Truenat™ HLA-B27 chip, and the test was initiated.  At the end of the test run, a result was displayed on the Truelab® analyzer screen showing whether HLA-B27 was “DETECTED” or “NOT DETECTED”, along with the threshold cycle value(Ct value) of the internal positive control(IPC) and the validity of the test.