Collection of specimens
First void urine was collected from male and female patients who attended the clinics of NSACP (n = 126) and Colombo South Teaching Hospital, Kalubowila, Sri Lanka (n = 200), from 19th March 2018 to 28th October 2019 (total n = 326). Criteria for recruitment were age ≥ 18 years, symptoms suggestive of urogenital infection (i.e. abnormal urethral/ cervical/vaginal discharge, increased white cells in discharge, co-infection with Neisseria gonorrhoeae) and asymptomatic patients with risk factors such as multiple partners, men who have sex with men, male and female sex workers, the clients of sex workers and contacts of patients with cervicitis and urethritis. Patients who had passed urine less than two hours before recruitment and who had taken antibiotics during the previous three weeks were excluded. For a CT prevalence rate of 13.9% symptomatic and 9.2% asymptomatic patients (De Silva et al. 2018), the calculated sample size was 184 symptomatic and 129 asymptomatic patients, with a margin of error of 0.05 and a 95% confidence interval (statistical formula http://www.calculator.net/sample-size-calculator.html). Prior to physical examination of patients, 30 ml of first void urine was collected and stored at 2-8oC, for not more than seven days before processing. Aliquots of fresh voided urine were stored at -80oC for repeat testing during optimization.
Positive and negative control urine
The positive control urine sample tested positive, both by a nested PCR (Somani et al. 2000) and the Artus® C. trachomatis Plus RG Real time PCR (Qiagen, Germany); the negative control urine sample was negative by both these tests. The positive/negative control urine samples were aliquoted and stored at -20oC and were used for all experiments.
Crude DNA extraction for LAMP (method-1)
Crude DNA extraction for LAMP was as described by Choopara et al. (2016) with a few modifications. Urine (1.5 ml) was centrifuged at 14,000g for 30 minutes. Supernatant was discarded and 40µl of urine deposit was heated with 20µl of 10mM Tris-HCl and 1mM EDTA buffer (pH 7.5) at 95°C for 5 minutes using a heating block (Choopara et al. 2016). It was cooled on ice until the liquid cleared on top and centrifuged at 17,000g for one minute. Crude extracts were prepared on the same day as the assay and the supernatant was used for the LAMP test.
Crude DNA extraction for LAMP (method-2)
To increase the sensitivity of the LAMP further, modifications were done to method − 1. Stored aliquots of urine (1.5ml) were thawed rapidly in a water bath at 37oC, followed by centrifugation at 14,000g for 30 minutes at room temperature. The supernatant was discarded and 200µl of phosphate buffered saline (PBS; pH 7.4) was added to dilute the urinary inhibitors, and centrifuged at 14,000 g for 30 minutes at room temperature. Following removal of the supernatant the pellet was dissolved in 200µl of PBS and heated at 95°C for 5 minutes with 100µl of Tris-EDTA (pH 7.4) buffer. Tubes were cooled on ice and centrifuged at 17,000g for 1 minute. The supernatant was used for LAMP.
Extraction of DNA from urine by QIAamp Viral RNA Kits
This kit is recommended for the use of DNA extraction from bacteria in urine (QIAamp Viral RNA Kits 2023). Extraction was performed according to the manufacturer’s instructions for the spin protocol. Hundred and forty microliters of urine were lysed and added to the spin columns with buffer and centrifuged. DNA was eluted in the given elution buffer. Aliquots of DNA were stored at -20oC for repeat assays.
Real-time PCR (rPCR)
DNA templates were prepared from patient urine using the QIAmp viral RNA mini kit (Qiagen, Germany). The Artus® C. trachomatis Plus RG Real time PCR (Qiagen, Germany) was performed according to the manufacturer’s instructions with the provided internal control. Real-time PCR results were used as the reference for determining the sensitivity and specificity of the LAMP assay.
Nested PCR
A nested PCR described by Somani et al. (2000) was used for confirming the rPCR results of the positive and negative controls. DNA extracted with QIAamp Viral RNA Mini kit was used and the nested PCR was performed as described by Somani et al. (2000) targeting the CT omp A gene.
LAMP-Sybr Gold
Positive and negative controls were used for optimizing the LAMP assay. A set of six published primers (Somboonna, 2016; petty patent 1503002132) FIP, BIP, F3, B3, FLP, BLP (Integrated DNA Technologies, USA), targeting the ompA gene of CT, was utilized for the LAMP assay (Choopara et al. 2016). Optimal LAMP assay conditions were determined for the following components; Betaine (0.5M, 0.8M), MgSO4 (6-10mM), Bsm DNA polymerase (4U, 6U, 8U), template volume (5–9µl), incubation temperature (55oC-60oC) and reaction time (40 and 60 minutes), using gel electrophoresis for detection. Three independent tests were performed for each component (Supplementary material, Fig. 1–4).
The optimized LAMP conditions for a 25 µl LAMP reaction were, 7 µl crude extract (method-1) in 1X buffer (20 mM Tris-HCl [pH 8.8], 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% (v/v) Tween 20), 1.6 µM FIP and BIP primers, 0.2 µM F3 and B3 primers, 1.4 µM FLP and BLP primers (Somboonna, 2016; petty patent 1503002132), 1.4mM deoxynucleoside triphosphate (Qiagen, Germany) each, 0.8M Betaine (Sigma Aldrich, USA), 6mM MgSO4, 8U Bsm polymerase large fragment (Thermo Scientific, USA). Before adding the crude extract, 12.5 µl of liquid paraffin was layered on top of the master mix and the template was deposited below this layer. One microliter of 10X Sybr™ Gold nucleic acid gel stain (Invitrogen, USA; Cat. No. S11494) was suspended from the inside of the lid of the reaction tube. It was incubated at 56°C for 60 min and the reaction was stopped by heating to 80oC for two minutes. At the end of incubation, the Sybr™ Gold stain (Invitrogen, USA; Cat. No. S11494) was incorporated in the reaction mix by vortexing. Positive reactions were detected by bright yellow flourescence under 320 nm UV illumination.
Analytical sensitivity and specificity
CT DNA extracted with QIAmp viral RNA mini kit (Qiagen, Germany), was quantitated using NanoDrop® Spectrophotometer. Analytical sensitivity of the optimized LAMP-Sybr Gold was determined using negative control urine, spiked with 10-fold serial dilutions of quantitated CT DNA (7.49 ng – 7.49x10− 4 fg; 8.06x109 − 8.06x10− 1 copies). The LAMP product was visualized using 320 nm UV light and 1.5% gel electrophoresis stained with Sybr™ Gold stain (Invitrogen, USA; Cat. No. S11494). Specificity of primers was confirmed by performing LAMP-Sybr Gold with negative control urine spiked with DNA from herpes simplex virus type 2, Neisseria gonorrhoeae, Trichomonas vaginalis, Candida albicans (obtained from NSACP, Sri Lanka) and Mycoplasma genitalium DNA (kind donation from Dr. Jørgen Skov Jensen, Statens Serum Institut, Copenhagen).
Diagnostic sensitivity and specificity
LAMP-Sybr Gold with crude extract (method-1) was evaluated using 326 first void urine samples collected from 189 symptomatic patients and 137 asymptomatic high risk patients. The investigator performing the LAMP assay was blinded to the results of rPCR assay which was the reference test. Results were recorded by visualizing the color change with the UV lamp.