Study design, participants and sample size
A retrospective study was conducted using archived conjunctival swab samples collected from children in trachoma-endemic communities in northern Tanzania to assess the performance of the DjinniChip. In February 2012, we recruited a cohort of children from three adjacent trachoma endemic villages in Kilimanjaro and Arusha regions, Northern Tanzania. All children, between the ages 3 and 11 years, who were normally resident in one of the three villages, were eligible for inclusion (n =666) [7]. 616 children were enrolled and the cohort was initially followed-up every 3 months for 4 years, for a total of 17 time points. Two conjunctival swab samples were collected consecutively from the right eye at each time point. Timepoint 14 (August 2015) was selected for this evaluation and 428 children were seen, 9 months after the last mass community treatment with antibiotic as detailed by the SAFE strategy. Conjunctival swabs were collected as previously described using sterile polyester-tipped swabs [7,8]. The first swab was collected into 250µl RNA later and was used elsewhere [7–9], whilst the second swab was collected dry and stored at -80°C until used in this study. 0.5% of swabs collected were ‘air’ control swabs in order to test for field and laboratory contamination. These samples were collected by passing a swab 10 cm from a participant's everted eye, these were labelled and processed identically to participant samples.
The study sample size was determined by the number of DjinniChips manufactured and shipped to KCRI-BL (Tanzania) for the study (n = 600) and the number of participants seen at timepoint 14 (n= 428). Based on the observed infection prevalence 9 months after MDA treatment round 1 (timepoint 7) and 2 (time point 11) the expected prevalence of ocular infection was estimated at 8%. The precision of the prevalence estimate based on this sample size at the 95% confidence level is 5%. Alternatively, assuming the index test (DjinniChip) achieves sensitivity and specificity of 95%, a sample size of 428 would achieve an odds of diagnosing a true (qPCR) positive of 62% (95% CI 51-72%) and an odds of 1% (95% CI 0 -2%) of a false negative.
The WHO endorsed SAFE strategy (surgery for trichiasis [in-turned eyelashes], antibiotics, facial cleanliness, and environmental improvement) was implemented in the study villages as approved by the Tanzanian Ministry of Health (MoH). Education about environmental improvements and facial hygiene was provided by the field team, free trichiasis surgery was offered, and all members of the three villages (including study participants) were offered azithromycin for trachoma control immediately following timepoints 3, 7 and 11 specimen collection in August of the years 2012, 2013, and 2014.
DNA extraction
Dry clinical samples were thawed and 400µl of lysis-amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1% Tween® 20, 1% Triton X, pH 8.8) was added to the tube. Samples were vortexed for 30 seconds, pulse centrifuged, and incubated for 10 minutes at 95°C for sample lysis. Lysates were then pulse centrifuged and incubated at room temperature for 10 minutes. For the positive control (PC), 46µl of lysis-amplification buffer was spiked with 4µl C. trachomatis DNA (10,000 omcB copies/µl) derived from C. trachomatis L2/434 which was originally cultured and prepared at the London School of Hygiene & Tropical Medicine (LSHTM). For the negative control (NC), 50 µl of lysis-amplification buffer was used. One hundred microliters of sample lysate was used for DNA extraction and C. trachomatis detection by quantitative PCR (qPCR) and 25µl was used for C. trachomatis detection by DjinniChip (see “DjinniChip detection of C. trachomatis”). Remaining sample lysate was stored at -20oC.
qPCR detection of C. trachomatis
One hundred microliters of sample lysate was mixed 1:1 with 100µl of sterile PBS and DNA was extracted using the QIAamp DNA mini kit (Qiagen Ltd, Manchester, UK) following the manufacturer’s instructions. DNA was eluted into 60µl of buffer AE and stored at 4oC prior to qPCR testing. C. trachomatis was detected using a reference qPCR assay, which has previously been evaluated against droplet digital PCR and Artus (Qiagen Ltd, Manchester, UK) C. trachomatis diagnostic assays for ocular samples [10]. The triplex qPCR assay detects chlamydial chromosomal (omcB) and plasmid (pORF2) targets and a human endogenous control gene (RPP30). Samples were tested in duplicate, with each 20µl reaction containing 1X TaqMan Multiplex Master Mix (Thermo Fisher Scientific, Inchinnan, UK), 300 nM of all primers and probes and 4 µl DNA. Samples were considered C. trachomatis positive if RPP30 in combination with pORF2 and/or omcB targets amplified in <40 cycles in either or both replicates. qPCR data were processed in STATA v15 and target concentrations were calculated by extrapolating from a standard curve. Clinical information from participants at timepoint 14 was available to the assessors of the reference qPCR test but the result of the index test was masked until the laboratory study was completed.
Droplet digital PCR (ddPCR) detection of C. trachomatis
ddPCR was used to detect and quantify C. trachomatis infectious load in 12 archived conjunctival clinical samples and in Quality Control for Molecular Diagnostics (QCMD, Glasgow, UK) panel samples for the preliminary evaluation of DjinniChips. Dry, frozen conjunctival swabs from 4 C. trachomatis-negative and 8 C. trachomatis-positive individuals, previously determined by an in-house 16S rRNA qPCR [11], were incubated for 10 minutes at room temperature in 400 µl elution buffer (1x PBS or 1% Triton X-100 + 10 mM Tris-HCl [pH7]). 200µl of eluate was frozen for DjinniChip evaluation. DNA was extracted from the remaining 200µl using the Blood and Serum DNA Isolation Kit (BioChain Institute Inc, Newark, CA, USA). For QCMD samples, DNA was extracted using the QIAamp Mini DNA Extraction (Qiagen Ltd, Manchester, UK). C. trachomatis plasmid and genome were quantified as previously described [10].
Preparation of DjinniChips
DjinniChip is an integrated diagnostic platform which amplifies DNA from the chromosomal porB gene of C. trachomatis using loop-mediated isothermal amplification (LAMP). The chips were produced by injection-moulding of cyclic olefin copolymer in Kunststoff-Zentrum in Leipzig (Germany). The reagents for LAMP were freeze-dried in the channel of the chip. Each portion of reaction mix contained 300 nmol MgSO4 , 16 U glycerol-free Bst 3.0 polymerase (New England Biolabs (Frankfurt am Main, Germany), 25 mmol dA, dG, dC, 22.5 mmol dU, 0.5 mmol dT (Jena Bioscience,Jena, Germany), 10 pmol of primer B3 and F3, 80 pmol primer BIP, 60 pmol unlabelled primer FIP, 20pmol 5’-biotin-labelled FIP, 40 pmol 5’-FAM-labelled primer LB and 40 pmol 5’-biotin-labelled primer LF (Eurofins Genomics, Ebersberg, Germany), 0.25 mg/ml (w/v) BSA and 6% trehalose (Sigma Aldrich, Taufkirchen, Germany). The primer sequences are provided in Table 1. The freeze-dried reagents were overlayed with a 4.3 x 35 mm PES filter membrane (Merck Millipore, Darmstadt, Germany), 0.22 µm pore size. A lateral flow test strip DetectLine Basic (Amodia, Braunschweig, Germany) was placed in the chip, and the bottom surface of the chip was sealed with Masterclear® real-time PCR Film (Eppendorf, Hamburg, Germany). DjinniChips were manufactured <6 weeks prior to testing and were stored in vacuum-sealed sachets at 4°C until use. The quality of each batch of the chips was verified by testing a positive (0.001 ng C. trachomatis DNA) and a negative control on random chips from the batch.
DjinniChip detection of C. trachomatis
Twenty-five microliters of sample lysate (see “DNA extraction” section method) was mixed with 25µl lysis-amplification buffer and the entire 50µl was added to the DjinniChip. Sample extract was driven by capillary effect into the reaction channel, dissolving the lyophilized reagents. The inlets of the chip were sealed with Air-O-Seal membrane (4titude, Berlin, Germany). Following incubation on the flat bed of a heat block for 35 min at 65°C for the LAMP reaction, the reaction was stopped by placing the DjinniChip on a flat ice block (cooling battery) for 2-5 minutes. The amplified product was then manually pumped towards the integrated lateral flow strip (LFS) by injecting 180µl chromatographic buffer (Amodia, Braunschweig, Germany) into the sample inlet. Biotin- and fluorescein (FAM)-labelled amplicons allowed LFS detection after 10 minutes incubation at room temperature, by use of gold nanoparticles attached to anti-fluorescein-antibodies (Figure 1). A positive control signal (C) and signal at the test zone (T) corresponded to C. trachomatis in the patient sample. Results were recorded electronically and photographic records were taken of each test result.
Samples were analysed using a second DjinniChip if a) the negative no-template control (NC) was weakly or strongly positive for the analysed batch of samples, or b) if the test band was only weakly positive (Figure S1). In either of these cases positive samples were re-tested, with additional positive and negative controls. The outcome of the second test was used as the final result: if the NC was negative but the sample was weakly positive again, the sample was determined positive. Clinical information and the reference test results were masked to the assessors of the Djinnichip results. The study was conducted and reported according to the STARD guidelines for diagnostic studies (Additional file 1. Table S1.)[12]
Mock samples
In order to determine the resilience and usability of the DjinniChip, 100 mock samples were tested in field conditions. Mock samples consisted of a 50µl suspension of C. trachomatis A2497 elementary bodies at 4 different concentrations (below) in PBS which were added to sterile polyester-tipped swabs and stored dry in 1.5 ml tubes at -80°C for a minimum of one week. C. trachomatis A2497 was grown in HEp-2 cells and purified as previously described and quantified by ddPCR [13]. Four field sites were selected; two humid and warm and two hot, dry and dusty. Results were compared to replicate mock samples tested on DjinniChips at Kilimanjaro Clinical Research Institute-Biotechnology Laboratory (KCRI-BL). At each of the five sites, 20 mock samples ranging in C. trachomatis concentration were tested (quadruplicates of negative control, 160 copies/swab, 800 copies/swab, 3200 copies/swab, 16000 copies/swab); 10 duplicates by an experienced lab technician and 10 duplicates by a field nurse who received basic training. A heat block (Thermo Fisher Scientific, Inchinnan, UK) powered from a 12V direct-current car battery was used to heat samples at mobile field sites. DjinniChip testing was performed in the open boot of a stationary Land Rover. The field team drove to each field site (selected to provide a range of typical trachoma environmental conditions), where testing was performed in the open boot of the stationary vehicle. Samples were transported on ice in a cool box and reactions were stopped on the surface of flat ice blocks. Results (including photograph), temperature, humidity and time to process 10 tests was recorded. Remaining sample extract was frozen at -20oC prior to DNA extraction (Qiagen Ltd, Manchester, UK) and qPCR testing for C. trachomatis.