Comparison of loop-mediated isothermal amplification (LAMP) and PCR for the diagnosis of infection with Trypanosoma brucei ssp. in equids in The Gambia

Abstract

Introduction: Infection of equids with Trypanosoma brucei (T. brucei) ssp. is of socioeconomic importance across sub-Saharan Africa as the disease often progresses to cause fatal meningoencephalitis. Loop-mediated isothermal amplification (LAMP) has been developed as a cost-effective molecular diagnostic test and is potentially applicable for use in field-based laboratories.

Part i: Threshold levels for T. brucei ssp. detection by LAMP were determined using whole equine blood specimens spiked with known concentrations of parasites. Results were compared to OIE antemortem gold standard of T. brucei-PCR (TBR-PCR).

Results i: Threshold for detection of T. brucei ssp. on extracted DNA from whole blood was 1 parasite/ml blood for LAMP and TBR-PCR, and there was excellent agreement (14/15) between tests at high (1 x 103/ml) concentrations of parasites. Detection threshold was 100 parasites/ml using LAMP on whole blood (LWB). Threshold for LWB improved to 10 parasites/ml with detergent included. Performance was excellent for LAMP at high (1 x 103/ml) concentrations of parasites (15/15, 100%) but was variable at lower concentrations. Agreement between tests was weak to moderate, with the highest for TBR-PCR and LAMP on DNA extracted from whole blood (Cohen's kappa 0.95, 95% CI 0.64-1.00).

Part ii: A prospective cross-sectional study of working equids meeting clinical criteria for trypanosomiasis was undertaken in The Gambia. LAMP was evaluated against subsequent TBR-PCR.

Results ii: Whole blood samples from 321 equids in The Gambia were processed under field conditions. There was weak agreement between LWB and TBR-PCR (Cohen's kappa 0.34, 95% CI 0.19-0.49) but excellent agreement when testing CSF (100% agreement on 6 samples).

Conclusions: Findings support that LAMP is comparable to PCR when used on CSF samples in the field, an important tool for clinical decision making. Results suggest repeatability is low in animals with low parasitaemia. Negative samples should be interpreted in the context of clinical presentation.

Fig 1. Sample table demonstrating design of in vitro experiment with whole blood containing serial dilutions of trypanosomes at a range of packed cell volumes.

Fig 2. Threshold study: Test sensitivity at varying parasite concentrations in blood.

The ability of various tests to detect trypanosomes at concentrations varying from 1000 trypanosomes/ml to 1 trypanosome/ml was assessed. The results are depicted in the form of a heat map with detection ranging from 100% to 0% across the various samples and tests. LWB: LAMP on whole blood template; LSDS: LAMP on whole blood treated with SDS detergent; LEX: LAMP on DNA extracted from whole blood; LFTA: LAMP on DNA extracted from FTA cards; PCREX: TBR-PCR on DNA extracted from whole blood; PCRFTA: TBR-PCR on DNA extracted from FTA cards.

Fig 3. LAMP test result at range of PCVs at a concentration of 1000 parasites/ml in processed blood template.

From left to right; negative control, 10% (+), 20%(+), 30% (+), 40% (+), 50% (+), positive control. Fluorescent result is visible in all test tubes. A small amount of cellular debris is visible at the bottom of the 50% tube (upper panel). This is confirmed by imaging in a UV transilluminator, where the positive results show fluorescence (lower panel).

Fig 4. Threshold study: Number of test positives at a range of packed cell volumes at between 10 and 1000 parasites/ml in processed blood template.

The number of positive results across the range of packed cell volumes (PCV; 10% to 50% at increments of 10%) is depicted by the height of the bars. Colour of the stacked bar represents type of test. Variability at the different packed cell volumes is present but generally inconsistent, with higher numbers of positive results at 10% and 50%. LWB: LAMP on whole blood template; LSDS: LAMP on whole blood treated with SDS detergent; LEX: LAMP on DNA extracted from whole blood; LFTA: LAMP on DNA extracted from FTA cards; PCREX: TBR-PCR on DNA extracted from whole blood; PCRFTA: TBR-PCR on DNA extracted from FTA cards.

Fig 5. Threshold study: Chart showing cumulative positive test results over 3 rounds of analysis at concentrations above 10 parasites/ml (15 tests repeated in triplicate; n = 45).

The number of test positive samples increases (between 1 and 3 additional positive samples) for all tests other than LSDS over 3 test replicates. Tests on DNA extracted from whole blood (PCREX and LEX) and LAMP on detergent treated blood (LSDS) appear to detect higher numbers of positive samples overall compared to those on DNA extracted from FTA cards (LFTA and PCRFTA) or LAMP on a whole blood template (LWB). LWB: LAMP on whole blood template; LSDS: LAMP on whole blood treated with SDS detergent; LEX: LAMP on DNA extracted from whole blood; LFTA: LAMP on DNA extracted from FTA cards; PCREX: TBR-PCR on DNA extracted from whole blood; PCRFTA: TBR-PCR on DNA extracted from FTA cards.

Fig 6. Field study: Diagram of samples analysed.

Total number of included field-acquired samples was 372 after exclusions. LAMP analysis on whole blood (LWB), DNA extraction from whole blood and FTA card application were performed in the field. LEX, LFTA, PCREX and PCRFTA were performed in the laboratory. Comparisons between results (Cohen’s kappa) were performed between LWB and laboratory tests, and also between LAMP and PCR results from the same DNA samples. LWB: LAMP on whole blood template; LSDS: LAMP on whole blood treated with SDS detergent; LEX: LAMP on DNA extracted from whole blood; LFTA: LAMP on DNA extracted from FTA cards; PCREX: TBR-PCR on DNA extracted from whole blood; PCRFTA: TBR-PCR on DNA extracted from FTA cards.

Fig 7. Field study: Chart to show cumulative positive results compared to number of repeat LAMP assays on a single sample.

The number of individuals with a positive test results (red) is plotted against the total number of test replicates performed (blue, LAMP test on whole blood; LWB). The number of positive individuals increases up to 4 test replicates but starts to plateau between 2 and 4 test replicates.