Using detergent-enhanced LAMP for African trypanosome detection in human cerebrospinal fluid and implications for disease staging

Abstract

Objective Where human African trypanosomiasis (HAT) patients are seen, failure to microscopically diagnose infections by Trypanosoma brucei gambiense in blood smears and/or cerebrospinal fluid (CSF) in the critical early stages of the disease is the single most important factor in treatment failure, a result of delayed treatment onset or its absence. We hypothesized that the enhanced sensitivity of detergent-enhanced loop-mediated isothermal amplification (LAMP) will allow for point of care (POC) detection of African trypanosomes in the CSF of HAT patients where the probability for detecting a single parasite or parasite DNA molecule in 1 μL of CSF sample is negligible by current methods. Methodology We used LAMP targeting the multicopy pan- T . brucei repetitive insertion mobile element (RIME LAMP) and the Trypanosoma brucei gambiense 5.8S rRNA-internal transcribed spacer 2 gene (TBG1 LAMP). We tested 1 μL out of 20 μL sham or Triton X-100 treated CSFs from 73 stage-1 and 77 stage-2 HAT patients from the Central African Republic and 100 CSF negative controls. Results Under sham conditions, parasite DNA was detected by RIME and TBG1 LAMP in 1.4% of the stage-1 and stage-2 gambiense HAT CSF samples tested. After sample incubation with detergent, the number of LAMP parasite positive stage-2 CSF’s increased to 26%, a value which included the 2 of the 4 CSF samples where trypanosomes were identified microscopically. Unexpected was the 41% increase in parasite positive stage-1 CSF’s detected by LAMP. Cohen’s kappa coefficients for RIME versus TBG1 LAMP of 0.92 (95%CI: 0.82–1.00) for stage-1 and 0.90 (95%CI: 0.80–1.00) for stage-2 reflected a high level of agreement between the data sets indicating that the results were not due to amplicon contamination, data confirmed in χ 2 tests (p<0.001) and Fisher’s exact probability test (p = 4.7e -13 ). Conclusion This study detected genomic trypanosome DNA in the CSF independent of the HAT stage and may be consistent with early CNS entry and other scenarios that identify critical knowledge gaps for future studies. Detergent-enhanced LAMP could be applicable for non-invasive African trypanosome detection in human skin and saliva or as an epidemiologic tool for the determination of human (or animal) African trypanosome prevalence in areas where chronically low parasitemias are present.

Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections

“…RIME and SRA positive samples were considered T. brucei rhodesiense, whilst if they were SRA negative, they could have been T. brucei brucei or T. brucei gambiense. LAMP sensitivity for this sequence was much greater than that for sensitivity by PCR (0.1 to 1000 trypanosomes/mL) [27].…”

“…The increased sensitivity associated with heat treatment could be due to faster DNA strand separation resulting in a more efficient LAMP test. Using heat-treated samples without compromising sensitivity eliminates the need for DNA extraction and further shortens the time to having results [27] also reducing the cost of and need for extensive laboratory infrastructure [25].…”

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

“…Positive test results were observed using LAMP on a whole blood template (LWB) with a detection limit of 100 parasites/ml which was improved to 10 parasites/ml by the addition of SDS (LSDS) in the laboratory threshold study. Inclusion of detergents causing cell lysis has been described in whole blood template, CSF and prior to application of blood to FTA cards and improves sensitivity of LAMP [26,44]. This step was not included in the field study here due to the increased risk of sample contamination.…”

“…However, LAMP on SDS treated samples has been used for T.b. gambiense detection in human CSF with promising results [44], and could be evaluated in field-based studies in equids in the future.…”

Sleeping Sickness at the Crossroads

“…The introduction of rapid diagnostic tests is a true advancement, but also lacks the specificity needed to make a final treatment decision [31]. Additional tools were recently developed but so far only introduced to a limited extent into routine use (e.g., loop-mediated isothermal amplification (LAMP) [32,33]; immune trypanolysis test [34]), which will both play a role in the "end-game". The question, however, is how such tests will be used in the future, and in what settings.…”