Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP).

Dennis J Grab,Liam J Morrison,Oriel M M Thekisoe,Olga V Nikolskaia,J Stephen Dumler,Noboru Inoue,Wendy Gibson

doi:10.1371/journal.pntd.0001249

Abstract

BackgroundThe loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.Methodology/Principal FindingsFor proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.Conclusions/SignificanceThis simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.

Highlights

Tsetse fly-transmitted African trypanosomes are major pathogens of humans and livestock
loop-mediated isothermal amplification (LAMP) with genomic parasite DNA Based on experiments repeated at least 3 times, LAMP assays successfully amplified T. b. rhodesiense DNA within 55–60 min at 62uC (RIME LAMP) or 63uC (PSEUDO-serum resistance associated (SRA) LAMP)
As rhodesiense IL1852 was spiked into human cerebrospinal fluid (CSF) without and with 0.5% Triton X-100 (+Tx)

Summary

Introduction

Tsetse fly-transmitted African trypanosomes are major pathogens of humans and livestock. Gambiense) cause human African trypanosomiasis (HAT, commonly called sleeping sickness). Gambiense (West and Central African HAT) or weeks to months with T. b. Rhodesiense HAT, but melarsoprol leads to severe and fatal encephalitis in about 5–10% of recipients despite treatment for this condition [3,4,5]. Where HAT is endemic, accurate staging is critical, because failure to treat CNS involvement leads to death, yet inappropriate CNS treatment exposes an early-stage patient unnecessarily to highly toxic and life-threatening drugs. The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by adding detergent to the samples prior to LAMP assay

Methods

Results

Conclusion