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PloS one2020; 15(8); e0237187; doi: 10.1371/journal.pone.0237187

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

Abstract: 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. 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). 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). 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. 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). 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.
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Publication Date: 2020-08-24 PubMed ID: 32833981PubMed Central: PMC7444819DOI: 10.1371/journal.pone.0237187Google Scholar: Lookup
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  • Comparative Study
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research investigates the effectiveness of Loop-mediated isothermal amplification (LAMP) compared to PCR (Polymerase chain reaction) for diagnosing the infection of Trypanosoma brucei in equids in The Gambia. The results show that the LAMP technique is comparable to PCR when applied to Cerebrospinal Fluid (CSF) samples in field conditions, providing an affordable and reliable tool for clinical decisions in areas with limited resources.

Methodology

  • The researchers used whole equine blood specimens with known concentrations of Trypanosoma brucei parasites to determine the detection threshold for LAMP, the low-cost molecular diagnostic test.
  • The results of LAMP were compared to the established gold standard for Trypanosoma brucei diagnosis, T. brucei-PCR (TBR-PCR).
  • The performance of LAMP and its agreement with TBR-PCR at high and lower concentrations of parasites was also studied.

Results

  • The study revealed that the detection threshold for Trypanosoma brucei using LAMP and TBR-PCR on extracted DNA from whole blood was 1 parasite/ml blood, showing excellent agreement between the tests at high parasite concentrations.
  • Detection threshold improved to 10 parasites/ml when a detergent was included in the LAMP process.
  • The performance of LAMP was found to be excellent at high parasite concentrations, but was inconsistent at lower concentrations.

Field Study

  • A cross-sectional study was also conducted in The Gambia on working equids showing clinical signs of trypanosomiasis.
  • Whole blood samples from 321 equids were processed under field conditions and cross-checked with LAMP and TBR-PCR.
  • The study showed weak agreement between the two methods on whole blood samples but excellent agreement on CSF samples.

Conclusion and Implications

  • The findings support the use of LAMP as a comparable, cost-effective alternative to PCR when used on CSF samples in field conditions.
  • However, the results suggest repeatability is low in animals with low parasitaemia levels, therefore negative samples should be interpreted in the context of the animal’s clinical presentation.

Cite This Article

APA
Gummery L, Jallow S, Raftery AG, Bennet E, Rodgers J, Sutton DGM. (2020). Comparison of loop-mediated isothermal amplification (LAMP) and PCR for the diagnosis of infection with Trypanosoma brucei ssp. in equids in The Gambia. PLoS One, 15(8), e0237187. https://doi.org/10.1371/journal.pone.0237187

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 15
Issue: 8
Pages: e0237187
PII: e0237187

Researcher Affiliations

Gummery, Lauren
  • Weipers Centre Equine Hospital, School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Jallow, Saloum
  • Gambia Horse and Donkey Trust, Sambel Kunda, The Gambia.
Raftery, Alexandra G
  • Weipers Centre Equine Hospital, School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Bennet, Euan
  • Weipers Centre Equine Hospital, School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Rodgers, Jean
  • Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Sutton, David G M
  • Weipers Centre Equine Hospital, School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

MeSH Terms

  • Animals
  • Cross-Sectional Studies
  • DNA, Protozoan / blood
  • DNA, Protozoan / genetics
  • Female
  • Gambia
  • Horse Diseases / parasitology
  • Horses / parasitology
  • Male
  • Molecular Diagnostic Techniques / economics
  • Molecular Diagnostic Techniques / methods
  • Nucleic Acid Amplification Techniques / economics
  • Nucleic Acid Amplification Techniques / methods
  • Polymerase Chain Reaction / economics
  • Polymerase Chain Reaction / methods
  • Prospective Studies
  • Sensitivity and Specificity
  • Trypanosoma brucei brucei / genetics
  • Trypanosomiasis, African / diagnosis
  • Trypanosomiasis, African / parasitology
  • Trypanosomiasis, African / veterinary

Conflict of Interest Statement

LAMP kits and LAMP incubator for field use were provided free of charge by Joseph M N’dungu and FIND Diagnostics. These parties had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare that they have no competing interests.

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