Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by Leishmania infantum When Compared to PCR.

Emilia García,Joseph Mathu Ndung’u,Israel Cruz,Sheila Ortega,Carmen Chicharro,Eugenia Carrillo,Ana Victoria Ibarra-Meneses,Carmen Sánchez,Javier Moreno

doi:10.3390/microorganisms9030610

Abstract

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Leishmania Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by L. infantum. A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by Leishmania nested PCR (LnPCR) were tested by: (i) the Loopamp™ Leishmania Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval—CI: 96.0–100.00) sensitivity and specificity of 97.7% (95% CI: 92.2–100) on VL samples, and 100% (95% CI: 99.1–100) sensitivity and 100.0% (95% CI: 98.8–100.0) specificity on CL samples. The Loopamp time-to-positivity (Tp) obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR’s cycle threshold (Ct). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to L. infantum. The excellent correlation between the Tp and Ct should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.

Highlights

Leishmaniasis is a vector-borne parasitic disease caused by more than 20 species of Leishmania
We demonstrated the analytical performance of LoopampTM Leishmania detection kit [13]
The results of the repeat Leishmania nested PCR (LnPCR) were the same as when the samples were first tested, confirming the integrity of the samples selected for this study

Summary

Introduction

Leishmaniasis is a vector-borne parasitic disease caused by more than 20 species of Leishmania. Cutaneous leishmaniasis (CL) is the most common, and about 0.6 to 1 million new cases are reported annually, these are caused by many different Leishmania species. Visceral leishmaniasis (VL) can be lethal without early treatment, is caused by the species Leishmania donovani and L. infantum; between 50,000 and 90,000 new cases occur annually, and being lethal without treatment makes it one of the deadliest parasitic disease [1]. To determine the concordance and Cohen’s κ coefficient analysis, we defined true positive, true negative, false positive, and false negative samples among the different tests The correlation between qPCR Ct/parasite load and LAMP Tp was estimated using Pearson’s coefficient. The correlations between the LAMP Tp, the qPCR Ct and parasite load, were determined using the same GraphPad Prism v.7.0 package

Methods

Results

Discussion

Conclusion