Utility of the Loop-Mediated Isothermal Amplification Assay for the Diagnosis of Visceral Leishmaniasis from Blood Samples in Ethiopia

Abstract

Rapid and accurate diagnosis of visceral leishmaniasis (VL) is needed to initiate prompt treatment to reduce morbidity and mortality. Here, we evaluated the performance of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of VL from blood in an endemic area in Ethiopia. LAMP was positive in 117/122 confirmed VL cases and negative in 149/152 controls, resulting in a sensitivity of 95.9% (95% CI: 90.69–98.66) and a specificity of 98.0% (95% CI: 94.34–99.59), respectively. The sensitivity of the LAMP assay was 95.0% (95% CI: 88.61–98.34) in HIV-negatives and 100% (95% CI: 85.18–100.0) in HIV-positives. Compared with microscopy, LAMP detected 82/87 (94.3%, 95% CI: 87.10–98.11) of the microscopy+ cases and was negative in 11/27 (40.7%, 95% CI: 22.39–61.20) of the microscopy− cases. Compared with the rK39 serology, LAMP detected 113/120 (94.2%, 95% CI: 88.35–97.62) of the rK39+ cases and was negative in 149/154 (96.8%, 95% CI: 92.59–98.94) of the rK39− cases. However, when compared with microscopy only, rK39 detected 83/87 (95.4%, 95% CI: 88.64–98.73) of the microscopy+ cases and negative in only 12/27 (44.4%, 95% CI: 25.48–64.67) of the microscopy– cases. There was an excellent agreement between rK39 and LAMP (Kappa = 0.91, 95% CI: 0.86–0.96). Furthermore, an algorithm using rK39 followed by LAMP would yield a sensitivity of 99.2% (95%CI: 95.52–99.89) and a specificity of 98.0% (95% CI: 94.34–99.59). The findings demonstrate that LAMP assay is an accurate and rapid molecular assay for VL diagnosis, including in HIV-1 coinfected patients, in an endemic setting.

Comparison of the Diagnostic Performances of Five Different Tests in Diagnosing Visceral Leishmaniasis in an Endemic Region of Ethiopia

“…The LAMP assay was performed using the LoopAmp Leishmania kit as per the manufacturer's instructions (Eiken Chemical Co., Tokyo, Japan) [44]. The kit uses 6 primers that target conserved Leishmania gene segments kDNA and 18S rRNA, respectively [13]. The LAMP assay utilized Bst polymerase, which has auto-DNA denaturing and amplification properties [33].…”

“…In most VL-endemic, resource-limited countries, the diagnosis of VL relies on a combination of the patient's clinical presentation and rapid serological tests and/or microscopy of Giemsa-stained tissue aspirates [12][13][14]. Despite its poor performance and risky specimen collection procedures, microscopy is often still used as the reference standard for VL diagnoses in many endemic countries.…”

PCR for detection of Leishmania donovani from microscopically negative tissue smears of suspected patients in Gondar, Ethiopia

“…Such algorithm would prioritize bone marrow samples, and patients with splenomegaly and especially low WBC values to be tested as they have a higher chance of being PCR positive. Alternatively, an affordable and simple test (e.g., loopmediated isothermal amplification [28] or a novel RDT) on a non-invasive sample like peripheral blood would be very useful to include in the diagnostic algorithm in VL endemic areas for early diagnosis and treatment of VL patients.…”

Comparison of the Diagnostic Performance of Five Different Tests to Diagnose Visceral Leishmaniasis in an Endemic Region of Ethiopia

“…The LAMP assay was performed using LoopAmp Leishmania kit (Eiken Chemical Co., Tokyo, Japan) [44]. The kit employed 6 primers that target conserved Leishmania gene segment [13]: kDNA and 18S rRNA. LAMP assay utilized Bst polymerase, which has auto-DNA denaturing and amplification properties [33].…”

“…In most VL-endemic resource-limited countries, the diagnosis of VL relies on a combination of the patient's clinical presentation and rapid serological tests and/or microscopy of Giemsa-stained tissue aspirate/s [12][13][14]. Despite its poor performance and risky specimen collection procedures, microscopy is still often used as the reference standard for VL diagnosis in many endemic countries.…”