Abstract
BackgroundConfirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp Leishmania Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results.Methodology/Principal findingsThe Loopamp Leishmania Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite Leishmania rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%.Conclusions/SignificanceDue to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.
Highlights
Either from spleen, bone marrow or lymph node, remains the Gold Standard for parasitological confirmation in patients suspected of visceral leishmaniasis (VL), and is often used for detection of relapses, and as a test of cure
Nucleic acid amplification tests (NAAT) are sensitive for the detection of Leishmania parasites in blood; in Visceral leishmaniasis (VL)-endemic settings, most NAAT are restricted to well-equipped laboratories
We have evaluated this kit in Sudan and obtained a sensitivity of 97.6% and specificity of 99.1%, using DNA obtained from peripheral blood through a simple boil and spin method
Summary
Visceral leishmaniasis (VL), known as kala-azar, is a vector-borne disease caused by parasitic protozoa of the Leishmania donovani complex, which are transmitted by female sandflies. VL affects mainly children and young adults, and can be fatal if left untreated It is characterized by fever, weight loss, wasting, and splenomegaly; lymphadenopathy is especially frequent in VL patients in Sudan, where it can be the only clinical manifestation accompanying fever [1]. Despite its limited sensitivity (52–65%), examination of lymph node aspirates is preferred over bone marrow or spleen aspirates because it is easier and safer, and can be performed by paramedical staff. Examination of bone marrow or splenic aspirates improves sensitivity (75–95%), but this requires experienced personnel, and should be performed in hospitals where blood transfusion is available, which might be needed in case of an adverse event. Bone marrow and splenic aspiration, are rarely performed in Sudan due to technical and logistical difficulties, and are limited to reference laboratories [3,5,6]
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