A new PCR–ELISA for diagnosis of visceral leishmaniasis in blood of HIV-negative subjects

Abstract

The PCR-ELISA represents a promising advance for diagnosis of visceral leishmaniasis (VL) in blood samples. However, the method has been validated mostly with HIV-positive patients who are known to have high levels of parasitaemia. We developed a new PCR-ELISA assay for specific detection of Leishmania in patients' blood and validated it in Nepalese subjects with clinically suspected VL, almost all of whom were HIV-negative. For blood samples, PCR-ELISA was more sensitive (83.9%) than conventional PCR (73.2%), and demonstrated 100% and 87.2% specificity when using healthy controls who had never travelled to a VL-endemic area and controls from a VL-endemic area as references, respectively. We have demonstrated the ability of PCR-ELISA to detect parasites in blood of HIV-negative patients. The method could be used for epidemiological as well as clinical purposes, as it reduces the need for traumatic bone marrow sampling and risky spleen aspiration.

Diagnosis of leishmaniasis

“…A PCR-ELISA was used to diagnose VL in HIVnegative patients; peripheral blood was used and yielded a high sensitivity [104]. A similar PCR-based technique was applied in the diagnosis of VL in HIV patients, with good results [28].…”

Molecular Dipstick Test for Diagnosis of Sleeping Sickness

“…Venous blood on EDTA was obtained from 28 healthy human Belgian blood donors who had never visited a HAT-endemic country (6).…”

Molecular Diagnosis of Leishmaniasis: Current Status and Future Applications

“…The method was claimed to be 100 times more sensitive than conventional PCR in the detection of Trypanosoma brucei (11), but thus far, this has-to our knowledge-not been applied to leishmaniasis diagnosis yet. Simplification of detection has been attempted by PCR-enzyme-linked immunosorbent assay (ELISA), a "reverse hybridization" method based on the capture of PCR amplicons by specific probes immobilized in ELISA microtiter wells and colorimetric visualization (6). High sensitivity was observed in blood samples from HIVnegative VL patients (6).…”

“…Simplification of detection has been attempted by PCR-enzyme-linked immunosorbent assay (ELISA), a "reverse hybridization" method based on the capture of PCR amplicons by specific probes immobilized in ELISA microtiter wells and colorimetric visualization (6). High sensitivity was observed in blood samples from HIVnegative VL patients (6). However, in PCR-ELISA, detection is still dependent on sophisticated equipment (i.e., an ELISA plate reader).…”

“…This application is required for differential diagnosis before initiating therapy, and the performances of PCR have consistently been shown to be better than microscopy or parasite culture, particularly in samples with low parasite loads (e.g., in ML patients [9]) or in samples from less intrusive sources, such as blood (4) and conjunctiva (24). The contribution of PCR also appears to be particularly relevant for the diagnosis of leishmaniasis in patients coinfected with HIV (2,4,6). Parasite detection by PCR for confirmation of a clinical cure appears to be important in VL (17) but should be further explored in CL since up to 80% of patient scars remain PCR positive, even 8 years after their clinical cure (22).…”