“…Previous studies have shown that Leishmania antigens can be detected in urine, however both the sensitivity (28-82%) and specificity (49-53%) of this method were unacceptably low (Galluzzi et al, 2018;Abeijon et al, 2019;Mondal et al, 2019). An alternative immunological based assay is to detect antibody anti Leishmania rK39 antigen with sensitivity of 67-100% (Pagliano et al, 2016;van Griensven and Diro, 2019), however, this immunological assay is unable to differentiate between current and historical infection (Saliba et al, 2019). Regular PCR is sensitive, but cannot be used to monitor the parasite load during medical treatment, and so is not appropriate for ongoing patient/disease management, or evaluating the treatment efficacy (Mesa et al, 2020).…”
Leishmaniasis is still a serious neglected tropical disease that may cause death in infected individuals. At present, the clinical diagnosis and treatment monitoring still rely on parasitological culture and microscopy that needs experienced technicians. The low sensitivity and inconvenience of microscopic examination could cause misdiagnosis and relapse of leishmaniasis. There is an urgent need for developing a sensitive and easily operated diagnostic method for the diagnosis and disease management of leishmaniasis. Thus, a quantitative real-time PCR (qPCR) based on the conversed regions of kinetoplast minicircle DNA (mkDNA) of Leishmania spp. was developed to detect different species of Leishmania. The designed mkDNA-based qPCR was able to detect as low as one copy of Leishmania mkDNA or DNA from single parasite. It also detected Pan-Leishmania protozoa including Leishmania donovani, Leishmania infantum and Leishmania major without cross-reaction with other pathogen DNAs available in our lab. This method was clinically applied to quantitatively detect skin lesion samples from 20 cutaneous leishmaniasis (CL) and bone marrow and/or PBMC samples from 30 current and cured visceral leishmaniasis (VL) patients, and blood samples from 11 patients with other infections and 5 normal donors as well. Total 20 skin lesion samples from current CL patients and 20 bone marrow and/or PBMC samples from current VL patients were all detected as positive with qPCR without cross-reaction with samples from patients with malaria, brucellosis and dengue or normal donors. Two VL patients with parasite converted to microscopically negative after treatment were detected positive with qPCR. The patients with bigger skin lesion in CL and higher level of immunoglobulin or splenomegaly in VL, had the higher parasite load detected by qPCR. The parasite load was significantly reduced after treatment. In conclusion, the mkDNA-based qPCR assay that we developed in this study can be used not only for diagnosis of both cutaneous and visceral leishmaniasis with high sensitivity and specificity, but also for evaluating the severity and treatment efficacy of this disease, presenting a rapid and accurate tool for clinical surveillance, treatment monitoring and the end point determination of leishmaniasis.
“…Previous studies have shown that Leishmania antigens can be detected in urine, however both the sensitivity (28-82%) and specificity (49-53%) of this method were unacceptably low (Galluzzi et al, 2018;Abeijon et al, 2019;Mondal et al, 2019). An alternative immunological based assay is to detect antibody anti Leishmania rK39 antigen with sensitivity of 67-100% (Pagliano et al, 2016;van Griensven and Diro, 2019), however, this immunological assay is unable to differentiate between current and historical infection (Saliba et al, 2019). Regular PCR is sensitive, but cannot be used to monitor the parasite load during medical treatment, and so is not appropriate for ongoing patient/disease management, or evaluating the treatment efficacy (Mesa et al, 2020).…”
Leishmaniasis is still a serious neglected tropical disease that may cause death in infected individuals. At present, the clinical diagnosis and treatment monitoring still rely on parasitological culture and microscopy that needs experienced technicians. The low sensitivity and inconvenience of microscopic examination could cause misdiagnosis and relapse of leishmaniasis. There is an urgent need for developing a sensitive and easily operated diagnostic method for the diagnosis and disease management of leishmaniasis. Thus, a quantitative real-time PCR (qPCR) based on the conversed regions of kinetoplast minicircle DNA (mkDNA) of Leishmania spp. was developed to detect different species of Leishmania. The designed mkDNA-based qPCR was able to detect as low as one copy of Leishmania mkDNA or DNA from single parasite. It also detected Pan-Leishmania protozoa including Leishmania donovani, Leishmania infantum and Leishmania major without cross-reaction with other pathogen DNAs available in our lab. This method was clinically applied to quantitatively detect skin lesion samples from 20 cutaneous leishmaniasis (CL) and bone marrow and/or PBMC samples from 30 current and cured visceral leishmaniasis (VL) patients, and blood samples from 11 patients with other infections and 5 normal donors as well. Total 20 skin lesion samples from current CL patients and 20 bone marrow and/or PBMC samples from current VL patients were all detected as positive with qPCR without cross-reaction with samples from patients with malaria, brucellosis and dengue or normal donors. Two VL patients with parasite converted to microscopically negative after treatment were detected positive with qPCR. The patients with bigger skin lesion in CL and higher level of immunoglobulin or splenomegaly in VL, had the higher parasite load detected by qPCR. The parasite load was significantly reduced after treatment. In conclusion, the mkDNA-based qPCR assay that we developed in this study can be used not only for diagnosis of both cutaneous and visceral leishmaniasis with high sensitivity and specificity, but also for evaluating the severity and treatment efficacy of this disease, presenting a rapid and accurate tool for clinical surveillance, treatment monitoring and the end point determination of leishmaniasis.
“…It is useful for quick screening and serodiagnosis of disease in epidemiologic studies in endemic areas. The efficacy of an immunoblotting/Western blotting method for the diagnosis of VL is very high 95,96 . Immunoblotting has a satisfactory performance in the diagnosis of CL, 79 and this test can be used, as an aid, for proper diagnosis of VL 90,97 .…”
Section: Diagnosis Of Visceral Leishmaniasismentioning
Leishmania donovani (a causative agent of visceral leishmaniasis) poses a serious health threat to the human population which is fatal if left untreated. The life cycle of Leishmania alternates between vertebrate host and Phlebotomine fly as intermediate ones. Due to the difficulties linked to vector (sandfly) control and the lack of an effective vaccine, the control of leishmaniasis relies mostly on chemotherapy. Unfortunately, the prevalence of parasites becoming resistant to the first‐line drug pentavalent antimonial (SbV)/sodium antimony gluconate (SAG) and some other anti‐leishmanial drug is increasing in several parts of the world. With the alarming rise of drug resistance and other issues related to VL, there is an urgent need to focus on early detection and quick diagnosis of VL case. Therefore, we have reviewed most of the methods used in the diagnostic process of VL. Along with existing diagnostic methods, developing more effective and sensitive diagnostic methods and biomarkers is also vital for enhancing VL identification and control programs. This review gathers the comprehensive information on diagnostics methods of VL under a single umbrella that could be the prominent tools for the development of rapid, accurate and cost‐effective diagnostic kits for VL which can be used in field conditions.
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