Research paperImmunodetection and molecular determination of visceral and cutaneous Leishmania infection using patients' urine
Introduction
Leishmaniases are vector-borne diseases, caused by obligate protistan parasites from the genus Leishmania (Trypanosomatida: Trypanosomatidae). They are endemic in large areas of the tropics, subtropics and the Mediterranean basin, spanning >98 countries and territories. There are almost 350 million people at risk and 12 million cases, with an estimated worldwide annual incidence of 0.7–1.2 million cases of cutaneous leishmaniasis (CL) and 0.2–0.4 million cases of visceral leishmaniasis (VL) (Alvar et al., 2012). Leishmania parasites are transmitted to vertebrates by the bite of infected female phlebotomine sandflies, and are frequently hosted by canids, rodents, marsupials, mongooses, bats and hyraxes (Akhoundi et al., 2016, Akhoundi et al., 2017).
In Iran, both VL and CL are identified important public health problems. The causative agents of VL in Iran are L. donovani and L. infantum. The latter is sporadically reported throughout the country in four main endemic foci, located in the north-western (Ardabil and Azerbaijan-e- Sharqi) and southern Iran (Fars and Bushehr) (Akhoundi et al., 2013). Zoonotic Cutaneous Leishmaniasis (ZCL) due to L. major, is a zoonotic disease with rodents as reservoirs. It occurs mainly in rural areas of 15 out of the 31 provinces of Iran (Akhoundi et al., 2013). The most prevalent endemic foci of ZCL are located in Turkmen Sahara and Lotf Abad, in north-east of Iran, Abardezh Varamin, Esfahan and Yazd, in center of Iran, Fars and Sistan-Baluchestan in south and south-east and Ilam and Khuzestan in south-west of Iran (Nadim and Seyedi-Rashti, 1971; Mohebali et al., 2004). Anthroponotic Cutaneous Leishmaniasis (ACL) due to L. tropica, mainly anthroponotic in nature, occurs in Tehran, Khorasan-e-Razavi in the north-east and Fars and Kerman provinces in the south of Iran (Nadim and Seyedi-Rashti, 1971; Mohebali et al., 2004).
The diagnosis of leishmaniasis is puzzled, by the diversity of the clinical pathology profiles of the disease; varies from simple cutaneous lesions to visceral forms. Variability in the clinical presentation is also reported, within each clinical form (VL, CL). Parasitological confirmation via culture or molecular techniques is often complex, invasive, and requires a sophisticated laboratory infrastructure. Molecular methods, including PCR-based approaches are highly sensitive and specific (Akhoundi et al., 2017), and they required sophisticated laboratory infrastructures. In addition, a substantial proportion of healthy individuals has parasite DNA in the blood, detected by PCR (Bhattarai et al., 2009). Serological tests like indirect fluorescent antibody test (IFAT), Western blot, or direct agglutination test (DAT), are used for leishmaniasis diagnosis (Mikaeili et al., 2007; Sarkari et al., 2008; Castellano et al., 2010). But these immunodetection methods present drawbacks because they detect antibodies, still present after a cure, as well as past or present asymptomatic infection.
Currently, all rapid diagnostic tests (RDT) rely on the principle of the antibody detection (see Boelaert et al., 2014). RDT were specifically developed for the field, used in VL-endemic areas. They also can be used, if sufficiently accurate, for the early diagnosis of VL, at peripheral and central levels of the health system (Boelaert et al., 2014). The most sensitive and specific RDT for VL is the rK39 immunochromatographic test (ICT) with 91.9% sensitivity and 92.4% specificity (Boelaert et al., 2014). In the recent years, rK-39 ICT has been carried out using patient urine samples and showed high sensitivity (ranging from 95 to 97.7%) and specificity (93.3 to 100%) (Musawwir Khan et al., 2010; Singh et al., 2013; Ghosh et al., 2016). In addition, qPCR analysis of the VL urine showed good prospect for Leishmania DNA detection (Pessoa-E-Silva et al., 2016).
In the majority of countries endemic for leishmaniasis, more than one human pathogenic Leishmania species are reported. The higher diversity of Leishmania including eight different pathogenic species is reported in Brazil (Akhoundi et al., 2017). Nevertheless, mentioned methods cannot discriminate between the infecting Leishmania species.
Because urine samples are easy to collect and process, therefore we decided to test the efficiency of Western blot, using patients' urine, (i) to diagnose Leishmania infection and (ii) to evaluate the sensitivity of such approaches for serological and molecular diagnosis.
Section snippets
Materials and methods
Clinic and samples
A prospective study was conducted between March 2015 and June 2017 on the patients referring to Dehloran, Mehran and Meshkin shar health centers, in the north-west and south-west of Iran. Serum, biopsy and urine samples were collected from individuals, with symptoms reminiscent of leishmaniasis (CL and VL). A total of 37 patients were selected: 23 with cutaneous lesions (8 ACL and 15 ZCL) and 14 suspected having VL. For CL, personal information, lesion duration, type of
Results
A total of, 37 patients (22 males and 15 females) were examined, including 23 patients with cutaneous lesions (15 patients infected by L. major and eight by L. tropica) and 14 patients infected by L. infantum (Table 1). In addition, samples from three healthy individuals were added, as control.
Studied patients had an age, ranging from 4 to 69 years old. For CL patients, the most abundant lesions affected patients who were 10 to 20 years old (32.4%). The lesions were frequently observed on the
Discussion
The diagnosis of leishmaniasis in health care is often based on clinical symptoms and if possible, it is confirmed on the basis of the presence and identification of the parasite. CL symptoms are easily identified by cutaneous lesions, compared to clinical symptoms indicative of a VL, but cutaneous manifestations of leishmaniasis are not so specific and neither a clear indication of CL. Misdiagnosis is common with other diseases like Virchow's hanseniasis, paracoccidioidomycosis, syphilis,
Conclusion
The use of easy-to-obtain samples, such as urine for serological and molecular assessments of Leishmania infection can be the alternative choice to replace with some invasive methods e.g. traumatic bone marrow and risky spleen aspiration sampling. The application of serological and molecular tests using patient urine is feasible. It is a comfortable, practical and fast diagnostic complement comparing to routine conventional techniques and their limitations. Antigen-based urine analysis can be
Acknowledgments
The authors are grateful to the personnel of Dehloran, Mehran and Mehskin shahr health centers for their contributions and assistances in the collection of human samples.
This study was supported partially by Grant No. 952006-96 dedicated by Ilam University of medical sciences. Moreover, this work was supported financially by the EDENext grant (FP7-261504 EDENext). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References (72)
- et al.
Leishmania infections: molecular targets and diagnosis
Mol. Asp. Med.
(2017) - et al.
Canine leishmaniasis in south-east of France: screening of Leishmania infantum antibodies (western blotting, ELISA) and parasitaemia levels by PCR quantification
Vet. Parasitol.
(2009) - et al.
Diagnosis of Mediterranean visceral leishmaniasis by detection of leishmania-related antigen in urine and oral fluid samples
Acta Trop.
(2017) - et al.
Detection of Leishmania donovani soluble antigen and antibody in the urine of visceral leishmaniasis patients
Trans. R. Soc. Trop. Med. Hyg.
(1987) - et al.
Detection of urinary antigens and their seroreactivity with serum of patients in Leishmania donovani infection
Asian Pac J Trop Med
(2011) - et al.
Detection by western blot of four antigens characterizing acute clinical leishmaniasis due to Leishmania infantum
Trans. R. Soc. Trop. Med. Hyg.
(1995) - et al.
New world cutaneous leishmaniasis: updated review of current and future diagnosis and treatment
J. Am. Acad. Dermatol.
(2010) - et al.
First detection of Leishmania infantum kinetoplast DNA in hair of wild mammals: application of qPCR method to determine potential parasite reservoirs
Acta Trop.
(2013) - et al.
Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR
J. Microbiol. Methods
(2016) - et al.
Western blot analysis as an aid for the diagnosis of cutaneous leishmaniasis due to Leishmania major
Trans. R. Soc. Trop. Med. Hyg.
(2012)
Antigenuria in visceral leishmaniasis: detection and partial characterisation of a carbohydrate antigen
Acta Trop.
Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis
Vet. Parasitol.
Anti- Leishmania IgA in urine samples from dogs with clinical leishmaniasis
Vet. Parasitol.
Identification and diagnostic utility of leishmnia infantum proteins found in urine samples from patients with visceral leishmaniasis
Clin. Vaccine Immunol.
Diagnostic potential of western blot analysis of sera from dogs with leishmaniasis in edndemic areas significance of the pattern
Am. J. Trop. Med. Hyg.
Molecular characterization of Leishmania spp. in reservoir hosts in endemic foci of zoonotic cutaneous leishmaniasis in Iran
Folia Parasitol.
A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies
PLoS Negl. Trop. Dis.
Leishmaniasis worldwide and global estimates of its incidence
PLoS ONE
Utility of western blot analysis for the diagnosis of cutaneous leishmaniasis
Iran. J. Parasitol.
A new perspective on cutaneous leishmaniasis-implications for global prevalence and burden of disease estimates
PLoS Negl. Trop. Dis.
PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar
Tropical Med. Int. Health
Rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease
Cochrane Database Syst. Rev.
Studies on immunity to Asiatic cholera; the absorption of immune globulin from the bowel and its excretion in the urine and feces of experimental animals and human volunteers
J. Infect. Dis.
Acute renal failure in visceral leishmaniasis
Am. J. Nephrol.
Evaluation of electro-eluted antigens in the serological diagnosis of cutaneous leishmaniasis
Ann. Trop. Med. Parasitol.
R: A Language and Environment for Statistical Computing
Evaluation of anti-Leishmania antibodies in Turkish patients with visceral leishmaniasis using western blotting
Türk. Parazitol. Derg.
The ade4 package: implementing the duality diagram for ecologists
J. Stat. Softw.
Congenital kala-azar and leishmaniasis in the placenta
Am. J. Trop. Med. Hyg.
Hand Book on Isolation. Characterization and Cryopreservation of Leishmania
Design and validation of real time PCR: quantitative diagnosis of common Leishmania species in Iran
Arch. Iran Med.
Leishmania infantum DNA detection in urine from patients with visceral leishmaniasis and after treatment control
Am. J. Trop. Med. Hyg.
Occurrence of Leishmania DNA in urines of dogs naturally infected with leishmaniasis
Vet. Res. Commun.
Development of a LAMP assay for detection of Leishmania infantuminfection in dogs using conjunctival swab samples
Parasit. Vectors
Use of noninvasive markers to detect Leishmania infection in asymptomatic human immunodeficiency virus-infected patients
J. Clin. Microbiol.
Evaluation of diagnostic performance of rK28 ELISA using urine for diagnosis of visceral leishmaniasis
Parasit. Vectors
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