Elsevier

Infection, Genetics and Evolution

Volume 63, September 2018, Pages 257-268
Infection, Genetics and Evolution

Research paper
Immunodetection and molecular determination of visceral and cutaneous Leishmania infection using patients' urine

https://doi.org/10.1016/j.meegid.2018.05.021Get rights and content

Abstract

The diagnosis of leishmaniasis relies mainly on the use of invasive processes, to collect the biological material for detecting Leishmania parasites. Body fluids, which can be collected by non-invasive process, would greatly facilitate the leishmaniasis diagnosis. In the present study, we investigated the potency of urine immunoblotting to diagnose cutaneous and visceral leishmaniasis and we compared with routine molecular methods. A total of 80 samples, including 40 sera and their 40 corresponding urine samples were collected from 37 suspected patients with cutaneous and visceral leishmaniasis, and 3 healthy individuals (as control), in Ilam and Ardabil provinces of Iran. All sera and urine samples were analyzed, using immunoblotting. The confirmation of leishmaniasis infection was performed, using conventional and quantitative PCRs as well as by sequencing the amplicons. Among 37 suspected patients, 23 patients presented cutaneous lesions (CL) and 14 exhibited clinical symptoms reminiscent of visceral leishmaniasis (L. infantum). Among cutaneous patients, 15 were positive for zoonotic cutaneous leishmaniasis (L. major), and eight for anthroponotic cutaneous leishmaniasis (L. tropica). Molecular quantification of Leishmania parasites was performed on sera, urines and cutaneous biopsies of CL and VL patients, demonstrating that parasite load is lower in urines, compared to sera or biopsy. DNA can be detected in 20 out of 23 (86.9%) CL urine samples and in 13 out of 14 (92.8%) VL urine samples. Immunodetection analysis demonstrates that 22 out of 23 (95.6%) sera from CL patients and all patients suspected with VL are positive. For urine samples, 18 out of 23 (78.2%) urine of CL patients and 13 out of 14 (92.8%) urine of VL patients were positive, using Western blot. Therefore, immunodetection and molecular analysis using urine samples can be used as a diagnostic tool for surveying cutaneous and visceral leishmaniasis.

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)

  • B. Sarkari et al.

    Antigenuria in visceral leishmaniasis: detection and partial characterisation of a carbohydrate antigen

    Acta Trop.

    (2002)
  • L. Solano-Gallego et al.

    Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis

    Vet. Parasitol.

    (2007)
  • F. Todolí et al.

    Anti- Leishmania IgA in urine samples from dogs with clinical leishmaniasis

    Vet. Parasitol.

    (2009)
  • C. Abeijon et al.

    Identification and diagnostic utility of leishmnia infantum proteins found in urine samples from patients with visceral leishmaniasis

    Clin. Vaccine Immunol.

    (2012)
  • M.J. Aisa et al.

    Diagnostic potential of western blot analysis of sera from dogs with leishmaniasis in edndemic areas significance of the pattern

    Am. J. Trop. Med. Hyg.

    (1998)
  • M. Akhoundi et al.

    Molecular characterization of Leishmania spp. in reservoir hosts in endemic foci of zoonotic cutaneous leishmaniasis in Iran

    Folia Parasitol.

    (2013)
  • M. Akhoundi et al.

    A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies

    PLoS Negl. Trop. Dis.

    (2016)
  • J. Alvar et al.

    Leishmaniasis worldwide and global estimates of its incidence

    PLoS ONE

    (2012)
  • M. Ashrafmansouri et al.

    Utility of western blot analysis for the diagnosis of cutaneous leishmaniasis

    Iran. J. Parasitol.

    (2015)
  • F. Bailey et al.

    A new perspective on cutaneous leishmaniasis-implications for global prevalence and burden of disease estimates

    PLoS Negl. Trop. Dis.

    (2017)
  • N.R. Bhattarai et al.

    PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar

    Tropical Med. Int. Health

    (2009)
  • M. Boelaert et al.

    Rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease

    Cochrane Database Syst. Rev.

    (2014)
  • W. Burrows et al.

    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.

    (1948)
  • F. Caravaca et al.

    Acute renal failure in visceral leishmaniasis

    Am. J. Nephrol.

    (1991)
  • L.R. Castellano et al.

    Evaluation of electro-eluted antigens in the serological diagnosis of cutaneous leishmaniasis

    Ann. Trop. Med. Parasitol.

    (2010)
  • R. Core Team

    R: A Language and Environment for Statistical Computing

    (2014)
  • N. Dayangac et al.

    Evaluation of anti-Leishmania antibodies in Turkish patients with visceral leishmaniasis using western blotting

    Türk. Parazitol. Derg.

    (2004)
  • S. Dray et al.

    The ade4 package: implementing the duality diagram for ecologists

    J. Stat. Softw.

    (2007)
  • I.A. Eltoum et al.

    Congenital kala-azar and leishmaniasis in the placenta

    Am. J. Trop. Med. Hyg.

    (1992)
  • D. Evans et al.

    Hand Book on Isolation. Characterization and Cryopreservation of Leishmania

    (1989)
  • M. Fekri Soofi Abadi et al.

    Design and validation of real time PCR: quantitative diagnosis of common Leishmania species in Iran

    Arch. Iran Med.

    (2016)
  • R. Fisa et al.

    Leishmania infantum DNA detection in urine from patients with visceral leishmaniasis and after treatment control

    Am. J. Trop. Med. Hyg.

    (2008)
  • A. Franceschi et al.

    Occurrence of Leishmania DNA in urines of dogs naturally infected with leishmaniasis

    Vet. Res. Commun.

    (2007)
  • C.H. Gao et al.

    Development of a LAMP assay for detection of Leishmania infantuminfection in dogs using conjunctival swab samples

    Parasit. Vectors

    (2015)
  • J.A. García-García et al.

    Use of noninvasive markers to detect Leishmania infection in asymptomatic human immunodeficiency virus-infected patients

    J. Clin. Microbiol.

    (2006)
  • P. Ghosh et al.

    Evaluation of diagnostic performance of rK28 ELISA using urine for diagnosis of visceral leishmaniasis

    Parasit. Vectors

    (2016)
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