• View in gallery

    Human antibody response against Leishmania tropica and sand fly saliva. Human serum samples from Korazim and Tiberias, two Leishmania foci in northern Israel, and samples from the sand fly–free area, the Czech Republic (CZ), were screened for the presence of antibodies against (A) Leishmania tropica and against saliva of three sand fly species: (B) Phlebotomus arabicus, (C) Phlebotomus sergenti, and (D) Phlebotomus papatasi. Asterisks above y axes indicate significant differences (P < 0.05) between Leishmania-PCR–positive (open triangle) and Leishmania-PCR–negative (open circle) individuals from the same locality. Asterisks below y axes indicate significant differences (P < 0.05) between localities (regardless the Leishmania status). Samples were evaluated in triplicates and are presented as mean values.

  • 1.

    Jacobson RL, 2003. Leishmania tropica (Kinetoplastida: Trypanosomatidae)—a perplexing parasite. Folia Parasitol (Praha) 50: 241250.

  • 2.

    Handler MZ, Patel PA, Kapila R, Al-Qubati Y, Schwartz RA, 2015. Cutaneous and mucocutaneous leishmaniasis clinical perspectives. J Am Acad Dermatol 73: 897910.

    • Search Google Scholar
    • Export Citation
  • 3.

    Gandacu D, Glazer Y, Anis E, Karakis I, Warshavsky B, Slater P, Grotto I, 2014. Resurgence of cutaneous leishmaniasis in Israel, 2001–2012. Emerg Infect Dis 20: 16051611.

    • Search Google Scholar
    • Export Citation
  • 4.

    Vinitsky O, Ore L, Habiballa H, Dar MC, 2010. Geographic and epidemiologic analysis of the cutaneous leishmaniasis outbreak in northern Israel, 2000–2003. Isr Med Assoc J 12: 652656.

    • Search Google Scholar
    • Export Citation
  • 5.

    Singer SR, Abramson N, Shoob H, Zaken O, Zentner G, Stein-Zamir C, 2008. Ecoepidemiology of cutaneous leishmaniasis outbreak, Israel. Emerg Infect Dis 14: 14241426.

    • Search Google Scholar
    • Export Citation
  • 6.

    Talmi-Frank D, Jaffe CL, Nasereddin A, Warburg A, King R, Svobodova M, Peleg O, Baneth G, 2010. Leishmania tropica in rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis. Am J Trop Med Hyg 82: 814818.

    • Search Google Scholar
    • Export Citation
  • 7.

    Svobodova M, Volf P, Votypka J, 2006. Experimental transmission of Leishmania tropica to hyraxes (Procavia capensis) by the bite of Phlebotomus arabicus. Microbes Infect 8: 16911694.

    • Search Google Scholar
    • Export Citation
  • 8.

    Jacobson RL 2003. Outbreak of cutaneous leishmaniasis in northern Israel. J Infect Dis 188: 10651073.

  • 9.

    Svobodova M 2006. Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, northern Israel. Emerg Infect Dis 12: 18601868.

    • Search Google Scholar
    • Export Citation
  • 10.

    Savioli L, Velayudhan R, 2014. Small bite, big threat: World Health Day 2014. East Mediterr Health J 20: 217218.

  • 11.

    Lestinova T, Rohousova I, Sima M, de Oliveira CI, Volf P, 2017. Insights into the sand fly saliva: blood-feeding and immune interactions between sand flies, hosts, and Leishmania. PLoS Negl Trop Dis 11: e0005600.

    • Search Google Scholar
    • Export Citation
  • 12.

    de Vries HJC, Reedijk SH, Schallig H, 2015. Cutaneous leishmaniasis: recent developments in diagnosis and management. Am J Clin Dermatol 16: 99109.

    • Search Google Scholar
    • Export Citation
  • 13.

    Elsafi SH, Evans DA, 1989. A comparison of the direct agglutination test and enzyme-linked immunosorbent assay in the serodiagnosis of leishmaniasis in the Sudan. Trans R Soc Trop Med Hyg 83: 334337.

    • Search Google Scholar
    • Export Citation
  • 14.

    Al-Salem WS 2014. Detection of high levels of anti-alpha-galactosyl antibodies in sera of patients with old world cutaneous leishmaniasis: a possible tool for diagnosis and biomarker for cure in an elimination setting. Parasitology 141: 18981903.

    • Search Google Scholar
    • Export Citation
  • 15.

    Costa LE 2016. New serological tools for improved diagnosis of human tegumentary leishmaniasis. J Immunol Methods 434: 3945.

  • 16.

    Celeste BJ, Sanchez MCA, Ramos-Sanchez EM, Castro LGM, Costa FAL, Goto H, 2014. Recombinant Leishmania infantum heat shock protein 83 for the serodiagnosis of cutaneous, mucosal, and visceral leishmaniases. Am J Trop Med Hyg 90: 860865.

    • Search Google Scholar
    • Export Citation
  • 17.

    Rohousova I, Ozensoy S, Ozbel Y, Volf P, 2005. Detection of species-specific antibody response of humans and mice bitten by sand flies. Parasitology 130: 493499.

    • Search Google Scholar
    • Export Citation
  • 18.

    Kravchenko V, Wasserberg G, Warburg A, 2004. Bionomics of phlebotomine sandflies in the Galilee focus of cutaneous leishmaniasis in northern Israel. Med Vet Entomol 18: 418428.

    • Search Google Scholar
    • Export Citation
  • 19.

    Volf P, Rohousová I, 2001. Species-specific antigens in salivary glands of phlebotomine sandflies. Parasitology 122: 3741.

  • 20.

    Drahota J, Lipoldova M, Volf P, Rohousova I, 2009. Specificity of anti-saliva immune response in mice repeatedly bitten by Phlebotomus sergenti. Parasite Immunol 31: 766770.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Serological Evaluation of Cutaneous Leishmania tropica Infection in Northern Israel

View More View Less
  • 1 Department of Parasitology, Charles University, Faculty of Science, Prague, Czech Republic;
  • 2 School of Veterinary Medicine, Hebrew University, Rehovot, Israel;
  • 3 Department of Pediatrics, Carmel Medical Center, Haifa, Israel;
  • 4 Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University - Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israel

Leishmania spp. are medically important unicellular parasites transmitted by phlebotomine sand flies. The World Health Organization recently highlighted the importance of reliable diagnostic tools for leishmaniasis. Our study of human infection was conducted in two endemic foci of Leishmania tropica in the Galilee region, northern Israel. Elevated anti-Leishmania antibodies were present in the majority (78.6%) of L. tropica-PCR positive individuals. Moreover, the enzyme-linked immunosorbent assay showed high sensitivity, specificity, and negative and positive predictive values (ranging between 73% and 79%), thus fulfilling the basic requirement for future development of a serodiagnostic and screening tool. The anti-sand fly saliva antibodies used as biomarkers of exposure reflected the composition of the local sand fly fauna as well as the abundance of individual species. High levels of antibodies against vector salivary proteins may further indicate frequent exposure to sand flies and consequently a higher probability of Leishmania transmission.

Leishmania spp. (Trypanosomatidae) are the causative agents of leishmaniases, a group of diseases with various manifestations attributed to the genotype of the infecting Leishmania species and host immune status. Leishmania tropica typically causes a chronic disease characterized by slow-developing, self-healing cutaneous lesions that may eventually result in persisting atrophic scars. However, human cases of recidivating or visceral manifestations due to L. tropica have also been reported.1,2 In Israel, L. tropica is the dominant causative agent of cutaneous leishmaniasis (CL) and is a contributor to the resurgence of CL together with Leishmania major in the last decade.3 Several outbreaks have been reported in northern Israel since 2003 when the incidence of CL peaked at 41 cases per 100,000 in the Kinneret subdistrict.3,4 Although L. tropica infection tends to be an urban, anthroponotic infection, in Israel it is zoonotic in nature.57 Moreover, there are two recognized vectors of L. tropica in Israel—Phlebotomus sergenti and Phlebotomus arabicus—making the epidemiology of this disease even more complex.8,9

The World Health Organization recently indicated the serious and increasing threat of leishmaniasis and highlighted the importance of reliable diagnostic tools.10 The goal of our study was to assess whether people diagnosed with L. tropica infection also produce detectable serum antibody levels to this parasite. We evaluated the serologic reaction of residents from two different localities in the Kinneret subdistrict to L. tropica antigen as well as to sand fly saliva of three local species to demonstrate their exposure to vector sand flies by detection of anti-sand fly saliva antibodies as proven biomarkers of exposure.11

Human sera were collected between May 2008 and September 2010 in an L. tropica endemic area in the Galilee region (northern Israel) at two locations, Korazim (north of Lake Kinneret) and Tiberias (south of Lake Kinneret). Of the 25 individual serum samples (Supplemental Table 1), 14 (group A) were obtained from Leishmania-PCR–positive individuals, and 11 (group B) were collected from apparently healthy individuals who were family members or neighbors of patients without history of any lesion consistent with CL. The control group (group C) consisted of 43 individuals from the Czech Republic, a sand fly–free area without autochthonous leishmaniasis. An informed consent was obtained from participants. Ethical approval was obtained from the Institutional Review Boards of the Carmel Medical Center (Haifa, Israel) (CMC-0052-07), Meir Medical Center (k-0057-08), Kupat Holim Clatit HMO (Israel), and Charles University (Czech Republic).

Anti-L. tropica antibody levels were measured using enzyme-linked immunosorbent assay (ELISA). Plates were coated overnight at 4°C with 75 ng/well with L. tropica antigen (MHOM/IL/2005/LRC-L1239). Plates were washed with PBS-0.05% Tween 20 and blocked with 2% milk for 1 hour at 25°C. Sera were diluted 1:50 in 2% milk and incubated for 1 hour at 37°C, followed by incubation with biotin-coupled protein G diluted 1:250 (Adar Biotech) for 2 hours at 37°C, followed by streptavidin–HRP (Jackson) diluted 1:250 in 2% milk. The plates were developed with 2,2-azinobis 3-ethylbenzthiazolinesulfonic, and absorbance was read at 405 nm.

Cut-off values were determined as three standard deviations from the control (group C) mean. Specificity, sensitivity, and positive and negative predictive values of the ELISA were calculated using four categories: true positives (individuals positive for both Leishmania-PCR and anti-L. tropica antibodies), false positives (Leishmania-PCR–negative individuals positive for anti-L. tropica antibodies), false negatives (Leishmania-PCR–positive individuals negative for anti-L. tropica antibodies), and true negatives (individuals negative for both Leishmania-PCR and anti-L. tropica antibodies). Statistical significance was analyzed using the nonparametric Wilcoxon rank-sum test or Wilcoxon signed-rank test for difference in medians. Statistical analyses were performed using NCSS 6.0.21 and R softwares (http://cran.r-project.org/).

Overall seroprevalence of anti-L. tropica antibodies among the samples was 56%. Among Leishmania-PCR–positive individuals (group A), a total of 78.6% showed elevated anti-L. tropica antibodies. In general, individuals from group A revealed significantly higher (P = 0.02) antibody levels compared with Leishmania-PCR–negative individuals (group B) (Figure 1A, Table 1).

Figure 1.
Figure 1.

Human antibody response against Leishmania tropica and sand fly saliva. Human serum samples from Korazim and Tiberias, two Leishmania foci in northern Israel, and samples from the sand fly–free area, the Czech Republic (CZ), were screened for the presence of antibodies against (A) Leishmania tropica and against saliva of three sand fly species: (B) Phlebotomus arabicus, (C) Phlebotomus sergenti, and (D) Phlebotomus papatasi. Asterisks above y axes indicate significant differences (P < 0.05) between Leishmania-PCR–positive (open triangle) and Leishmania-PCR–negative (open circle) individuals from the same locality. Asterisks below y axes indicate significant differences (P < 0.05) between localities (regardless the Leishmania status). Samples were evaluated in triplicates and are presented as mean values.

Citation: The American Journal of Tropical Medicine and Hygiene 98, 1; 10.4269/ajtmh.17-0370

Table 1

Serology data

Locationnanti-leishanti-ARAanti-SERanti-PAP
Korazim127122
 Group A55122
 Group B72000
Tiberias137021
 Group A96011
 Group B41010
Total2514143

Data are presented as the number of individuals seropositive for anti-Leishmania tropica immunoglobulin G (IgG) (anti-leish), anti-Phlebotomus arabicus IgG (anti-ARA), anti-Phlebotomus sergenti IgG (anti-SER), and anti-Phlebotomus papatasi IgG (anti-PAP). Total number of residents in a given locality or in an assigned group is indicated (n); group A = Leishmania-PCR–positive individuals; group B = Leishmania-PCR–negative individuals. Cut-off values were determined as three standard deviations from the mean optical densities (OD) of the control sera (group C). The cut-off values were as follows: anti-L. tropica, OD = 0.092; anti-P. arabicus IgG, OD = 0.261; anti-P. sergenti IgG, OD = 0.297; and anti-P. papatasi IgG, OD = 0.303.

Current serologic tests for CL are restricted because of the poor humoral response provoked by the infection and the consequential low sensitivity.12,13 However, our study showed that 78.6% of tested CL patients exhibited elevated anti-L. tropica antibodies. Our ELISA performed with high sensitivity and high positive predictive value (both 79%), indicating a minimal number of false-positive samples. Specificity and negative predictive value were both 73% achieving minimal false-negative error rate, demonstrating its potential as a complementary diagnostic tool. In general, serologic assays usually do not distinguish between active and former infection; thus, the group assigned as false positive may actually include individuals with quiescent infection.

Higher sensitivity (91%) was found in another study for chemiluminescent ELISA detecting Leishmania anti-α-Gal immunoglobulin G (IgG) as a diagnostic marker of L. tropica infection in humans. Moreover, anti-α-Gal antibodies showed promising features as a marker distinguishing patients from cured individuals.14 Recently, Costa et al.15 introduced three antigens that performed in an ELISA with 100% sensitivity and specificity using sera of patients infected with Leishmania braziliensis, highlighting no cross-reactions with sera of Trypanosoma cruzi– or Leishmania infantum–infected patients. Another example of an already successfully tested serodiagnostic tool for CL is ELISA based on recombinant L. infantum heat shock protein 83. This test was performed without significant cross-reactivity to sera from patients with Chagas disease, toxoplasmosis, and malaria, but was unable to distinguish the infecting Leishmania species.16

In this study, we tested individuals also for anti-sand fly saliva antibodies, a well-proven biomarker of exposure to estimate the risk of human infection.11,17 Serum samples were tested for the presence of IgG antibodies against the saliva of P. arabicus, P. sergenti, and Phlebotomus papatasi sand flies. The anti-sand fly saliva antibodies were measured by ELISA as described in Supplemental Figure 1 using human sera diluted 1:50 and peroxidase-conjugated goat anti-human IgG (Sigma-Aldrich, Czech Republic) diluted 1:2,500. Antigens for the sand fly saliva tests were prepared from colonies of P. arabicus and P. sergenti, species serving as L. tropica vectors in Israel,8,9 originated from the same locality where human samples were tested.

The study included human sera collected from inhabitants of Tiberias and Korazim that differ in composition of sand fly fauna and sand fly vector species. Tiberias has been identified as a new emerging focus of L. tropica in early 2000.4,8 Phlebotomus sergenti, the vector species, is the most abundant sand fly, comprising more than 90% of caught specimens.9 Conversely, P. papatasi is a minor species (2%) and P. arabicus is absent in Tiberias.9 The presence of antisaliva antibodies in the serum of Tiberias residents (Table 1) corresponds well with the sand fly species composition; none of the samples reacted with the salivary antigen of P. arabicus.9

Korazim, another emerged L. tropica focus, is located in the northern Galilee region, and although only 10–15 km northeast from Tiberias, it has some different characteristics. The sand fly fauna consists of seven sand fly species, including P. sergenti (up to 30% of collected sand flies), P. arabicus (22%), and P. papatasi (2.5%).8,9,18 Two species have been found infected in this area—P. sergenti and P. arabicus—the latter identified as a new vector of L. tropica.8,9 Similar to Tiberias, the antibodies against sand fly saliva in human sera reflected the occurrence of sand fly species present in Korazim (Table 1). Moreover, patients from Korazim presented higher levels of anti-P. arabicus IgG than healthy individuals from the same place (P < 0.05, Figure 1B), supporting the employment of antivector antibodies as a risk marker of L. tropica infection.

To exclude the possible cross-reactivity of antisaliva antibodies with heterologous antigens, we used a mouse model. In accordance with our previous results,17,19,20 mouse antibodies elicited by bites of a single sand fly species were highly species specific (Supplemental Figure 1). The results from Tiberias indicate similar species specificity also for human sera; none of the sera showed reactivity with salivary antigen of P. arabicus, the species that is absent in this focus.9

In conclusion, our study demonstrated elevated anti-Leishmania antibodies present in the majority of L. tropica–infected patients, thus fulfilling the basic requirement for future serodiagnostic tool. We have also shown that anti-sand fly saliva antibodies reflected the composition of the local sand fly fauna and thus could be used as a biomarker of exposure to the vector sand fly in epidemiological studies. Further studies are required to reinforce the diagnostic potential of anti-sand fly saliva antibodies as a risk marker of L. tropica infection.

Supplementary Material

Acknowledgments:

C. L. J. holds the Michael and Penny Feiwel Chair in Dermatology.

REFERENCES

  • 1.

    Jacobson RL, 2003. Leishmania tropica (Kinetoplastida: Trypanosomatidae)—a perplexing parasite. Folia Parasitol (Praha) 50: 241250.

  • 2.

    Handler MZ, Patel PA, Kapila R, Al-Qubati Y, Schwartz RA, 2015. Cutaneous and mucocutaneous leishmaniasis clinical perspectives. J Am Acad Dermatol 73: 897910.

    • Search Google Scholar
    • Export Citation
  • 3.

    Gandacu D, Glazer Y, Anis E, Karakis I, Warshavsky B, Slater P, Grotto I, 2014. Resurgence of cutaneous leishmaniasis in Israel, 2001–2012. Emerg Infect Dis 20: 16051611.

    • Search Google Scholar
    • Export Citation
  • 4.

    Vinitsky O, Ore L, Habiballa H, Dar MC, 2010. Geographic and epidemiologic analysis of the cutaneous leishmaniasis outbreak in northern Israel, 2000–2003. Isr Med Assoc J 12: 652656.

    • Search Google Scholar
    • Export Citation
  • 5.

    Singer SR, Abramson N, Shoob H, Zaken O, Zentner G, Stein-Zamir C, 2008. Ecoepidemiology of cutaneous leishmaniasis outbreak, Israel. Emerg Infect Dis 14: 14241426.

    • Search Google Scholar
    • Export Citation
  • 6.

    Talmi-Frank D, Jaffe CL, Nasereddin A, Warburg A, King R, Svobodova M, Peleg O, Baneth G, 2010. Leishmania tropica in rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis. Am J Trop Med Hyg 82: 814818.

    • Search Google Scholar
    • Export Citation
  • 7.

    Svobodova M, Volf P, Votypka J, 2006. Experimental transmission of Leishmania tropica to hyraxes (Procavia capensis) by the bite of Phlebotomus arabicus. Microbes Infect 8: 16911694.

    • Search Google Scholar
    • Export Citation
  • 8.

    Jacobson RL 2003. Outbreak of cutaneous leishmaniasis in northern Israel. J Infect Dis 188: 10651073.

  • 9.

    Svobodova M 2006. Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, northern Israel. Emerg Infect Dis 12: 18601868.

    • Search Google Scholar
    • Export Citation
  • 10.

    Savioli L, Velayudhan R, 2014. Small bite, big threat: World Health Day 2014. East Mediterr Health J 20: 217218.

  • 11.

    Lestinova T, Rohousova I, Sima M, de Oliveira CI, Volf P, 2017. Insights into the sand fly saliva: blood-feeding and immune interactions between sand flies, hosts, and Leishmania. PLoS Negl Trop Dis 11: e0005600.

    • Search Google Scholar
    • Export Citation
  • 12.

    de Vries HJC, Reedijk SH, Schallig H, 2015. Cutaneous leishmaniasis: recent developments in diagnosis and management. Am J Clin Dermatol 16: 99109.

    • Search Google Scholar
    • Export Citation
  • 13.

    Elsafi SH, Evans DA, 1989. A comparison of the direct agglutination test and enzyme-linked immunosorbent assay in the serodiagnosis of leishmaniasis in the Sudan. Trans R Soc Trop Med Hyg 83: 334337.

    • Search Google Scholar
    • Export Citation
  • 14.

    Al-Salem WS 2014. Detection of high levels of anti-alpha-galactosyl antibodies in sera of patients with old world cutaneous leishmaniasis: a possible tool for diagnosis and biomarker for cure in an elimination setting. Parasitology 141: 18981903.

    • Search Google Scholar
    • Export Citation
  • 15.

    Costa LE 2016. New serological tools for improved diagnosis of human tegumentary leishmaniasis. J Immunol Methods 434: 3945.

  • 16.

    Celeste BJ, Sanchez MCA, Ramos-Sanchez EM, Castro LGM, Costa FAL, Goto H, 2014. Recombinant Leishmania infantum heat shock protein 83 for the serodiagnosis of cutaneous, mucosal, and visceral leishmaniases. Am J Trop Med Hyg 90: 860865.

    • Search Google Scholar
    • Export Citation
  • 17.

    Rohousova I, Ozensoy S, Ozbel Y, Volf P, 2005. Detection of species-specific antibody response of humans and mice bitten by sand flies. Parasitology 130: 493499.

    • Search Google Scholar
    • Export Citation
  • 18.

    Kravchenko V, Wasserberg G, Warburg A, 2004. Bionomics of phlebotomine sandflies in the Galilee focus of cutaneous leishmaniasis in northern Israel. Med Vet Entomol 18: 418428.

    • Search Google Scholar
    • Export Citation
  • 19.

    Volf P, Rohousová I, 2001. Species-specific antigens in salivary glands of phlebotomine sandflies. Parasitology 122: 3741.

  • 20.

    Drahota J, Lipoldova M, Volf P, Rohousova I, 2009. Specificity of anti-saliva immune response in mice repeatedly bitten by Phlebotomus sergenti. Parasite Immunol 31: 766770.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Gad Baneth, Koret School of Veterinary Medicine, The Hebrew University, P.O. Box 12, Rehovot 7610001, Israel. E-mail: gad.baneth@mail.huji.ac.il

Financial support: This work was partially supported by Charles University, the Czech Republic (UNCE 204013, http://www.cuni.cz/UKEN-1.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors’ addresses: Iva Rohoušová, Tatiana Spitzová, and Petr Volf, Department of Parasitology, Charles University, Faculty of Science, Prague, Czech Republic, E-mails: kolarova2011@gmail.com, tatiana.spitzova@gmail.com, and volf@cesnet.cz. Dalit Talmi-Frank and Gad Baneth, Koret School of Veterinary Medicine, The Hebrew University, Rehovot, Israel, E-mails: dalitvet@gmail.com and gad.baneth@mail.huji.ac.il. Michaela Vlková, Genome Plasticity and Disease Group, Mater Medical Research Institute, Translation Research Institute Level 4, Woolloongabba, Queensland, Australia, E-mail: michaelakindlova84@gmail.com. Koranit Rishpon, Psychiatric Department, Rambam Medical Center, Haifa, Israel, E-mail: k_rishpon@rambam.health.gov.il. Charles L. Jaffe, Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University - Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israel, E-mail: cjaffe@cc.huji.ac.il. Moshe Ephros, Department of Pediatrics, Carmel Medical Center, Haifa, Israel, E-mail: mefrat@technion.ac.il.

Save