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Detection of Leishmania (Mundinia) macropodum (Kinetoplastida: Trypanosomatidae) and heterologous Leishmania species antibodies among blood donors in a region of Australia with marsupial Leishmania endemicity

Open AccessPublished:October 11, 2022DOI:https://doi.org/10.1016/j.ijid.2022.10.006

      Abstract

      Objectives

      The Australian Leishmania (Mundinia) macropodum parasite causes cutaneous leishmaniasis (CL) among marsupial species. While CL is a major public health burden globally, it is not clear if humans are naturally exposed to the unique L. macropodum. To assess whether humans have an IgG response to L. macropodum, we examined anti-Leishmania antibodies among humans residing in a region of marsupial Leishmania endemicity in Australia.

      Methods

      Employing a serological enzyme-linked immunosorbent assay, we characterized Leishmania-specific IgG and IgG subclass responses to soluble Leishmania antigen (SLA) from L. macropodum and other Leishmania species (L. donovani, L. major and L. mexicana) in 282 blood donor samples.

      Results

      We found 20.57% of individuals demonstrated a positive total IgG response to L. macropodum. For individuals with antibodies to SLA from one Leishmania species, there was no increased likelihood of recognition to other Leishmania species. For samples with detectable L. macropodum IgG, IgG1 and IgG2 were the prevalent subclasses detected.

      Conclusions

      It is not yet clear whether the IgG antibody detection in this study reflects exposure to Leishmania parasites or a cross-reactive immune response that was induced against an unrelated immunogen. Future studies should investigate whether L. macropodum can result in a viable infection in humans.

      Graphical Abstract

      Keywords

      Introduction

      Cutaneous leishmaniasis (CL) is one of the three main clinical forms of the leishmaniases, a complex group of vector-borne diseases with a diverse spectrum of clinical presentation (). Infection is acquired through the bite of phlebotomine sandflies carrying Leishmania (Kinetoplastida: Trypanosomatidae) parasites (
      • Bates PA.
      Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies.
      ). Approximately 20 Leishmania parasite species can cause CL in humans with varying degrees of clinical severity ranging from sub-clinical (often referred to as asymptomatic) and self-resolving lesions, to chronic infection that results in severe tissue destruction and disfigurement. CL causes major public health burden across the world; estimated to affect more than 1 million people annually ().
      The transmission dynamics of Leishmania are complicated and highly dependent on the infecting Leishmania species. Humans can act as the primary reservoir host (anthroponotic transmission), or when non-human species are involved, the transmission is zoonotic. The main Leishmania species responsible for CL are Leishmania major, L. tropica and L. aethiopica (Old World species), and L. amazonensis, L. mexicana, L. braziliensis and L. guyanensis (New World species). With several etiological Leishmania species causing variable clinical manifestations, the epidemiology of CL is complicated (
      • Panahi E.I.
      • Stanisic D.S.
      • Peacock C.J.
      • Herrero L.
      Protective and Pathogenic Immune Responses to Cutaneous Leishmaniasis.
      ).
      In 2001 a novel species, Leishmania (Mundinia) macropodum, was identified as endemic in the tropical region of the Northern Territory (NT), Australia, and shown to cause clinical manifestations of CL in marsupial species (
      • Dougall A
      • Shilton C
      • Low Choy J
      • Alexander B
      • Walton S
      New reports of Australian cutaneous leishmaniasis in Northern Australian macropods.
      ;
      • Rose K
      • Curtis J
      • Baldwin T
      • Mathis A
      • Kumar B
      • Sakthianandeswaren A
      • et al.
      Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.
      ). L. macropodum is suspected to be transmitted by Forcipomyia (Lasiohelea) Kieffer (Diptera: Ceratopogonidae) (
      • Dougall AM
      • Alexander B
      • Holt DCDC
      • Harris T
      • Sultan AH
      • Bates PA
      • et al.
      Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia.
      ;
      • Panahi E
      • Shivas M
      • Hall-Mendelin S
      • Kurucz N
      • Rudd PA
      • De Araujo R
      • et al.
      Utilising a novel surveillance system to investigate species of Forcipomyia (Lasiohelea) (Diptera: Ceratopogonidae) as the suspected vectors of Leishmania macropodum (Kinetoplastida: Trypanosomatidae) in the Darwin region of Australia.
      ), a day-biting phlebotomine sandfly. F. (Lasiohelea) has not been implicated in the transmission of other medically important Leishmania species (
      • Ready PD.
      Biology of Phlebotomine Sand Flies as Vectors of Disease Agents.
      ). Locally acquired human cases of CL have not been reported or diagnosed in Australia, leading to the assumption that humans are not infected by this parasite. To date, the possibility of human exposure to L. macropodum in the NT had not been formally investigated.
      The gold standard for diagnosis of clinical CL in any vertebrate is based on direct microscopic examination of skin lesion smears or biopsies, in combination with culturing parasites from lesions of infected hosts (
      • Aronson N
      • Herwaldt BL
      • Libman M
      • Pearson R
      • Lopez-Velez R
      • Weina P
      • Carvalho EM
      • Ephros M
      • Jeronimo S
      • Magill A.
      Diagnosis and Treatment of Leishmaniasis: Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH).
      ). It has been reported that aysmptomatic human CL is the most common outcome following the bite of an Leishmania -infected sandfly (
      • Andrade-Narvaez FJ
      • Loría-Cervera EN
      • Sosa-Bibiano EI
      • Van Wynsberghe NR.
      Asymptomatic infection with American cutaneous leishmaniasis: epidemiological and immunological studies.
      ). The absences of clinical lesions in these individuals makes diagnosis a challenge. Molecular methods (polymerase chain reaction; PCR) and/or serological methods (enzyme-linked immunosorbent assay (ELISA), immunochromatography test, direct agglutination test and western blot) have all played a supportive role in detecting asymptomatic infection or prior exposure to leishmania parasites (Mannan et al., 2021,
      • Ibarra-Meneses AV
      • Carrillo E
      • Nieto J
      • Sánchez C
      • Ortega S
      • Estirado A
      • Latasa Zamalloa P
      • Sanz JC
      • García-Comas L
      • Ordobás M
      • Moreno J
      Prevalence of asymptomatic Leishmania infection and associated risk factors, after an outbreak in the south-western Madrid region, Spain, 2015.
      ). There is no consensus on the best method to formally diagnosis asymptomatic CL. Currently, a postivitve response to the the Montenegro skin test (MST) is considered the most practical marker for asymptomatic CL and the term asymptomatic CL infection is used with caution requiring an individual to have a positive MST and be living in an endemic area of CL without clinical signs/symptoms (
      • Andrade-Narvaez FJ
      • Loría-Cervera EN
      • Sosa-Bibiano EI
      • Van Wynsberghe NR.
      Asymptomatic infection with American cutaneous leishmaniasis: epidemiological and immunological studies.
      ).
      Leishmania parasites are known as persistent parasites, i.e. they can reside indefinitely within phagocytic cells, even when symptoms of CL have resolved (
      • Mendonça MG
      • de Brito MEF
      • Rodrigues EHG
      • Bandeira V
      • Jardim ML
      • Abath FGC.
      Persistence of Leishmania Parasites in Scars after Clinical Cure of American Cutaneous Leishmaniasis: Is There a Sterile Cure?.
      ). The challenge of identifying asymptomatic individuals and the potential role of these individuals in the Leishmania transmission cycle makes transfusion-transmissible Leishmania a possibility in endemic areas (
      • Aliaga L
      • Ceballos J
      • Sampedro A
      • Cobo F
      • López-Nevot MÁ
      • Merino-Espinosa G
      • et al.
      Asymptomatic Leishmania infection in blood donors from the Southern of Spain.
      ;
      • Cardo LJ.
      Leishmania: risk to the blood supply.
      ;
      • Ferreira-Silva MM
      • Teixeira LAS
      • Tibúrcio MS
      • Pereira GA
      • Rodrigues V
      • Palis M
      • et al.
      Socio-epidemiological characterisation of blood donors with asymptomatic Leishmania infantum infection from three Brazilian endemic regions and analysis of the transfusional transmission risk of visceral leishmaniasis.
      ;
      • França A de O
      • Castro VL
      • de Junior
      • MS da CL
      • Pontes ERJC
      • Dorval MEC.
      Anti-Leishmania antibodies in blood donors from the Midwest region of Brazil.
      ;
      • Fukutani KF
      • Figueiredo V
      • Celes FS
      • Cristal JR
      • Barral A
      • Barral-Netto M
      • et al.
      Serological survey of Leishmaniainfection in blood donors in Salvador, Northeastern Brazil.
      ;
      • Jimenez-Marco T
      • Riera C
      • Girona-Llobera E
      • Guillen C
      • Iniesta L
      • Alcover M
      • et al.
      Strategies for reducing the risk of transfusion-transmitted leishmaniasis in an area endemic for Leishmania infantum: A patient- And donor-targeted approach.
      ;
      • Pérez-Cutillas P
      • Goyena E
      • Chitimia L
      • De la Rúa P
      • Bernal LJ
      • Fisa R
      • et al.
      Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: A study of environmental, demographic and social risk factors.
      ;
      • Riera C
      • Fisa R
      • Lpez-Chejade P
      • Serra T
      • Girona E
      • Jimnez M
      • et al.
      Asymptomatic infection by Leishmania infantum in blood donors from the Balearic Islands (Spain).
      ;
      • Sarkari B
      • Gadami F
      • Shafiei R
      • Motazedian MH
      • Sedaghat F
      • Kasraian L
      • et al.
      Seroprevalence of Leishmania infection among the healthy blood donors in kala-azar endemic areas of Iran.
      ).
      Given serological assays are not able to delineate current asymptomatic infection, previous now cleared symptomatic infection, or merely previous parasite exposure, serological surveys alone are not recommended as the sole method for diagnosing asymptomatic CL, regardless of whether an individual is from an endemic region (
      • Aronson N
      • Herwaldt BL
      • Libman M
      • Pearson R
      • Lopez-Velez R
      • Weina P
      • Carvalho EM
      • Ephros M
      • Jeronimo S
      • Magill A.
      Diagnosis and Treatment of Leishmaniasis: Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH).
      ). However, the complexity of validated tests such as the MST, raises concerns that asymptomatic infections are going undiagnosed and asymptomatic infection is viewed as a concern for global Leishmania elimination programs (Mannan et al., 2021). Given the simplicity of serological assays such as ELISAs, further research into their role in supporting CL diagnosis is being investigated (
      • Deepachandi B
      • Weerasinghe S
      • Gunathilake H
      • Andrahennadi TP
      • Wickramanayake MN
      • Siri S
      • Chandrasekharan V
      • Soysa P
      • Siriwardana Y.
      Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis.
      ;
      • Sarkari B
      • Ashrafmansouri M
      • Hatam G
      • Habibi P
      • Abdolahi Khabisi S.
      Performance of an ELISA and indirect immunofluorescence assay in serological diagnosis of zoonotic cutaneous leishmaniasis in iran.
      ). ELISAs have been developed using a range of Leishmania antigens such as SLA, recombinant proteins and synthetic peptides, all which can influence the sensitivity and specificity of the assay.
      In Australia, symptomatic cases of L. macropodum CL have been identified and the parasites have been isolated from marsupial species (
      • Dougall A
      • Shilton C
      • Low Choy J
      • Alexander B
      • Walton S
      New reports of Australian cutaneous leishmaniasis in Northern Australian macropods.
      ;
      • Rose K
      • Curtis J
      • Baldwin T
      • Mathis A
      • Kumar B
      • Sakthianandeswaren A
      • et al.
      Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.
      ). Additionally, we have shown that several marsupial species from the L. macropodum endemic region of Australia are IgG positive to SLA derived from L. macropodum, despite showing no clinical signs of an active infection (Panahi et al., data not published). Given that humans are known hosts of medically important Leishmania species, it is important to determine whether humans may be infected with L. macropodum.
      As the first step to this end, plasma samples were tested for the presence of antibodies that recognized SLA derived from L. macropodum and three other Leishmania species. Where IgG was detected, IgG subclass responses were also examined.

      Materials and Methods

      Study design and sample collection

      This study was carried out using archived samples from ARC Lifeblood donors (n=282) residing in the NT, which is considered a marsupial L. macropodum-endemic region. Samples were collected at the capital city (Darwin) blood collection centre in 2016 and were provided deidentified for this study. Demographic characteristics of blood donors included their age and sex. Previous clinical history was unavailable.
      Negative controls (NC; n=12) were recruited from South East Queensland (SEQ), a region in Australia that is non-endemic for L. macropodum as i) no local cases of CL have been reported in humans or animals, and ii) to our knowledge it is a region where the suspected transmitting-vector, F. (Lasiohelea) sp., is absent. Eligibility criteria for NC were: i) no travel history to the NT or to a recognised Leishmania-endemic country and ii) no previous clinical history of leishmaniasis.
      For donor samples, whole blood was collected in plasma preparation tubes (BD Vacutainer PPT plasma preparation tube 5 mL, Becton Dickinson) followed by centrifugation at 1000 × g for 10 minutes. Plasma was collected and stored in aliquots at –80°C. For SEQ NC, whole blood was collected in heparin plasma collection tubes (BD Vacutainer lithium heparin tube 6 mL, Becton Dickinson) and centrifuged at 1000 × g for 10 minutes. Plasma was collected and stored in aliquots at –80°C.

      Preparation of Soluble Leishmania Antigen (SLA)

      SLA was prepared from cultured L. macropodum, L. major, L. donovani or L. mexicana promastigotes (
      • Dougall AM
      • Alexander B
      • Holt DCDC
      • Harris T
      • Sultan AH
      • Bates PA
      • et al.
      Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia.
      ;
      • Rose K
      • Curtis J
      • Baldwin T
      • Mathis A
      • Kumar B
      • Sakthianandeswaren A
      • et al.
      Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.
      ). Leishmania promastigotes were grown at 26°C in Grace's Insect medium (Gibco) containing 20% heat-inactivated foetal bovine serum (iFBS), 2mM L-glutamine, 100 u/mL penicillin and 100 ug/mL streptomycin. SLA was prepared and standardised across Leishmania species using 1×108 promastigotes per mL at their stationary phase. Briefly, promastigotes were washed three times in cold 1 × phosphate buffered saline, pH 7 (PBS; Gibco) at 3000 × g for 15 minutes at 4°C. Following washing, the parasite suspension was submitted to five freeze-thaw cycles between dry ice and a 37°C water bath. The parasite suspension was subsequently sonicated on ice with three pulses of 30 seconds (Sonicator 150). Finally, the suspension was centrifuged at 8000 × g for 20 minutes at 4°C. The supernatant was collected and stored at –80°C.

      Enzyme-linked immunosorbent assay (ELISA)

      For the detection of anti-Leishmania antibodies, a three-step detection in-house enzyme-linked immunosorbent assay (ELISA) was developed. Briefly, 96-well polystyrene plates (Costar, Thermo fisher Scientific) were coated with diluted SLA in bicarbonate coating buffer, pH 9.6, and incubated overnight at 4℃. After washing with 0.05% Tween20/PBS (Sigma), plates were blocked with 200 µL 3% BSA/0.05% Tween20/PBS buffer and incubated for one hour at 37℃. Following three washes, plasma (diluted 1:50 in 3% BSA/0.05% Tween20/PBS) was added to the plates in duplicate and incubated for one hour at 37℃. The plates were washed three times again, and 100 µL of a goat anti-human IgG (Fc specific) secondary antibody (Merck Millipore, Australia) was added at 1:10,000 in 3% BSA/0.05% Tween20/PBS buffer. Plates were incubated for one hour at 37℃. Following further washing, 100 µL of a tertiary antibody, an anti-goat IgG (whole molecule) horseradish peroxidase (HRP) conjugate (Merck Millipore, Australia) was added at 1:30,000 in 3% BSA/0.05% Tween20/PBS buffer and plates were incubated for one hour at 37℃. Finally, plates were washed four times and 100 µL of 3,3k,5,5k-tetramethylbenzidine substrate solution (TMB; Invitrogen, Thermo Fisher Scientific) was added to the plates for 5 minutes. The reaction was stopped by the addition of 100 µL 2N HCL and absorbance was measured at 450 nm using a Tecan infite2000 PRO plate reader. NC samples were included in triplicate on each plate and the seropositivity cut-off was established as the mean optical density (OD) of NC plus 3 standard deviations above the mean. The serological experiments were repeated three times, yielding comparable results.
      Samples that were categorized as IgG positive (in all three assays) for each Leishmania parasite were further assessed for IgG subclass responses with the three-step detection ELISA as described above but with modifications. Following plasma sample incubations, mouse anti-human IgG1, 2, 3 or 4 (1:1,000; IgG1 clone HP6069; IgG2 clone HP6002; IgG3 clone HP6047; IgG4 clone HP6025; Invitrogen, Thermo Fisher Scientific) was added and plates were incubated for one hour at 37℃. Bound IgG subclasses were detected with a goat anti-mouse IgG-HRP conjugated antibody (1:5,000; Abcam) for one hour at 37℃. Plates were developed with TMB for 20 minutes prior to reading. Again, NC samples were included in triplicates on each plate and serological experiments were repeated thrice.

      Statistical analysis

      To examine relationships between seropositivity, age and sex, a number of statistical tests were undertaken. First, a chi-squared test was used to determine whether males or females were more likely to be positive to any parasite species. Then an analysis of variance (ANOVA) was used to test relationships between age and seropositivity for any parasite species. Finally, to determine whether an individual was more likely to be seropositive for a given parasite species, an ANOVA was used. We also examined whether there was significant variation in the IgG subclass response within parasites (e.g., for L. macropodum IgG1-4) and between parasites (e.g., IgG1 across all parasites). To do so we used a generalised linear model (GLM) to assess the number of IgG subclass responses either above or below the OD threshold for each parasite species and subclass. To determine the most prevalent IgG subclass detected amongst IgG positive blood donors a cut-off for positivity was based on NC. All analyses were undertaken in R and we considered a relationship to be significant if p-value = <0.05.

      Results

      Detection of IgG that recognises SLA derived from Leishmania species

      A total of 282 blood donors residing in the NT were screened for IgG against SLA derived from the following four Leishmania parasite species, L. macropodum, L. major, L. mexicana and L. donovani. Plasma from ninety-six (34.04%) blood donors contained IgG that recognised SLA from at least one Leishmania species. IgG positivity was highest for SLA derived from L. macropodum (20.57%), followed by L. donovani (13.12%), L. major (9.57%) and L. mexicana (6.03%) (Table 1). Of the 58 individuals IgG positive for L. macropodum, 34 tested positive for L. macropodum alone (Table 2), whereas 20 showed positive IgG for one or more other species. Twenty-four blood donors (8.51%) were IgG positive for ≥ two Leishmania species, five blood donors (1.77%) were IgG positive for ≥ three Leishmania species and four blood donors (1.42%) were IgG positive for all four Leishmania species (Table 1).
      Table 1IgG positivity for soluble Leishmania antigen derived from L. macropodum, L. major, L. mexicana and L. donovani among blood donors residing in the Northern Territory, Australia
      IgG positivity was determined by Leishmania species SLA-specific ELISA
      .
      Number testedPositive
      Number%95% CI
      L. macropodum IgG
      Overall2825820.5716.26—25.67

      Age group
      18 - 24511121.5712.49—34.63
      25 - 34521528.8518.33—42.27
      35 - 44531222.6413.45—35.53
      45 - 5450918.009.77—30.80
      55 - 6447612.775.98—25.17
      ≥ 6529517.247.60—34.55
      Sex
      Male1492617.4512.20—24.34
      Female1333224.0617.59—31.99
      L. major IgG
      Overall282279.576.66—13.57
      Age group
      18 - 245123.921.08—13.22
      25 - 345247.693.03—18.17
      35 - 4453815.097.85—27.05
      45 - 5450816.008.34—28.51
      55 - 644748.513.36—19.93
      ≥ 652913.450.61—17.18
      Sex
      Male1491812.087.78—18.29
      Female13396.773.60—12.36
      L. mexicana IgG
      Overall282176.033.80—9.44
      Age group
      18 - 245111.960.35—10.30
      25 - 345247.693.03—18.17
      35 - 445347.552.97—17.86
      45 - 545036.002.06—16.22
      55 - 644748.513.36—19.93
      ≥ 652913.450.61—17.18
      Sex
      Male149138.725.17—14.35
      Female13343.011.18—7.48
      L. donovani IgG
      Overall2823713.129.67—17.56
      Age group
      18 - 245147.843.09—18.50
      25 - 34521019.2310.80—31.90
      35 - 4453713.216.55—24.84
      45 - 5450918.009.77—30.80
      55 - 644748.513.36—19.93
      ≥ 6529310.343.58—26.39
      Sex
      Male1492214.779.96—21.34
      Female1331511.286.95—17.78
      1 IgG positivity was determined by Leishmania species SLA-specific ELISA
      Table 2Total positive blood donors for each Leishmania species and number of blood donors showing positive results to other species
      IgG positivity was determined by Leishmania species SLA-specific ELISA. Columns list first species positive. Rows the subsection of IgG positive to other species.
      .
      First SecondL. macropodumL. majorL. mexicanaL. donovani
      L. macropodumNA11/275/1720/37
      L. major11/58NA17/1710/37
      L. mexicana4/589/27NA7/37
      L. donovani20/5810/277/17NA
      Total positive58271737
      1 IgG positivity was determined by Leishmania species SLA-specific ELISA. Columns list first species positive. Rows the subsection of IgG positive to other species.
      There was no significant difference between males and females in terms of the presence of IgG that recognised SLA, except for L. mexicana, where males had significantly higher IgG positivity rates than females (p-value = 0.04). There was also no significant relationship between age and IgG positivity for any Leishmania species (Table 1).

      IgG subclass responses to SLA among blood donors with detectable IgG from a marsupial Leishmania endemic region of Australia

      Donors that were IgG positive to SLA from a given Leishmania species were further tested to determine their IgG subclass profile (Figure 1). IgG1 and IgG2 were the predominant subclasses detected for L. macropodum (26/58 IgG1, 26/58 IgG2) and L. donovani (15/37 IgG1, 14/37 IgG2) whereas IgG2 was solely predominant for L. major (10/12 IgG2) and L. Mexicana (9/17 IgG2).
      Figure 1
      Figure 1IgG subclass profiles for Leishmania species among SLA-IgG positive blood donors residing in Northern Territory, Australia. IgG subclass responses were determined by Leishmania species soluble Leishmania antigen (SLA)-specific ELISA. The y-axis represents the optical density (OD) measured at 450nm. The cut-off value for positivity is indicated as a horizontal line (black) and represents the mean+3 standard deviation of negative control samples from a non-endemic region (Southeast QLD). Each circle represents a single individual screened for IgG subclass (IgG1-4) responses to SLA derived from four Leishmania species. Experiments were repeated thrice, and this figure represents the results from one assay.
      Using an ANOVA, L. macropodum was the only parasite species that had a significant difference in percentage positivity between antibody subclass responses (p-value = 0.0151). There was no significant difference in the remaining subclass responses between Leishmania species.
      Comparing between species within each subclass, IgG3 and IgG4 showed significantly lower OD values to IgG1 (p-value = 0.049, p-value = 0.011) across all species. IgG2 was not different to IgG1 (p-value = 1). There was no significant difference within antibody subclass responses for L. donovani (p-value = 0.5), L. major (p-value = 0.413) or L. mexicana (p-value = 0.669).
      Irrespective of parasite, IgG3 responses were significantly below the optical density (OD) threshold (p-value = 0.0452) indicating limited IgG3 response to any Leishmania species. IgG4 appeared show higher OD readings for L. macropodum when comparing between species, though this was not statistically significant (p-value = 0.08394).

      Discussion

      This is the first serosurvey conducted in Australia examining the prevalence of IgG responsiveness and the IgG subclass profile to SLA derived from four Leishmania species among blood donors residing in a region of Australia known to be endemic for marsupial CL. Serosurveys are routinely used to evaluate and confirm Leishmania parasite exposure in humans (
      • Ibarra-Meneses AV
      • Corbeil A
      • Wagner V
      • Onwuchekwa C
      • Fernandez-Prada C.
      Identification of asymptomatic Leishmania infections: a scoping review.
      ). In this study we examined anti-Leishmania antibodies among blood donors residing in a region of marsupials Leishmania endemicity in Australia to assess whether humans have an IgG response to L. macropodum. We observed a 20.57% IgG positivity to L. macropodum, 13.12% to L. donovani, 9.57% to L. major and 6.03% to L. mexicana.
      Our results in blood donors represent a cross-section of the adult population residing in the NT, and therefore this study suggests that approximately 20% of this NT population may have IgG to L. macropodum. It was reported that the seroprevalence of anti-Leishmania (Viannia) braziliensis IgG among healthy blood donors residing in endemic countries of the New World was up to 11.4%, with 15.3% among the general population (Mannan et al., 2021). It is important to note that antibody detection is not necessarily a confirmation of current infection.
      Further limitations of the current study must also be considered. In endemic areas of human disease, surveys are regularly conducted to estimate the prevalence of asymptomatic Leishmania infection, which is currently estimated to be 20-60% (
      • Ibarra-Meneses AV
      • Corbeil A
      • Wagner V
      • Onwuchekwa C
      • Fernandez-Prada C.
      Identification of asymptomatic Leishmania infections: a scoping review.
      ;
      • Singh OP
      • Hasker E
      • Sacks D
      • Boelaert M
      • Sundar S.
      Asymptomatic Leishmania Infection: A New Challenge for Leishmania Control.
      ). However, there is no unified approach to define asymptomatic infection (reviewed in (
      • Ibarra-Meneses AV
      • Corbeil A
      • Wagner V
      • Onwuchekwa C
      • Fernandez-Prada C.
      Identification of asymptomatic Leishmania infections: a scoping review.
      )). The only currently accepted method of confirming human infection in areas of Leishmania endemicity, is parasitological diagnosis through direct parasite examination (
      • Aronson N
      • Herwaldt BL
      • Libman M
      • Pearson R
      • Lopez-Velez R
      • Weina P
      • Carvalho EM
      • Ephros M
      • Jeronimo S
      • Magill A.
      Diagnosis and Treatment of Leishmaniasis: Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH).
      ). Thus, the clinical significance of IgG positivity to SLA derived from L. macropodum in this study must remain uncertain.
      Currently, there is no commercially available, validated serological test for L. macropodum. We developed an in-house ELISA using SLA by adapting previously published protocols (
      • Dougall AM
      • Alexander B
      • Holt DCDC
      • Harris T
      • Sultan AH
      • Bates PA
      • et al.
      Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia.
      ;
      • Rose K
      • Curtis J
      • Baldwin T
      • Mathis A
      • Kumar B
      • Sakthianandeswaren A
      • et al.
      Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.
      ). As our SLA preparation is produced by disrupting whole promastigotes it is possible that it may contain antigens that cross-react with other pathogens with phylogenetic proximity (
      • Carvalho AMRS
      • Mendes TA de O
      • Coelho EAF
      • Duarte MC
      • Menezes-Souza D.
      New antigens for the serological diagnosis of human visceral leishmaniasis identified by immunogenomic screening.
      ). Cross-reactivity of Leishmania with pathogens such as Babesia canis (
      • Oliveira TMF de S
      • Furuta PI
      • Carvalho D de
      • Machado RZ
      Study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test.
      ), Ehrlichia canis (
      • Costa LE
      • Salles BCS
      • Santos TTO
      • Ramos FF
      • Lima MP
      • Lima MIS
      • et al.
      Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis.
      ), and Trypanosoma cruzi (
      • Menezes-Souza D
      • Mendes TA de O
      • Gomes M de S
      • Bartholomeu DC
      • Fujiwara RT.
      Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope.
      ), and cross-reactivity between Leishmania species is widely acknowledged to cause false-positive results. Additionally, during the recruitment of healthy controls, we were only able to recruit 12 participants with no travel history to leishmania endemic areas. Whereas we acknowledge that the lower number may have impacted the specificity of the assay, the OD values for the 12 controls demonstrated close grouping adding reassurance our results.
      Cross-reactivity as a possible explanation for our results cannot be ruled out. However, from human serosurveys across Australia, neither B. canis, E. canis, T. cruzi nor other Leishmania species are circulating and locally acquired among humans in the Northern Territory of Australia (
      • Faddy HM
      • Rooks KM
      • Irwin PJ
      • Viennet E
      • Paparini A
      • Seed CR
      • et al.
      No evidence for widespread Babesia microti transmission in Australia.
      ;
      • Jackson Y
      • Pinto A
      • Pett S.
      Chagas disease in Australia and New Zealand: risks and needs for public health interventions.
      ). Whereas Trichomonas vaginalis is present in the NT, the prevalence in women is reported far higher than men, and our results show no gender bias, suggesting cross reactivity with Trichomonas is unlikely to have occurred. L. macropodum is known to circulate endemically in marsupials in this area and thus exposure to this species is more plausible than exposure to those with known serological cross-reactivity. Further, cross-reactivity with other Leishmania species in this sample is unlikely to be significant. We screened all blood donor samples against three additional Leishmania species and although we detected antibodies to all three Leishmania species within the study samples, samples were not statistically likely to be IgG positive to more than one species and therefore no correlation between IgG positivity to L. macropodum and the other Leishmania species was identified. The possibility for cross-reactivity to non-related antigens was not investigated in this study.
      The detection of IgG that recognized SLA from non-endemic Leishmania species in individual samples probably reflects overseas exposure. As this study used archived and unidentified blood donor samples, we were unable to obtain data on clinical history, occupation or travel history of donors and it was therefore not possible to confirm the likelihood of an overseas exposure. Without the ability to collect further information, it was also not possible to determine risk factors nor timeline for exposure to L. macropodum for individual donors.
      Previous studies on Leishmania species have examined IgG subclass profiles to explain the diverse clinical manifestations by identifying specific elevated and suppressed antibodies as potential markers in early diagnosis as well to understand subclinical CL. In this study, L. macropodum IgG1 and IgG2 were the most prevalent subclasses. From murine models infected with L. major and L. donovani, a clear relationship has been demonstrated between TH2 interleukin induced (IL)-4/IgG1 with disease progression, and TH1 induced interferon (IFN)-γ/IgG2 with resistance and protective immunity (
      • Afrin F
      • Ali N.
      Isotype Profiles of Leishmania donovani-Infected BALB/c Mice: Preferential Stimulation of IgG2a/b by Liposome-Associated Promastigote Antigens.
      ;
      • Anam K
      • Afrin F
      • Banerjee D
      • Pramanik N
      • Guha SK
      • Goswami RP
      • et al.
      Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.
      ;
      • Bretscher PA
      • Wei G
      • Menon JN
      • Bielefeldt-Ohmann H.
      Establishment of Stable, Cell-Mediated Immunity that Makes “Susceptible” Mice Resistant to Leishmania major.
      ), however this relationship is not well understood in humans or may not be applicable during infection with L. macropodum. The presence of specific IgG subclasses has been suggested to be associated with different clinical forms of CL (
      • Magalhães A
      • Carvalho LP
      • Costa R
      • Pita MS
      • Cardoso TM
      • Machado PRL
      • et al.
      Anti-Leishmania IgG is a marker of disseminated leishmaniasis caused by Leishmania braziliensis.
      ). Patients with CL (caused by L. mexicana and L. braziliensis) have shown to have a strong cell-mediated immunity with a predominance of IgG1 and IgG2 subclasses whereas patients with mucocutaneous leishmaniasis (MCL) have a predominance of IgG3, but in both cases, they have been linked with a TH1 reactivity. In comparison, during diffuse leishmaniasis (DL) an elevated IgG4 antibody response has been demonstrated to be correlated with a TH2 response (
      • Anam K
      • Afrin F
      • Banerjee D
      • Pramanik N
      • Guha SK
      • Goswami RP
      • et al.
      Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.
      ;
      • Castes M
      • Cabrera M
      • Trujillo D
      • Convit J.
      T-cell subpopulations, expression of interleukin-2 receptor, and production of interleukin-2 and gamma interferon in human American cutaneous leishmaniasis.
      ;
      • Rodríguez V
      • Centeno M
      • Ulrich M.
      The IgG isotypes of specific antibodies in patients with American cutaneous leishmaniasis; relationship to the cell-mediated immune response.
      ). Here we detected a predominance of IgG1 and IgG2 among blood donors with detectable IgG to L. macropodum. However, without evidence of clinical history of blood donors, an association between IgG subclass responses and L. macropodum CL remains inconclusive.
      This is the first study of its kind assessing the prevalence of anti-L. macropodum IgG and IgG subclass responses in a healthy human population from a region of marsupial L. macropodum endemicity in Australia. This study suggests humans are exposed and mount an immune response to L. macropodum but cannot conclusively rule out an impact of SLA cross-reactivity on results. Future studies should therefore i) develop an assay using a recombinant protein that is validated for L. macropodum to increase the sensitivity and to confirm the specificity of IgG responses, and ii) evaluate whether IgG positivity indicates infection with L. macropodum in human hosts. In Australia, a clear link between symptomatic CL of marsupials and L. macropodum infection has been demonstrated. Therefore, the ongoing circulation of L. macropodum in the NT and the implication to public health is important to further investigate.

      Uncited References

      • Mannan S Bin
      • Elhadad H
      • Loc TTH
      • Sadik M
      • Mohamed MYF
      • Nam NH
      • et al.
      Prevalence and associated factors of asymptomatic leishmaniasis: a systematic review and meta-analysis.

      Declaration of Competing Interest

      The authors declare no conflict of interest.

      Acknowledgments

      We would like to acknowledge and thank Associate Professor Christopher Peacock from the University of Western Australia for kindly providing all Leishmania species isolates and his inputs.

      Funding

      Australian Governments fund Australian Red Cross Lifeblood to provide blood, blood products and services to the Australian community

      Author contributions

      Study design: E.P, D.I.S, M.K.Y, H.M.F, and L.J.H.
      Data collection: E.P, D.I.S, M.K.Y, H.M.F, and L.J.H.
      Data analysis: E.P, E.B.S and L.J.H.
      Writing: E.P, D.I.S, M.K.Y, H.M.F, and L.J.H.

      Ethical approval

      The study was conducted in accordance with Australian ethical guidelines and approved by the Griffith University Human Research Ethics Committee (GU HREC; GU Ref No: 2019/805, 24th October 2019) for the involvement of participants from the Southeast Queensland (SEQ) region and approved by the Australian Red Cross (ARC) Lifeblood Human Research Ethics Committee for access to archived donor plasma samples (Ref No: 08102019, 14th October 2019). Written informed consent was obtained from all subjects involved in the study.

      References

        • Afrin F
        • Ali N.
        Isotype Profiles of Leishmania donovani-Infected BALB/c Mice: Preferential Stimulation of IgG2a/b by Liposome-Associated Promastigote Antigens.
        J Parasitol. 1998; 84: 743-748https://doi.org/10.2307/3284581
        • Aliaga L
        • Ceballos J
        • Sampedro A
        • Cobo F
        • López-Nevot MÁ
        • Merino-Espinosa G
        • et al.
        Asymptomatic Leishmania infection in blood donors from the Southern of Spain.
        Infection. 2019; (00)https://doi.org/10.1007/s15010-019-01297-3
        • Anam K
        • Afrin F
        • Banerjee D
        • Pramanik N
        • Guha SK
        • Goswami RP
        • et al.
        Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.
        Clin Diagn Lab Immunol. 1999; 6 (1999): 231-235https://doi.org/10.1128/CDLI.6.2.231-235
        • Andrade-Narvaez FJ
        • Loría-Cervera EN
        • Sosa-Bibiano EI
        • Van Wynsberghe NR.
        Asymptomatic infection with American cutaneous leishmaniasis: epidemiological and immunological studies.
        Mem Inst Oswaldo Cruz. 2016; 111: 599-604https://doi.org/10.1590/0074-02760160138
        • Aronson N
        • Herwaldt BL
        • Libman M
        • Pearson R
        • Lopez-Velez R
        • Weina P
        • Carvalho EM
        • Ephros M
        • Jeronimo S
        • Magill A.
        Diagnosis and Treatment of Leishmaniasis: Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH).
        Clin Infect Dis. 2016; 63 (Dec 15): 1539-1557https://doi.org/10.1093/cid/ciw742
        • Bates PA.
        Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies.
        Int J Parasitol. 2007; 37: 1097-1106https://doi.org/10.1016/j.ijpara.2007.04.003
        • Bretscher PA
        • Wei G
        • Menon JN
        • Bielefeldt-Ohmann H.
        Establishment of Stable, Cell-Mediated Immunity that Makes “Susceptible” Mice Resistant to Leishmania major.
        Science. 1992; 257: 539-542https://doi.org/10.1126/science.1636090
        • Burza S
        • Croft SL
        • Leishmaniasis Boelaert M.
        Lancet. 2018; 392: 951-970https://doi.org/10.1016/S0140-6736(18)31204-2
        • Cardo LJ.
        Leishmania: risk to the blood supply.
        Transfusion. 2006; 46: 1641-1645https://doi.org/10.1111/j.1537-2995.2006.00941.x
        • Carvalho AMRS
        • Mendes TA de O
        • Coelho EAF
        • Duarte MC
        • Menezes-Souza D.
        New antigens for the serological diagnosis of human visceral leishmaniasis identified by immunogenomic screening.
        PLoS One. 2018; 13e0209599https://doi.org/10.1371/journal.pone.0209599
        • Castes M
        • Cabrera M
        • Trujillo D
        • Convit J.
        T-cell subpopulations, expression of interleukin-2 receptor, and production of interleukin-2 and gamma interferon in human American cutaneous leishmaniasis.
        J Clin Microbiol. 1988; 26 (1988): 1207-1213https://doi.org/10.1128/jcm.26.6.1207-1213
        • Costa LE
        • Salles BCS
        • Santos TTO
        • Ramos FF
        • Lima MP
        • Lima MIS
        • et al.
        Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis.
        Microb Pathog. 2017; 110: 14-22https://doi.org/10.1016/j.micpath.2017.06.020
        • Deepachandi B
        • Weerasinghe S
        • Gunathilake H
        • Andrahennadi TP
        • Wickramanayake MN
        • Siri S
        • Chandrasekharan V
        • Soysa P
        • Siriwardana Y.
        Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis.
        Int J Anal Chem. 2020; 2020 (Jul 15)9289651https://doi.org/10.1155/2020/9289651
        • Dougall A
        • Shilton C
        • Low Choy J
        • Alexander B
        • Walton S
        New reports of Australian cutaneous leishmaniasis in Northern Australian macropods.
        Epidemiol Infect. 2009; 137: 1516-1520https://doi.org/10.1017/S0950268809002313
        • Dougall AM
        • Alexander B
        • Holt DCDC
        • Harris T
        • Sultan AH
        • Bates PA
        • et al.
        Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia.
        Int J Parasitol. 2011; 41: 571-579https://doi.org/10.1016/j.ijpara.2010.12.008
        • Faddy HM
        • Rooks KM
        • Irwin PJ
        • Viennet E
        • Paparini A
        • Seed CR
        • et al.
        No evidence for widespread Babesia microti transmission in Australia.
        Transfusion. 2019; 59: 2368-2374https://doi.org/10.1111/trf.15336
        • Ferreira-Silva MM
        • Teixeira LAS
        • Tibúrcio MS
        • Pereira GA
        • Rodrigues V
        • Palis M
        • et al.
        Socio-epidemiological characterisation of blood donors with asymptomatic Leishmania infantum infection from three Brazilian endemic regions and analysis of the transfusional transmission risk of visceral leishmaniasis.
        Transfus Med. 2018; 28: 433-439https://doi.org/10.1111/tme.12553
        • França A de O
        • Castro VL
        • de Junior
        • MS da CL
        • Pontes ERJC
        • Dorval MEC.
        Anti-Leishmania antibodies in blood donors from the Midwest region of Brazil.
        Transfus Apher Sci. 2013; 49: 627-630https://doi.org/10.1016/j.transci.2013.07.009
        • Fukutani KF
        • Figueiredo V
        • Celes FS
        • Cristal JR
        • Barral A
        • Barral-Netto M
        • et al.
        Serological survey of Leishmaniainfection in blood donors in Salvador, Northeastern Brazil.
        BMC Infect Dis. 2014; 14: 422https://doi.org/10.1186/1471-2334-14-422
        • Ibarra-Meneses AV
        • Corbeil A
        • Wagner V
        • Onwuchekwa C
        • Fernandez-Prada C.
        Identification of asymptomatic Leishmania infections: a scoping review.
        Parasit Vectors. 2022; 15: 5https://doi.org/10.1186/s13071-021-05129-y
        • Ibarra-Meneses AV
        • Carrillo E
        • Nieto J
        • Sánchez C
        • Ortega S
        • Estirado A
        • Latasa Zamalloa P
        • Sanz JC
        • García-Comas L
        • Ordobás M
        • Moreno J
        Prevalence of asymptomatic Leishmania infection and associated risk factors, after an outbreak in the south-western Madrid region, Spain, 2015.
        Euro Surveill. 2019; 24 (May)1800379https://doi.org/10.2807/1560-7917.ES.2019.24.22.1800379
        • Jackson Y
        • Pinto A
        • Pett S.
        Chagas disease in Australia and New Zealand: risks and needs for public health interventions.
        Trop Med Int Heal. 2014; 19: 212-218https://doi.org/10.1111/tmi.12235
        • Jimenez-Marco T
        • Riera C
        • Girona-Llobera E
        • Guillen C
        • Iniesta L
        • Alcover M
        • et al.
        Strategies for reducing the risk of transfusion-transmitted leishmaniasis in an area endemic for Leishmania infantum: A patient- And donor-targeted approach.
        Blood Transfus. 2018; 16: 130-136https://doi.org/10.2450/2017.0201-16
        • Magalhães A
        • Carvalho LP
        • Costa R
        • Pita MS
        • Cardoso TM
        • Machado PRL
        • et al.
        Anti-Leishmania IgG is a marker of disseminated leishmaniasis caused by Leishmania braziliensis.
        Int J Infect Dis. 2021; 106: 83-90https://doi.org/10.1016/j.ijid.2021.02.016
        • Mannan S Bin
        • Elhadad H
        • Loc TTH
        • Sadik M
        • Mohamed MYF
        • Nam NH
        • et al.
        Prevalence and associated factors of asymptomatic leishmaniasis: a systematic review and meta-analysis.
        Parasitol Int. 2021; 81102229https://doi.org/10.1016/j.parint.2020.102229
        • Mendonça MG
        • de Brito MEF
        • Rodrigues EHG
        • Bandeira V
        • Jardim ML
        • Abath FGC.
        Persistence of Leishmania Parasites in Scars after Clinical Cure of American Cutaneous Leishmaniasis: Is There a Sterile Cure?.
        J Infect Dis. 2004; 189: 1018-1023https://doi.org/10.1086/382135
        • Menezes-Souza D
        • Mendes TA de O
        • Gomes M de S
        • Bartholomeu DC
        • Fujiwara RT.
        Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope.
        PLoS Negl Trop Dis. 2015; 9: e3426
        • Oliveira TMF de S
        • Furuta PI
        • Carvalho D de
        • Machado RZ
        Study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test.
        Rev Bras Parasitol Veterinária. 2008; 17: 7-11https://doi.org/10.1590/S1984-29612008000100002
        • Panahi E.I.
        • Stanisic D.S.
        • Peacock C.J.
        • Herrero L.
        Protective and Pathogenic Immune Responses to Cutaneous Leishmaniasis.
        Leishmaniasis - Gen. Asp. a Stigmatized Dis. 2022; 32 (volIntechOpen): 1854-1858https://doi.org/10.5772/intechopen.101160
        • Panahi E
        • Shivas M
        • Hall-Mendelin S
        • Kurucz N
        • Rudd PA
        • De Araujo R
        • et al.
        Utilising a novel surveillance system to investigate species of Forcipomyia (Lasiohelea) (Diptera: Ceratopogonidae) as the suspected vectors of Leishmania macropodum (Kinetoplastida: Trypanosomatidae) in the Darwin region of Australia.
        Int J Parasitol Parasites Wildl. 2020; https://doi.org/10.1016/j.ijppaw.2020.06.004
        • Pérez-Cutillas P
        • Goyena E
        • Chitimia L
        • De la Rúa P
        • Bernal LJ
        • Fisa R
        • et al.
        Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: A study of environmental, demographic and social risk factors.
        Acta Trop. 2015; 146: 127-134https://doi.org/10.1016/j.actatropica.2015.03.017
        • Ready PD.
        Biology of Phlebotomine Sand Flies as Vectors of Disease Agents.
        Annu Rev Entomol. 2013; 58: 227-250https://doi.org/10.1146/annurev-ento-120811-153557
        • Riera C
        • Fisa R
        • Lpez-Chejade P
        • Serra T
        • Girona E
        • Jimnez M
        • et al.
        Asymptomatic infection by Leishmania infantum in blood donors from the Balearic Islands (Spain).
        Transfusion. 2008; 48: 1383-1389https://doi.org/10.1111/j.1537-2995.2008.01708.x
        • Rodríguez V
        • Centeno M
        • Ulrich M.
        The IgG isotypes of specific antibodies in patients with American cutaneous leishmaniasis; relationship to the cell-mediated immune response.
        Parasite Immunol. 1996; 18: 341-345https://doi.org/10.1046/j.1365-3024.1996.d01-113.x
        • Rose K
        • Curtis J
        • Baldwin T
        • Mathis A
        • Kumar B
        • Sakthianandeswaren A
        • et al.
        Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.
        Int J Parasitol. 2004; 34: 655-664https://doi.org/10.1016/j.ijpara.2004.03.001
        • Sarkari B
        • Gadami F
        • Shafiei R
        • Motazedian MH
        • Sedaghat F
        • Kasraian L
        • et al.
        Seroprevalence of Leishmania infection among the healthy blood donors in kala-azar endemic areas of Iran.
        J Parasit Dis. 2015; 39: 545-549https://doi.org/10.1007/s12639-013-0393-3
        • Sarkari B
        • Ashrafmansouri M
        • Hatam G
        • Habibi P
        • Abdolahi Khabisi S.
        Performance of an ELISA and indirect immunofluorescence assay in serological diagnosis of zoonotic cutaneous leishmaniasis in iran.
        Interdiscip Perspect Infect Dis. 2014; (2014Epub 2014 Aug 11)505134https://doi.org/10.1155/2014/505134
        • Singh OP
        • Hasker E
        • Sacks D
        • Boelaert M
        • Sundar S.
        Asymptomatic Leishmania Infection: A New Challenge for Leishmania Control.
        Clin Infect Dis. 2014; 58: 1424-1429https://doi.org/10.1093/cid/ciu102