Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.


Introduction
Cutaneous leishmaniasis (CL) is caused by a variety of Leishmania (L.) species transmitted to humans by the bite of phlebotomine sandflies [1,2]. It presents as skin lesions on exposed parts of the body, leaving life-long scars and causing disfigurement and distress [1,2]. About 95% of CL cases occur in the Americas, the Mediterranean basin, the Middle East, and Central Asia, with an estimated incidence between 600,000 and 1 million new cases occurring worldwide annually [1]. In North African countries, the burden of the disease is high and three Leishmania species, associated to distinct eco-epidemiological patterns, namely L. infantum, L. major and L. tropica, are involved in Leishmania transmission [3][4][5]. However, L. major is by far the most frequent species, with more than 90% of registered cases in Algeria and Tunisia [5,6].
Cutaneous leishmaniasis caused by L. major, also known as zoonotic or rural zoonotic cutaneous leishmaniasis (ZCL), is a major public health problem in the North African region, including Tunisia, with thousands of cases occurring each year [5,6]. It is distributed in the arid and Saharan bioclimatic stages, where it follows an epidemic pattern with seasonal occurrence of cases [5,6]. Typical ZCL lesions evolve from papules to nodules to ulcerative lesions, with a central depression and a raised, indurate border [5]. They are typically multiple and located on limbs and tend to be exudative or "wet", large and complicated by superficial and secondary bacterial infections [5]. Most cases do not cause any clinical diagnostic difficulties, diagnosis often being suspected during epidemics on evocative clinical presentations in patients living in or coming from endemic areas [5][6][7]. However, occasionally patients present with unusual morphological forms of CL, which initially may elude diagnosis [8]. In Tunisia, parasitological confirmation of the diagnosis is highly recommended before engaging treatment [9]. It is mainly based on the direct microscopic identification of Leishmania amastigotes in Giemsa-stained dermal scrapings [10].
The diagnosis of CL is based on clinical features (supported by epidemiologic data) and laboratory testing. Numerous diagnostic methods have been described, including direct parasitological examination, molecular and immunological diagnostics [11,12]. Parasitological diagnosis, which is typically undertaken by direct microscopy, histopathology or culture of material from suspected lesions (obtained by scraping, needle aspiration, punch or biopsy), is still considered the gold standard in CL diagnosis because of its high specificity [11,12]. However, while these techniques are highly specific for diagnosing leishmaniasis, they are not sensitive enough [11,12]. Thus, the percent success for microscopic detection of Leishmania amastigotes in stained dermal scrapings varies depending on the number of parasites present and microscopist expertise, and is estimated around 60-80% for CL caused by L. major [6,10]. Likewise, culture on NNN medium is a less sensitive technique, which is moreover limited by the nonexceptional bacterial and fungal contamination [11,12]. To overcome these limits molecular diagnostic tests have been developed over the last decades, as these are assumed to have better sensitivity than traditional diagnostic methods [11,12]. In particular, PCRs, using either genomic or kinetoplast DNA (kDNA) and performed either as a single test or in a nested format or as a quantitative assay (qPCR), have been widely exploited [11,12]. Among these, PCR targeting kDNA minicircle is considered to be the most sensitive method for CL diagnosis, since there are about 10,000 copies of minicircles per parasite [13,14], whereas PCR assay amplifying the internal transcribed spacer 1 (ITS1) region of the rRNA genes has been shown to be a sensitive method that allows identification of almost all pathogenic Old World Leishmania species by restriction fragment length polymorphism (RFLP) [14]. These techniques are, however, only available in some specialized centers. Immunologic diagnostic methods are based on the detection of anti-Leishmania antibodies or Leishmania antigens. Although serologic tests are available for CL, they are not widely employed for CL diagnosis [11,12]. The CL Detect™ Rapid Test (InBios, Washington, DC, USA) targeting the peroxidoxin antigen produced by Leishmania amastigotes in skin lesions has been evaluated in various endemic settings with varying results [15,16]. On the other hand, it is now recognized that immunohistochemistry (IHC) detecting Leishmania antigens in tissue sections is a reliable complementary tool improving sensitivity and specificity of the histopathological diagnosis of CL, especially for lesions with low parasite burden [17,18]. Monoclonal [19,20] and polyclonal antibodies [18,[21][22][23][24][25] produced against Leishmania, as well as immune serum from dog naturally infected with Leishmania [26], were successfully used to detect Leishmania amastigotes and their antigens in routinely prepared histological sections. Though antibodies detecting Leishmania antigens were widely used in pathology diagnosis, they were applied only in the Americas on large series of noninvasive samples [27]. Their use on dermal scrapings from Old World CL remains rarely reported [28]. There is no information available about reliability of immunofluorescence assays to access diagnosis of Old World CL in comparison to other available methods.
The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens, to examine its specificity on amastigotes-infected macrophages and macrophage-derived amastigotes, and to assess its reliability on dermal scrapings in the routine diagnosis of CL in Tunisia.

Leishmania Isolates and Culture
All experiments were carried out using two Leishmania isolates GLC94 (MHOM/TN95/ GLC94) and LV59 (MHOH/TN07/LV59) collected from cutaneous and visceral cases and typed as L. major and L. infantum, respectively. These two isolates were available from the biobank of Leishmania species at Institute Pasteur of Tunis.
Cryopreserved Leishmania promastigotes were stabilized and cultured in RPMI 1640 medium (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Lonza), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. The RPMI culture was incubated at 26 • C and maintained by adding medium every 72 h. Metacyclic promastigotes were harvested during the stationary phase of culture after 5 days of incubation without culture media addition and purified through a discontinuous Ficoll gradient, as previously described [29].

Rabbit Anti-L. major Immune Serum
Anti-Leishmania antibodies were produced in 3-month-old Californian female rabbits by 5 biweekly intravenous injections of 10 8 living L. major metacyclic promastigotes, as previously described [30]. , 100 mM sodium pyruvate, 25 mM HEPES, 100 U/mL penicillin (Sigma Aldrich), 100 µg/mL streptomycin (Sigma Aldrich), 100 mM nonessential amino acids (Sigma Aldrich) and 10% FBS were plated on a Permanox ® slide (10 5 cells per well) in an 8-well chamber slide system (Nunc ® Lab-Tek, Sigma Aldrich) and allowed to adhere to the slides for 3 h at 37 • C, 5% CO 2 . Then, adherent macrophages were infected with Leishmania metacyclic promastigotes at a macrophages-to-parasite ratio of 1:10 overnight at 37 • C, 5% CO 2 . A control well of RAW 264.7 cells without parasites was set up. After overnight incubation, noninternalized promastigotes were removed by washing two times with RPMI, then culture medium was added, and infected macrophages were maintained at 37 • C in 5% CO 2 . After two days of incubation, Leishmania-amastigotes-infected macrophages were visualized by microscopic examination of Giemsa-stained slides (data not shown). This two day incubation period was used for all subsequent experiments.
Amastigotes were released from infected cells by using 0.05% SDS in PBS, as previously described by Jain et al. [31]. Briefly, Leishmania-amastigotes-infected macrophages were washed with PBS and then exposed to 1 mL of 0.05% SDS in PBS for 30 s. The total volume of parasite suspension was aspirated, immediately diluted by adding PBS and washed 3 times to remove SDS. Amastigotes were collected by centrifugation at 2500 rpm for 10 min and resuspended in serum saline 0.9%.

Reactivity of the Rabbit Anti-L. major Immune Serum
An indirect immunofluorescence (IF) assay using the rabbit anti-L. major immune serum followed by fluorescein-conjugated anti-rabbit IgG was tested on (i) L. major amastigotesinfected macrophages and (ii) corresponding derived amastigotes. Briefly, Leishmaniaamastigotes-infected macrophages were washed twice with RPMI, fixed with formaldehyde 3% for 15 min, permeabilized with PBS 0.2% Triton X-100 for 5 min and blocked with PBS 0.5% Tween-20 3% FBS for one hour at room temperature (RT), whereas macrophagederived free amastigotes were spotted on a slide and methanol fixed. Indirect IF used 20 µL of rabbit anti-Leishmania immune serum at 1/10 dilution for one hour at RT in darkness and fluorescein-conjugated anti-rabbit IgG (Invitrogen, Thermo Fisher) at 1/100 dilution for 30 min. Visualization was carried out by a Leica DM 5500, epi-illumination fluorescence microscope. Two kinds of negative controls were used in the experiments: (i) uninfected macrophages that were subjected to the entire immunofluorescence assay and (ii) infected macrophages and free amastigotes incubated with pre-immune rabbit sera as no primary antibody controls.

Optimization of Direct Immunofluorescence Assay (DIF) on Dermal Scrapings
Anti-L. major IgGs were purified from rabbit immune serum using protein A-Sepharose Column ® (abcam, Cambridge, UK then concentrated through an Amicon Ultra-4 filter (Millipore, Dramstadt, Germany) and quantified using BiCinchoninic acid (BC) assay (BC assay, Thermo Fischer). Purified IgGs were labeled to fluorescein isothiocyanate (FITC), as previously described [32]. Briefly, 0.2 volume of sodium carbonate buffer was added to the IgG solution to bring the pH to 9.0. Fifty microliters of FITC (Merck Millipore, Molsheim, France) at 1 mg/mL in dimethyl sulfoxide (DMSO) was prepared and added progressively to 1 mL of IgG (about 50 µg of FITC/3.2 mg IgG). The solution was incubated at +4 • C for 8 h with gentle rotation. Centrifugation through an Amicon Ultra-4 centrifugal Filter unit (Merck Millipore, Darmstadt, Germany) was used to remove unbound FITC and to concentrate the FITC-labeled IgG solution. Absorbance was read at 280 nm and 495 nm using a spectrofluorimeter. Fluorochrome-to-protein optical densities ratio (F/P) corresponded to 1+. The conjugate was stored in aliquots at −20 • C.
Methanol-fixed dermal smears from positive and negative controls were incubated with 0.2% Triton X-100 in PBS for 5 min, washed three times with PBS and incubated with 0.5% Tween-20 FBS 5% in PBS for 15 min at RT. DIF assay used 20 µL of FITC-labeled anti-L. major IgG at 1:100 dilution for one hour in the dark at RT. The slides were counterstained with Evans's blue (Invitrogen) and mounted in Dako mounting fluorescence medium, then observed at 40× magnification under epifluorescence microscopy (LeicaDM5500 capture station) and LSM880 confocal microscope (Carl Zeiss Microscopy GmbH). The DIF assay was conducted with and without using DAPI (Sigma Aldrich, St. Louis, MO, USA).

Evaluation of DIF Assay in the Diagnosis of CL and Comparison to ITS1-PCR
Direct immunofluorescence assay and ITS1-PCR were performed on 101 methanolfixed dermal smears from 59 confirmed CL lesions and 42 negative ones (Table 1). All slides were provided by the laboratory of Parasitology-Mycology, Pasteur Institute of Tunis and were leftover unstained slides prepared in the setting of routine diagnosis from 101 suspected CL patients. Diagnostic results from laboratory of Parasitology-Mycology, Pasteur Institute of Tunis are given on the basis of a combined reference test of microscopy and real-time kDNA PCR, which is considered positive if one of the two tests is positive, and negative if the two tests are negative, direct microscopy being systematically conducted and kDNA PCR being performed only in case of negativity of microscopy (Table 1) [10].
The DIF assay was performed as previously described without adding DAPI. The slides were counterstained with Evans's blue in saline and mounted with buffered glycerol (pH 8.0) and observed at 40× magnification under epi-fluorescence microscopy (Le-icaDM5500 capture station). After DIF assay, glycerol was wiped off with absolute ethanol and material forming the dry smear was scraped with a sterile scalpel and covered with 200 µL Qiagen Lysis buffer (Qiamp DNA Blood Mini Kit, Qiagen, Hilden, Germany) for 5 to 10 min before transfer to a 1.5 mL reaction tube. Enzymatic digestion was performed overnight with proteinase K, then DNA was extracted according to the manufacturer's recommendations. DNA elution was conducted in 50 µL AE buffer and used for ITS1 PCR, as previously described [33]. Leishmania species identification was achieved using restriction profile analysis in comparison to L. major, L. tropica and L. infantum reference isolates [34]. Parasitic load of dermal smears from microscopy-negative/kDNA qPCRpositive CL lesions was estimated by kDNA qPCR performed, as previously described by Mary et al. using a standard curve (from 10 4 to 0.01 parasites/µL) generated from a dilution series of Leishmania DNA extracted from 10 6 L. infantum promastigotes [35].

Statistical Analysis
Sensitivity and specificity of DIF assay and ITS1-PCR were computed considering results of routine diagnosis as a gold standard. Chi-squared test was used for comparison of proportions. The significance level was set at 5%.

Ethical Considerations
All experimentations on animals were approved by the Animal Ethics Committee of the Pasteur Institute of Tunis, Tunisia (Reference 2016/08/I/LR11IPT06/V3, 22 March 2019). Methanol-fixed slide smears were prepared from dermal scraping in the setting of routine diagnosis of CL and correspond to residual unstained slides. Slide smears were anonymized and tests were performed in blinded conditions. This work was carried out in accordance with the relevant guidelines and regulations, and does not provide any personal data.

Reactivity of the Rabbit Anti-L. major Immune Serum against In Vitro Infected Macrophages and Macrophage-Derived Amastigotes
The indirect IF test using rabbit anti-L. major immune serum was positive when applied on in vitro infected macrophages or on amastigotes ( Figure 1C,D). In contrast, in control experiments, (i) uninfected macrophages did not react with rabbit anti-L. major immune serum and (ii) infected macrophages did not react with the preimmunization rabbit serum ( Figure 1A,B).
Leishmania major-infected RAW cells showed different fluorescence images ( Figure 1C). The most representative was a dense speckled cytoplasmic fluorescence suggesting the presence of Leishmania-derived antigens into cytoplasm of infected host cells. Some RAW cells demonstrated brilliant surface fluorescence labeling and several amastigotes were revealed outside macrophages as fluorescent stained bodies. the presence of Leishmania-derived antigens into cytoplasm of infected host cells. Some RAW cells demonstrated brilliant surface fluorescence labeling and several amastigotes were revealed outside macrophages as fluorescent stained bodies.

Detection of Leishmania Antigens by Direct Immunofluorescence Staining of Dermal Scrapings
In dermal scrapings from confirmed L. major CL cases, the FITC-labeled anti-L. major IgG detected infected mononuclear phagocytic cells and Leishmania amastigotes ( Figure  2A-C), whereas, in negative controls, no fluorescence was shown ( Figure 2D). Infected mononuclear phagocytic cells were identified by a fluorescence cell pattern with Leishmania antigen appearing as a diffuse deposit into the cytoplasm (Figure 2A-C). Amastigotes were identified by their size and shape into phagocytes (Figure 2A,C).

Detection of Leishmania Antigens by Direct Immunofluorescence Staining of Dermal Scrapings
In dermal scrapings from confirmed L. major CL cases, the FITC-labeled anti-L. major IgG detected infected mononuclear phagocytic cells and Leishmania amastigotes (Figure 2A-C), whereas, in negative controls, no fluorescence was shown ( Figure 2D). Infected mononuclear phagocytic cells were identified by a fluorescence cell pattern with Leishmania antigen appearing as a diffuse deposit into the cytoplasm (Figure 2A-C). Amastigotes were identified by their size and shape into phagocytes (Figure 2A,C).

Reliability of Direct Immunofluorescence (DIF) Method Using FITC-Labeled Anti-L. major IgG in Comparison to Other Available Methods
The DIF assay was positive in 58 out 59 positive CL cases and negative in all negative ones, giving a sensitivity of 98.3% and a specificity of 100% in CL diagnosis. The DIF assay results 100% correlated with those of ITS 1-PCR. However, DIF was more sensitive than direct microscopy (98.3% versus 71.2%, p < 0.001) ( Table 1).
The DIF assay allowed diagnosis for 16 out of the 17 smears from CL lesions negative by microscopy and positive by kDNA qPCR. Parasite quantification from these latter slides was globally low with a median parasitic load of 60 parasites/smear and an interquartile range of 13-123 parasites/smear. The only negative slide by DIF assay had a very low parasitic load, which was estimated at four parasites/smear. ITS1 PCR-RFLP analysis of DNA extracted from the DIF-positive slides allowed species identification in 56 cases out of 58. These were 52 cases of L. major, two cases of L. infantum and two cases of L. tropica (Table 1).

Reliability of Direct Immunofluorescence (DIF) Method Using FITC-Labeled Anti-L. major IgG in Comparison to Other Available Methods
The DIF assay was positive in 58 out 59 positive CL cases and negative in all negative ones, giving a sensitivity of 98.3% and a specificity of 100% in CL diagnosis. The DIF assay results 100% correlated with those of ITS 1-PCR. However, DIF was more sensitive than direct microscopy (98.3% versus 71.2%, p < 0.001) ( Table 1).
The DIF assay allowed diagnosis for 16 out of the 17 smears from CL lesions negative by microscopy and positive by kDNA qPCR. Parasite quantification from these latter slides was globally low with a median parasitic load of 60 parasites/smear and an interquartile range of 13-123 parasites/smear. The only negative slide by DIF assay had a very low parasitic load, which was estimated at four parasites/smear. ITS1 PCR-RFLP analysis of DNA extracted from the DIF-positive slides allowed species identification in 56 cases out of 58. These were 52 cases of L. major, two cases of L. infantum and two cases of L. tropica (Table 1).

Discussion
Rabbit intravenous infection with live Leishmania metacyclic promastigotes was already used as a cheap and non-laborious way to produce anti-Leishmania amastigote antibodies [36,37]. Used in an indirect immunofluorescence assay, our anti-L. major serum demonstrated positive staining of L. major amastigotes released from infected RAW cells. On the other hand, we demonstrated in a previous study that these polyclonal IgG antibodies recognize the promastigote form of Leishmania and cross-react with other Leishmania species than L. major [30].
Applied on in vitro L. major-infected RAW macrophages the anti-L. major serum followed by FITC-conjugated goat anti-rabbit IgG showed fluorescence images suggestive of the presence of Leishmania-derived antigens into the cytoplasm and on the membrane of the host cells. The key to providing Leishmania antibody specificity in these experiments was the use of two kinds of negative controls, ensuring that uninfected macrophages are not recognized by the polyclonal antibodies and that fluorescence staining is produced only when the anti-L. major antibodies are used. The observed fluorescence images are in concordance with results of previous experimental studies, indicating that Leishmania infection of macrophages is followed by the appearance of Leishmania antigen from amastigote origin on the external surface membrane of macrophages [38,39]. Furthermore, other authors evaluated the distribution of Leishmania proteins in infected RAW cells and showed that some of these are secreted into parasitophorous vacuoles (PVs) and then traffic out PVs into the host cell cytosol and nucleus in vesicles of distinct morphologies [40].
Anti-Leishmania IgGs were purified and coupled to FITC. The latter remains the most commonly used amine-reactive fluorophore for fluorescence labeling, with an excellent fluorescence potential and with high-quality images [41]. Moreover, DIF assay offers the advantage over the indirect immunofluorescence procedure of reducing nonspecific background signal and limiting the possibility of antibody cross-reactivity through the use of conjugated primary antibodies. On dermal scrapings, the DIF assay was optimized using DAPI, a cell nuclear-specific dye. DAPI blue fluorescence allowed marked contrast with the green fluorescence of probed antigens and, more importantly, allowed localization of the cells present in the dermal scraping via their nuclei. This made it easier to visualize in dermal scrapings from confirmed L. major CL cases, mononuclear cells with intracytoplasmic fluorescent green structures, with size and shape corresponding to those of Leishmania amastigotes, but also fluorescent deposits likely corresponding to Leishmania-derived antigens. The presence of this intracytoplasmic leishmanian material has been previously documented by IHC on biopsies from L. tropica and L. braziliensis CL lesions [21,24]. Another IHC study has documented the intracellular presence of amastigote degradation products and soluble degradation components [41]. For CL, due to L. major, intracytoplasmic leishmanian material existence into macrophages is likely related to a dynamic and intense process of amastigote destruction and subsequent antigen processing [42,43].
Applied on dermal scrapings, the DIF assay was more sensitive than microscopy and as sensitive as ITS1 PCR. Furthermore, the gain in sensitivity of DIF and ITS1-PCR was achieved mainly in smears containing about 10-100 parasites. Our previous studies (carried out in the same epidemiological setting) already reported that this low parasite load was not detected by microscopy but was by conventional ITS1 PCR, a lower load not being detected by either technique [10]. This high sensitivity of the DIF assay, which reaches that of the molecular tool, corroborates results obtained with histopathological diagnosis [17,18,23], polyclonal antibodies produced against Leishmania showing higher Leishmania detection rate compared to monoclonal ones [26]. Thus when the parasite load is low and ordinary techniques such as Giemsa stains fail to detect the parasites, immunohistochemistry with anti-Leishmania antibodies has demonstrated a higher level of sensitivity in the identification of amastigotes [44,45]. Furthermore, in our study, most CL cases were caused by L. major and DIF assay stained, in addition to the parasites, amastigote-derived antigens into host cells, which may have enhanced sensitivity of the method.
On the other hand, the polyclonal anti-L. major IgGs enabled detection of L. infantum (n = 2) and L. tropica (n = 2) cases, which also explain their high sensitivity in detecting Leishmania. This result is in accordance with those of previous studies indicating antigenic cross-reactivity between Leishmania species. Thus, immune serum from dog naturally infected with L. chagasi was successfully used to diagnose American tegumentary leishmaniasis [26]. Moreover, no difference in fluoresecence staining was reported with antisera to L. tropica, L. major or L. Mexicana [41]. Further complementary study is, however, needed to better evaluate the test accuracy according to species. On the other hand, although the negative controls validated the specificity of polyclonal antibodies against Leishmania antigens in the experimental study, and the DIF assay applied to negative dermal scrapings did not visualize parasites and/or infected cells, the specificity of DIF in the diagnosis of CL needs to be evaluated by a dedicated protocol. The latter should include specimens of other infectious and noninfectious skin diseases whose lesions are clinically suggestive of CL [8,9].

Conclusions
Leishmania major is the main Leishmania species causing CL in Tunisia. Its lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection and delay patient management. Here, we described the development of a direct immunofluorescence (DIF) method that stains specifically, in addition to Leishmania amastigotes, their degraded residues and soluble components. The DIF assay applied to dermal scrapings showed fluorescence images suggestive of both parasites and infected cells and was more sensitive than microscopy and as sensitive as ITS1-PCR. Furthermore, the DIF assay using anti-L. major polyclonal IgG recognized the other Leishmania species present in Tunisia. Used as a second-line test, DIF could improve management of patients with a false-negative microscopy result.