In vitro growth of Leishmania parasites from biopsy samples of suspected cutaneous and visceral leishmaniasis cases in Sri Lanka: An observational study.

Sri Lanka reports a large focus of Leishmania donovani caused cutaneous leishmaniasis (CL). Subsequent emergence of visceral leishmaniasis (VL) was also reported recently. Expansion of the on-going disease outbreak and many complexities indicate urgent need to enhance early case detection methods. In vitro cultivation (IVC) of parasites causing visceral leishmaniasis (VL) is important for disease confirmation and to obtain sufficient quantities of parasites required in many scientific studies. IVC is carried out as a useful second line investigation for direct microscopy negative patients with CL in this setting. Along with the emergence of VL, current study was carried out to evaluate in vitro growth of local VL parasites and to identify their differences associated with in vitro growth characteristics. Routine parasitological diagnostic methods, i.e., light microscopy (LM), polymerase chain reaction (PCR) were used for confirmation of suspected cases. Lesion samples from 125 suspected CL cases and bone marrow or splenic aspirations from 125 suspected VL patients were used to inoculate IVCs. Media M199 (about 70 µl) supplemented with 15-20% of heat inactivated fetal bovine serum was used for initial culturing procedures in capillaries. Capillary cultures were monitored daily. Total of 44 different compositions/conditions were used for evaluating in vitro growth of VL causing parasite. Daily records on parasite counts, morphological appearance (size, shape, and wriggly movements) were maintained. In vitro transformation of Leishmania promastigotes to amastigotes and outcome of the attempts on recovery of live Leishmania from culture stabilates was also compared between CL and VL parasites. Proportion of cultures showing a transformation of promastigotes were 40/45 (88.9%) and 4/10 (40.0%) for CL and VL respectively. In the transformed cultures, parasites showing typical shape, size and movement patterns were less in VL (1/4, 25.0%) compared to CL (28/40, 70.0%). CL cultures showed a growth up to mass culturing level with mean duration of two weeks while it was about five weeks for VL cultures. Proportion of cultures that reached a parasite density of 1 × 106 cells/ml (proceeded to mass cultures) was significantly low in VL (4/10, 40%) as compared to CL (28/40, 70.0%). None of media compositions/conditions were successful for mass culturing of VL parasites while all of them were shown to be useful for growing CL strains. Also in vitro transformation to amastigote form and recovering of culture stabilates were not successful compared to CL. There were clear differences between in vitro growth of Leishmania parasites causing local CL and VL. Further studies are recommended for optimization of in vitro culturing of VL parasite which will be invaluable to enhance case detection in future.

Serological studies on rK39 negative Visceral Leishmaniasis in an endemic focus of Leishmania donovani induced Cutaneous Leishmaniasis.

Sri Lanka reports a focus of L. donovani induced cutaneous leishmaniasis (CL). Our more recent parasite and clinical studies and historical evidence point towards long term existence of Leishmania in the country, indicating a possible evolution leading to antigenic heterogenicity as well. In-house enzyme-linked immunosorbent assay (ELISA) that was developed during phase 1 study indicated >80% sero-positivity in local CL, while visceral leishmaniasis (VL) remained very rare with majority being negative when tested with rK39 assay. A novel serological tool was developed and sero-positivity of VL was assessed for the first time. The assay showed 100.0% sensitivity and 98.3% specificity for detection of VL. Samples were showed less positivity with established direct agglutination test (DAT) and rK39 strip test. The assay was less expensive than that of established rapid diagnostic tests (RDTs), culture and PCR assays. This assay may be useful in diagnosing clinical VL infections, detection of light microscopy (LM) negative patients, tracking post treatment stages, field screening of asymptomatic cases and in further serological studies.

First Evidence from Sri Lanka for Subphenotypic Diversity within L. donovani-Induced Classical Cutaneous Leishmaniasis.

Sri Lanka reports a large focus of Leishmania donovani-induced cutaneous leishmaniasis (CL) with CL as the main clinical entity. Two independent, long existed, and clinicoepidemiologically different transmission foci in the northern region (NR) and southern region (SR) were recently reported. Current project is an extension to this previous study. Clinical diversity within a profile of classical cutaneous leishmaniasis (CCL) in a focus of L. donovani-induced CL is described for the first time. Patients with laboratory confirmed CCL (n = 550) from NF and SF were evaluated. Lesions in both foci were found to have all classical developmental stages (small and large nodules, ulcerating nodules, and ulcers) and other identified changes (multiplication, ulceration, and enlargement). Main difference was in the proportions of lesions progressing in to each different stages, proportions of lesion undergoing the main changes, and in timing of these changes during the course of a lesion. Northern focus reported a smaller proportion of lesions showing enlargement and ulceration, and a longer period of time was also required for these changes when compared to same in southern focus. In northern focus, most lesions remained small and nonulcerating and showed a higher tendency to multiply while most lesions reported in southern focus enlarged and ulcerated rapidly and remained single. Current study also evidenced a wider spectrum in the rate and pattern of progression of a skin lesion and high individual variation which could mask these region-based differences. Parasitic, vector-related, or a host etiology is suggested. Slow progressing nonulcerating infections in North may be the result of a well-adopted parasite strain that coevolved with its host for a long period while inducing only a minimal host response. This could be one among many reasons for previously observed silent expansion in northern focus while southern focus remained more confined and stable over time. Small nonprogressive, nondisturbing lesions can play a major role as silent parasite reservoirs in a community. In addition, the laboratory detection rate declined significantly when lesions multiplied and enlarged indicating the need for early laboratory confirmation. Usefulness of identified features in clinical screening and management needs to be considered.

First Serological Study Revealing High Humoral Response and Evidence for Antigenic Heterogeneity in Leishmania donovani Induced CL in Sri Lanka.

Posing a threat to the ongoing leishmaniasis elimination efforts in the Indian subcontinent, L. donovani-induced cutaneous leishmaniasis (CL) has been recently reported in many countries. Sri Lanka reports a large focus of human cutaneous leishmaniasis (CL) caused by Leishmania donovani, a usually visceralizing parasite. Enhanced case detection, early treatment, and in-depth understanding of sequalae are required to contain the spread of disease. Visceralizing potential of dermotropic strains has not been fully ruled out. Sri Lankan strains have shown a poor response to established serological assays. The present concern was to develop an in-house serological assay and to determine the seroprevalence of CL for identifying visceralizing potential and its usefulness in enhancing case detection. Crude cell lysate of dermotropic L. donovani promastigotes-based indirect enzyme-linked immunosorbent assay (ELISA) was previously optimized. Assay was evaluated using sera from 200 CL patients, 50 endemic and 50 nonendemic healthy controls, 50 patients with other skin diseases, and 50 patients with other systemic diseases. Seroprevalence and clinicoepidemiological associations were analyzed. Assay was compared with light microscopy (LM) and in vitro culturing (IVC). Cost comparison was carried out. Seroprevalence of CL was 82.0%. The assay had 99.5% specificity, and all healthy controls were negative at 0.189 cut-off. Positive and negative predictive values were 99.4% and 84.7%, respectively. Positivity obtained in ELISA was comparable to LM and higher than that of IVC. Cost per patient was 3.0 USD for both ELISA and LM and 6.0 USD for IVC. Infections occurring in all age groups and both genders demonstrated >75.0% of seropositivity. Patients had lesions with different durations/types/sizes showed >70.0% of seropositivity. Study identified a high seroprevalence of L. donovani-induced CL for the first time, indicating potential for visceralization or transient serological response. This can be used as a second line test in LM-negative CL cases to enhance clinical case detection. Further studies are warranted to examine in-depth correlations, antigen profiles, comparison with other established serological tools, and usefulness in the detection of asymptomatic cases. (National patent LK/P/1/19697).

Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis.

Human leishmaniasis which is considered a neglected tropical parasitic disease presents in three main clinical forms (i.e., cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL)) that are mainly determined by its causative species. Leishmania donovani, the most virulent and visceralizing parasite, is increasingly reported to cause CL in many countries in the world. Although CL is generally not considered to evoke a humoral immune response except for a nonrobust and a variable response in minority of cases, VL is associated with a clear strong humoral response. However, humoral response in L. donovani-induced CL has not been well evaluated before. A suitable serology-based assay is an essential primary step in such a study. An indirect enzyme-linked immunosorbent assay (ELISA) based on Leishmania promastigote crude antigen (Ag) was designed and optimized in order to utilize in further serological studies on this new clinical entity. Optimization included quantification of crude Ag, checkerboard titration method for determination of optimal concentrations for coating Ag, human sera and secondary antibody (Ab) with suitable coating buffer, blocking buffer, and incubating temperatures. The selected coating buffer was 0.02 M phosphate buffer, pH 6.8, and the blocking buffer was 2% fetal bovine serum with 0.01 M phosphate-buffered saline. At least 1 µg of crude Ag was required for coating the ELISA plate, while 1 : 1000 serum was used as primary Ab. The optimized concentration of secondary Ab was 1 : 64000 which might be altered according to manufacturer recommendations. The assay specificity was pre-evaluated using sera (n = 20 from each category) from confirmed CL patients and controls (other skin diseases which mimic CL, other systemic diseases that mimic VL, nonendemic healthy controls, and endemic healthy controls). This procedure described an optimization procedure of an ELISA technique for detection of anti-Leishmania antibodies in patients with L. donovani caused CL.

Quantification of Soluble or Insoluble Fractions of Leishmania Parasite Proteins in Microvolume Applications: A Simplification to Standard Lowry Assay.

Protein quantification is often an essential step in any research field that involves proteins. Although the standard Lowry assay and its modifications are most abundantly used in protein quantification, the existing methods are rigid or often demonstrate nonlinearity between protein concentration and color intensity. A method for fast and accurate qualitative and/or quantitative determination of total soluble/insoluble proteins or micro-well plate immobilized proteins isolated from Leishmania parasites in microvolumes was described in the current study. Improvements in cost-effective techniques are necessary to increase the research outputs in resource-limited settings. This method is a modification to the established Lowry assay for protein quantification. Concentrations of unknown samples were calculated using a standard curve prepared using a standard series of bovine serum albumin (BSA). The optimized reagents were 2 N NaOH (sodium hydroxide), 2% Na2CO3 (sodium carbonate), 1% CuSO4 (copper sulfate), 2% KNaC4H4O6 (potassium sodium tartrate), and 2 N Folin and Ciocalteu's phenol. This modified protein assay was sensitive for quantifying Leishmania proteins in a total crude extract or in a soluble fraction within the approximate range of 10-500 µg/ml (1-50 µg/assay) and showed a linearity between color intensity and concentration of the protein. This is an easier, fast, and accurate method for quantifying proteins with microvolumes in a cost-effective manner for routine use in research laboratories in resource-limited settings.

A highly sensitive modified nested PCR to enhance case detection in leishmaniasis.

BACKGROUND: Human leishmaniasis is one of the major parasitic diseases with worldwide distribution. Sri Lanka is a recently established focus of leishmaniasis caused by a variant Leishmania donovani. Early case detection and management is a main approach identified for L. donovani control in the regional leishmaniasis elimination drive. Usefulness of light microscopy and in-vitro culture are limited in chronic, atypical or treated lesions though timely and accurate detection of all light microscopy/in-vitro culture negative cases of all forms of leishmaniasis is necessary for treatment. Timely treatment is important to minimize risk for death in visceral disease and undesired sequelae of long standing infection and illness on both patients and community. We described a 100% sensitive, Leishmania spp. specific modified version of a nested PCR (Mo-STNPCR) that also minimizes carry over and cross contaminations while facilitate investigation of light microscopy and in-vitro culture negative clinically suggestive cases of leishmaniasis. METHODS: Leishmania DNA was amplified using previously published P221: 5'-GGTTCCTTTCCTGATTTACG-3' and P332: 5'-GGCCGGTAAAGGCCGAATAG-3'outer primers followed by a nested reaction using P223: 5'-TCCCATCGCAACCTCGGTT-3' and P333: 5'-AAGCGGGCGCGGTGCTG-3' inner primers that by passes the requirement of tube handling between the two steps of the conventional nested PCR. Leishmania DNA was detected in a range of infected tissue material. Infected material from patients with cutaneous leishmaniasis (n = 30), visceral leishmaniasis (n = 10) and from a control group including patients with non-leishmanial skin diseases (n = 10), other systemic diseases (n = 10) and healthy individuals (n = 10) were examined with Mo-STNPCR. Results were further compared with those of light microscopy and in-vitro culture. RESULTS: Mo-STNPCR method was 100% sensitive and 100% specific for diagnosis of leishmaniasis. Light microscopy and in-vitro culture were positive in 75.0% (n = 30/40) and 72.5% (n = 29/40) samples respectively where combined results of them gave 87.5% (n = 35/40) sensitivity. Mo-STNPCR did not cross react with control samples. Furthermore, Mo-STNPCR reduces the risk of cross-contaminations and carry over contaminations since the full reaction is carried out without opening the tubes. Per patient cost was calculated as 22 USD while the same was 3 and 6 USD for light microscopy and in-vitro culture respectively. CONCLUSION: Mo-STNPCR method is a useful tool in detecting leishmaniasis in minority of cases that go undetected by first line investigations.