Posing a threat to the ongoing leishmaniasis elimination efforts in the Indian subcontinent,
Leishmaniasis, one of the neglected vector-borne parasitic diseases is caused by different species of the genus
Sri Lanka is a recent focus of human leishmaniasis in South Asia. This country reports a world’s large epidemic of CL caused by
Meanwhile,
Serological assays are increasingly used in detecting asymptomatic and clinical infections with leishmaniasis [
The current study evaluated an in-house ELISA, examined the serological response in
Sera from five major groups of patients were used for this study.
The absence of leishmaniasis in control samples was confirmed based on absent clinical picture and/or confirmed alternative diagnosis and response to appropriate treatment in NCL, EHC, and NEHC or based on negative clinical picture and/or negative LM/IVC/PCR of skin or bone marrow samples in CL, NCL, and NVL.
Venous blood (3 cc) was collected by trained medical or paramedical personnel after obtaining an informed written consent from each patient and control persons. Blood samples were incubated at room temperature for 30 minutes to 1 hour to allow the clotting of blood. Serum was separated by centrifugation at 2500 rpm for 10 minutes and aliquoted and stored at -20°C for later use.
A locally acquired, confirmed positive CL sample was selected from the sample cohort. If positive patients were with a history (within two years prior to diagnosis) of overseas travel, they were excluded from the study. M199 supplemented with 10% HI-FBS and 1% PenStrep (M199-complete media) was used for
Total crude extracts were prepared from harvested promastigotes of
ELISA was carried out using a previously optimized protocol [
Each sample was analyzed in duplicates, and the mean value of absorbance was considered as the final value. Only absorbance values closer to the second decimal point in duplicates were considered in calculating mean. Each ELISA plate was run with an air blank, five or more healthy controls and controls with and without conjugate. Normalization of day-to-day variations of the assay and test reproducibility was assessed according to an acceptance and rejection criteria defined using mean absorbance values of healthy controls (
Validation of ELISA was carried out according to approved guidelines described on fundamental validation parameters for immunoassays which were presented in U.S. Pharmacopeia Chapter 1225, Validation on Compendial Methods, 2009 and ICH Q2 (R1) on Validation of Analytical Procedures: Text and Methodology, 2005 [
The cut-off value for the assay was determined using a receiver operating characteristic curve (ROC curve). The area under curve (AUC) was determined using the ROC curve. AUC values closer to one are considered as tests with high diagnostic accuracy which reliably distinguishes positive and negative samples. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of assay were calculated using the ROC curve and
The reference standard used for quality controlling of assay was obtained from a patient with Indian VL. The serum was positive for the rK39 strip test. Linearity of the assay was determined using a standard curve constructed with six analyses of five different concentrations (concentration spanned from about 80%-120% of expected concentration range, i.e., 1 : 16000, 1 : 32000, 1 : 64000, 1 : 128000, and 1 : 512000) of reference standard
More than six determinations of three different matrices at three different concentrations were performed, and relative standard deviation was calculated to determine reproducibility/repeatability. Accordingly, ELISA values obtained for healthy controls within 20 different days were analyzed. Also, relative standard deviation (SD) and coefficient of variation (CV, <10 of CV was considered as highly accurate with high reproducibility/repeatability) of ELISA values for high and intermediate positive sera within 10 days were calculated.
The accuracy of the test was also determined in relation to gold standard, i.e., LM using standard calculation methods (Accuracy = true positives and true negatives/total number of samples). The mean reactivity of CL sera was further compared with other control groups, and statistical significance was calculated using SPSS version 25.0.
The smallest concentration of a measurand that can be reliably measured by test was determined using standard ELISA parameters, i.e., LOB, LOD calculated using standard equations,
According to manufacturer recommendations of ELISA reader used, the accurate range was typical value ±1% (0–2.0 Abs) at 405 nm.
Samples were stored in aliquots for avoiding repeating freeze-thawing cycles. Also, the same room conditions, temperature, and light/dark conditions were used for each run to increase the accuracy of the test. Samples, stock solutions, and other reagents were stable and used for more than two years without any deviation of ELISA readings by aliquoting and storing under recommended conditions.
To further validate ELISA, assay results were compared with classical parasitological diagnostic methods used for CL, i.e., LM and IVC. Also, cost analysis per patient was carried out according to approved guidelines [
Serological response was further compared with the epidemiological data of patients (i.e., age and sex) and clinical features of lesions (i.e., lesion duration, number, type, size, and site) using SPSS version 25.0.
Samples were collected upon written informed consent given by patients and healthy controls. Ethics approval for the study was obtained from the Ethics Review Committee, Faculty of Medicine, University of Colombo.
ROC curve for ELISA. ELISA absorbance that maximized the total of sensitivity and specificity was selected as the best cut-off value.
According to results obtained for ROC analysis, the best cut-off value was 0.189 of absorbance. At this cut-off, sensitivity is 82.0% and specificity is 99.5% (Table
Sensitivity and (1-specificity) for ELISA at different absorbance values. Italics show the best cut-off value.
Positive if greater than or equal to (ELISA absorbance values) | Sensitivity | 1-specificity | Specificity | Sensitivity +specificity |
---|---|---|---|---|
0.181 | 0.830 | 0.065 | 0.935 | 1.765 |
0.182 | 0.830 | 0.050 | 0.950 | 1.780 |
0.183 | 0.830 | 0.045 | 0.955 | 1.785 |
0.184 | 0.830 | 0.040 | 0.960 | 1.790 |
0.185 | 0.820 | 0.025 | 0.975 | 1.795 |
0.187 | 0.820 | 0.015 | 0.985 | 1.805 |
0.191 | 0.815 | 0.005 | 0.995 | 1.810 |
0.192 | 0.805 | 0.005 | 0.995 | 1.800 |
0.196 | 0.800 | 0.005 | 0.995 | 1.795 |
0.199 | 0.795 | 0.005 | 0.995 | 1.790 |
0.200 | 0.790 | 0.005 | 0.995 | 1.785 |
0.201 | 0.790 | 0.000 | 1.000 | 1.790 |
0.202 | 0.785 | 0.000 | 1.000 | 1.785 |
At 0.189 cut-off value obtained from the ROC curve, of 200 CL samples studied, in-house ELISA identified 164 seropositive cases (
Optical density distribution of ELISA. Variation of ELISA values of CL and control groups including NEHC, EHC, NCL, and NVL is shown at 0.189 cut-off level.
Diagnostic
Disease confirmatory status | |||||
---|---|---|---|---|---|
Positive for CL | Negative for CL | Total count | |||
In-house ELISA | Seropositive | 164 | 1 | 165 | |
Seronegative | 36 | 199 | 235 | ||
Total count | 200 | 200 | 400 | ||
According to the standard curve shown in Figure
Standard curve for a dilution series of reference standard. Five known concentrations of reference standard were used.
Variation of mean value of ELISA absorbance at 450 nm obtained for healthy controls.
CV of ELISA values for high and intermediate positive sera within 10 days period was about 2.6% and 3.5%, respectively (Table
Repeatability assay using high and low positive serum samples for 10 days.
Replicates | ELISA value for high positive serum | ELISA value for low positive serum |
---|---|---|
1 | 0.449 | 0.222 |
2 | 0.430 | 0.226 |
3 | 0.447 | 0.219 |
4 | 0.420 | 0.219 |
5 | 0.419 | 0.223 |
6 | 0.427 | 0.2055 |
7 | 0.441 | 0.207 |
8 | 0.419 | 0.22 |
9 | 0.418 | 0.2175 |
10 | 0.428 | 0.232 |
M | 0.430 | 0.219 |
SD | 0.011 | 0.008 |
CV | 2.6 | 3.5 |
The accuracy of the assay was determined as 90.8%.
Mean reactivity for sera from CL patients was statistically different to those from healthy individuals (NEHC and EHC) and patients with other diseases (NCL and NVL) (
Comparison of mean ELISA values obtained for sera of CL patients, healthy individuals (NEHC and EHC), patients with other skin diseases (NCL), and patients with other systemic diseases (NVL).
Serum samples | ELISA absorbance, | Confidence interval of 95% | ||
---|---|---|---|---|
Lower limit | Upper limit | |||
CL | 0.286 | 0.324 | ||
NEHC | 0.118 | 0.140 | ≤0.001 | |
EHC | 0.087 | 0.111 | ≤0.001 | |
NCL | 0.097 | 0.125 | ≤0.001 | |
NVL | 0.051 | 0.075 | ≤0.001 |
There were
Comparison of results obtained for ELISA with LM
LM positive | LM negative | Total count | ||
---|---|---|---|---|
ELISA | Seropositive | 86 | 12 | 98 |
Seronegative | 16 | 4 | 20 | |
Total count | 102 | 16 | 118 |
Comparison of results obtained for ELISA with IVC
Culture positive | Culture negative | Total count | ||
---|---|---|---|---|
ELISA | Seropositive | 58 | 40 | 98 |
Seronegative | 12 | 8 | 20 | |
Total count | 70 | 48 | 118 |
Patients presented during the period from 2002 to 2017. More than 75.0% of CL infections in each gender demonstrated seropositivity. The majority of infections occurring in all age groups also demonstrated a seropositivity of >75%. Lesions of short (<3 months), medium (4 to 6 months), and long durations (>6 months) were also associated with high seropositivity (72-92.0%), with lesions of very short duration (<3 months) also showing >70% seropositivity (Table
Clinicoepidemiological correlations with seropositivity in the study populations.
Seroprevalence measured by ELISA | ||||
---|---|---|---|---|
Clinicoepidemiological data | Seropositive count ( | Seronegative count ( | Total count ( | |
Age (years) | ||||
≤25 | 38/49 (77.6) | 11/49 (22.4) | 49 | 0.497 |
26 to 50 | 96/118 (81.4) | 22/118 (18.6) | 118 | |
>50 | 29/33 (87.9) | 4/33 (12.1) | 33 | |
Sex | ||||
Male | 133/160 (83.1) | 27/160 (16.9) | 160 | 0.257 |
Female | 30/40 (75.0) | 10/40 (25.0) | 40 | |
Lesion size | ||||
Up to 1 cm | 34/49 (69.4) | 15/49 (30.6) | 49 | 0.044 |
2-3 cm | 89/103 (86.4) | 14/103 (13.6) | 103 | |
>3 cm | 34/42 (81.0) | 8/42 (19.0) | 42 | |
Number of lesions | ||||
One lesion | 133/161 (82.6) | 28/161 (17.4) | 161 | 0.490 |
>one lesion | 30/39 (76.9) | 9/39 (23.1) | 39 | |
Duration of lesions | ||||
Up to 3 months | 46/64 (71.9) | 18/64 (28.1) | 64 | 0.017 |
4 to 6 months | 56/61 (91.8) | 5/61 (8.2) | 61 | |
>6 months | 44/55 (80.0) | 11/55 (20.0) | 55 | |
Type of lesions | ||||
Ulcerative# | 95/108 (88.0) | 13/108 (12.0) | 108 | 0.008 |
Nonulcerative# | 65/89 (73.0) | 24/89 (27.0) | 89 | |
Site of lesion | ||||
Head and neck area | 36/54 (66.7) | 18/54 (33.3) | 54 | 0.007 |
Arms | 81/94 (86.2) | 13/94 (13.8) | 94 | |
Other | 41/47 (87.2) | 6/47 (12.8) | 47 |
Both genders and a wide age range in CL cases responded satisfactorily with a high serological response. At the selected cut-off level, there were no age or gender-dependent significant reduction in seroprevalence (
This is the first time study reporting a high seroprevalence (82.0% at 99.5% specificity) in CL caused
This high level of seroprevalence could be due to a still unconfirmed potential for visceralization or a transient serological response associated with localized CL infections.
Some studies have rejected these possibilities of CL having lower seroprevalence in CL. As instances, in Svobodova et al., CL patients caused by
Analysis of clinicoepidemiological characteristics of patients further highlighted some associations of lesion site, type, duration, and gender of patients with seropositivity (Table
Low seropositivity in patients having lesions in the head and neck area may be due to a higher amount of lymph nodes of human body area are located in the head and neck area, leading to higher cell-mediated immunity compared to humoral immunity [
Diagnosis of CL and other clinical forms based on clinical presentations is challenging in tropical settings due to the presence of many other conditions with similar clinical profiles. With a high level of specificity, assay can be useful in differentiating leishmanial infections from other nonleishmanial conditions. PPV and NPV of ELISA were 99.4% and 84.7%, respectively. High PPV allows the assay to remain useful even when the prevalence of leishmaniasis is low or decreasing. A good NPV allows an accurate diagnosis at a high specificity.
In addition, this ELISA assay seems to be suitable in detecting CL infections in all studied age and gender categories with
ELISA assay was able to detect 75.0% of LM negative cases. Also, in ELISA negative cases, only 80.0% was detected by LM or 60.0% by IVC. Parasitological investigations can become negative in chronic, treated, atypical, or partially treated infections. However, in order to assess treatment response and cure, it is important to establish a laboratory-based diagnosis in all possible cases. Positivity obtained in ELISA was comparable to LM but higher than IVC. Since IVC needs invasive sample collection procedures, experts to handle, and it is highly possible with contaminations, ELISA can be used as an additional diagnostic method for local CL. Presumably, ELISA will replace IVC with the added advantage of low cost and noninvasiveness. Also, ELISA will be useful where infection cannot be detected by eliciting the presence of parasites or parts of them. Therefore, in-house ELISA could be used as a useful second line option in the detection of all LM-negative cases before expensive and complex IVC or molecular biological procedures are performed. Furthermore, assay cost is also comparable to that of LM, which is the first line investigation used in routine case detection in leishmaniasis. In addition, ELISA could further be performed without having to sample an infection site.
Asymptomatic infections comprise an important component that contributes to the silent onward propagation of disease in leishmanial endemic settings. Increasing efforts are made to study this clinical entity in affected countries, and most studies employ serological tools due to lack of obvious bodily sites of infection that hinder the researcher from collecting infected tissue for parasitological assessment. The usefulness of newly developed assay could be explored in the detection of preclinical infections in Sri Lanka and similar settings. Furthermore, this assay could be useful in the detection of other recently emerged visceral and mucosal leishmaniasis in Sri Lanka. Further understanding on immune dominant antigen profile, further associations with clinical and parasitological variations, and usefulness as an early detection or outcome prediction marker will be useful.
With several evidences associated with CL and seropositivity in the world as mentioned above, it is still unknown whether the seropositivity observed in local CL due to visceralization potential of the local parasite or immunogenic nature of the parasite. The developed ELISA will be useful as a second line investigation for increasing the successive case detection rate of local CL in the near future.
Data supporting the conclusions of this article are included within the article. Other data has not been made available as it was not part of the ethics application and due to patient confidentiality.
Content is solely the responsibility of the authors and does not necessarily represent the official views of the mentioned funding sources.
The authors declare that there are no conflicts of interest.
YS conceived and desgined the project, financed and guided BD, contributed to writing, and critically reviewed the final manuscript. YS, BD, MC and PS together designed the methods. BD carried out the laboratory work, data analysis, and wrote the first draft of paper. SW & SR contributed to sample preparation for assays., TPA and MNP contributed to further sample processing. NK provided administrative and logistical support. MC, PS, and VC provided laboratory facilities and technical guidance. SS revised the manuscript critically for important intellectual content. All authors agreed on the final manuscript.
We acknowledge Nilusha Priyanthi, Yasasmi R Gange, Sashika Dayananda, Thisari S Rathnayake, and Nirosha Pathirana for the project assistance. The research reported here was supported by the University of Colombo research grant (AP/3/2/2014/RG/13).