Cutaneous leishmaniasis in Bikaner, India: Clinicoepidemiological profile; parasite identification using conventional, molecular methods and CL Detect™ rapid test, a new Food and Drug Administration-approved test.

BACKGROUND AND
OBJECTIVE: Cutaneous Leishmaniasis (CL) is a dermal manifestation caused by various species of Leishmania. This is the most common Leishmanial syndrome seen worldwide and is emerging as and threatens to become an uncontrollable disease. The present study was planned to understand the current epidemiology of CL in the conventionally endemic area of Bikaner, Rajasthan. Species characterization was also done.
MATERIAL AND METHODS: The diagnostic modalities used in the study were microscopy, histopathologic examination, antigen testing and confirmation and species characterization by PCR. Field evaluation of the new antigen detection kit approved by FDA in 2016 CL Detect™ IC-RDT was done and its results compared with other available diagnostic tests.
RESULTS: A total of 14 cases with 25 skin lesions presented to the Skin OPD during the six month study period (January to June 2018). Out of these, 5 were males and 9 were females, average age being 32 yrs. Students and housewives of poor socio economic group were the most frequently affected groups. Lesions were found on the exposed areas of the body, mainly on the upper limbs and facial region. Majority of them were ulcerated plaques. Out of 14 cases under study, antigen test confirmed 7 positives; thereby yielding a positive rate of 50% against 71.4% for microscopy, 72.7% for PCR and a mere 38.5% for histopathology. Species characterization revealed Leishmania tropica as the causative organism. INTERPRETATION AND
CONCLUSION: The study indicates that the number of cases have declined substantially over the past decade in Bikaner. Despite being the oldest technique used, microscopy still is a good option for diagnosis. Combined with PCR, diagnostic accuracy and hence utility is increased multifold. In its current form, IC-RDT may not be very useful. Copyright:

INTRODUCTION

Leishmaniasis is a parasitic disease affecting a substantial number of people in about 98 countries. Around 310 million people are known to live under risk of infection with this neglected tropical disease. According to the World Health Organization (WHO), the annual incidence is estimated at 1–1.5 million cases of cutaneous leishmaniasis (CL) and 500,000 cases of the visceral form.[1] CL is the most common Leishmanial syndrome worldwide and is reported to affect about 1 million people every year.[12] It is a dermal manifestation caused by various species such as Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmania amazonensis, Leishmania braziliensis, Leishmania mexicana, and some others. The clinical manifestations range from spontaneously healing lesions to chronic and mutilating cutaneous or mucocutaneous ulcers. It may also cause significant scarring and disfigurement, which is often observed in poor and rural communities.[23]

Although historically known since times immemorial, the disease is continually evolving. In India, disease was classically known to occur in specific pockets in Thar Desert of Rajasthan State.[4] It is now spreading to areas hitherto unknown to harbor it. Cases of CL have been detected in Bihar, which was conventionally known as a domain for only the visceral form of the disease.[5] Newer foci are being found reported from Himachal Pradesh, Kerala, etc.[6789] In Rajasthan also, reporting of cases was originally restricted to Bikaner,[1011] but lately, new cases are being detected and reported from Udaipur, Ajmer, and Jaipur too.[121314] The species characterization in Indian CL is also an important aspect, particularly in view of the recent identification of Leishmania donovani causing CL in some parts of India.[1112131415] Even in neighboring countries like Sri Lanka, endemic foci of cases with L. donovani as the causative agent have been documented.[16]

The CL disease has acquired more importance with HIV coinfection. The incidence and the spectrum of Leishmanial infections in HIV coinfected patients is being frequently reported throughout the world.[71718] Thus, CL is an important emerging opportunistic infection and needs to be studied more in its entirety.

The diagnosis of CL conventionally depends on demonstration of the parasite in appropriately stained smears or skin biopsy specimens on direct microscopic examination, histopathology, and culture.[1920] These tests are however limited by their low sensitivities. Serological response is considered unreliable due to low production of specific antibodies.[1920] Newer modalities like polymerase chain reaction (PCR) are definitely more sensitive and specific but can still not be used as a point of care (POC) test and is mainly restricted to research laboratories.[1921] A test needs to be evaluated and be made available which gives a rapid diagnosis, is sensitive and specific and also easily applicable in field conditions. A new point-of-care rapid diagnostic test (RDT), CL Detect™ (In Bios International, Inc.), has been Food and Drug Administration (FDA) approved, for use in ulcerative CL skin lesions in 2016.[2223] This RDT along with microscopy and PCR may help us in more effective diagnosis of the disease.

The present study was thus planned to understand the current epidemiology and species identification of CL in Bikaner. Efforts were also made to do a field evaluation of this new FDA-approved antigen detection kit and compare its results with other available diagnostic tests.

MATERIALS AND METHODS

This was a prospective observational hospital-based study undertaken in a tertiary care Bikaner Government Medical College and hospital, Bikaner. The study included all patients attending the Dermatology OPD with lesions clinically suspected to be CL from January to June 2018 and who were willing to participate. Written informed consent was obtained from all patients. Individual record for each patient was maintained on a pro forma which included demographic data (age, sex, occupation, and area of residence) and clinical details of the lesion (site, size, type of lesion, number of lesions, duration of the lesion, and inflammatory signs). The history of similar lesions in family or neighbors and the presence of sandflies, rearing of animals was also noted and documented.

Ethical committee approval

Ethical clearance for the study was obtained by Institutional Ethical committee (No F1/Acad/MC/JU/18/5152).

Sample collection

All lesions clinically diagnosed to be of CL were documented. After cleaning the affected area, a 3–4 mm long nick was made at the edge of lesion with a sterile scalpel. Smear was made from the tissue aspirate on a glass slide, fixed, and sent for Giemsa staining. Tissue biopsy was taken from margin of all suspected lesions from all except one patient as the lesion was very close to medial canthus of eye. Giemsa staining and histopathological (H/P) examination was done, and presence of amastigote if any was recorded.

CL Detect™ Rapid Test was done as per manufacturer's instructions. The sample was collected by twisting a sterile dental broach at the border of the lesion. The broach was then placed into a sample cup containing three drops of lysis buffer. Material was made to stay in lysis buffer for at least 5–10 min after which 20 μl of the sample collected in lysis buffer was pipetted onto the sample pad of the test strip. The strip was then placed into a cup containing two to three drops of chase buffer. Validation of kit was provided by an inbuilt control line. The presence of control line served as verification of sufficient sample volume, proper sample flow, and was also the control for the reagents. The presence of test line indicated a positive result.[24]

DNA extraction and polymerase chain reaction amplification with Kinetoplast DNA-specific primer (Uni21/Lmj4)

DNA was isolated from the biopsy samples taken from the clinically suspected patients. In brief, total DNA was isolated from tissue samples using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), as per manufacturer's instructions. The concentration of the extracted DNA was then measured at 260 nm using a spectrophotometer, and the purity was calculated using 260/280 nm ratio. All DNA samples were stored at −20°C until further use. For PCR amplification, primer pair Uni21 (5’-GGGGTTGGTGTAAAATAGGCC-3’) and Lmj4 (5’-CTAGTTTCCCGCCTCCGAG-3’) was used.[25] A conventional PCR was performed in 30 μl reaction volumes containing 10 pmol each of the gene-specific forward and reverse primers, 10 μM of each dNTP, 2 mM MgCl2, 100 ng of genomic DNA, and 5 units of PfuTaq DNA polymerase (Bangalore Genei, Bangalore, India). After a preheating step of 5 min at 94°C, 36 cycles of denaturation (1 min at 94°C), annealing (1 min at 54°C), and elongation (1.5 min at 72°C) followed with, finally, an extension step of 10 min at 72°C was undertaken for each sample. Twenty-five μL of the PCR products (800 bp specific to L. tropica) were loaded on 1.8% agarose gel prepared in 1 × TAE buffer, containing ethidium bromide. This was visualized using gel documentation system (Systems and Controls, India). Either 100 base pairs (bp) or 1 kilobase (kb) pair DNA ladder (Fermentas Inc., USA) was loaded along with to visualize the amplified products. DNA of L. tropica was used as positive control, and nuclease-free water was used as a negative control.[26] Eleven randomly selected samples were sent for PCR (for confirmation and speciation) to AIIMS, New Delhi.

Statistical analysis

The presence of clinically diagnosed lesions and thereafter good response to therapy were taken as prerequisite to diagnose a confirmed case and this constituted the gold standard. Positive rates of each test were calculated using percentages. Further, t-test was applied to compare the results of all diagnostic tests used. P < 0.05 was considered to be statistically significant at 5% level of significance.

RESULTS

A total of 17 clinically diagnosed patients with CL had reported in the OPD of Bikaner Government Medical College, during the 6 months’ study period. Three of them did not agree to get the tests done and were not included in the study. Thus, 14 cases with 25 skin lesions were included in the study. Of these, 5 were males and 9 were females (females outnumbered males). These cases were in the age range of 10–56 years, average age being 32 years. All the cases presented within 1–5 months of noticing/onset of lesions. Positive family history was seen in 3 of 14 cases. Most of the cases belonged to rural background. Most frequently affected groups were students and homemakers of poor socioeconomic group. Two-thirds of the patients gave history of keeping domestic animals in their residences, which included cows, buffaloes, and dogs [Table 1].

Table 1

The clinicoepidemiological characteristics of cutaneous leishmaniasis patients

Variable	Category	n (%)
Age grouping in years	0-20	6 (42.9)
	21-40	5 (35.7)
	>40	3 (21.4)
Sex	Male	5 (35.7)
	Female	9 (64.3)
Habitat	Urban	1 (7.1)
	Rural	13 (92.9)
Occupation	Farmer	3 (21.4)
	Labour	1 (7.1)
	Service man	0
	House wife	4 (28.6)
	Student	6 (42.9)
Site of lesion*	Face and neck	7 (28)
	Upper limb	18 (72)
	Lower limb	0
	Trunk	0
Number of lesions	1	7 (50)
	2-3	4 (28.6)
	>3	3 (21.4)
History of duration of 1st appearance of 1st lesion/s	<1 month	0
	1-6 months	20 (80)
	>6 months	5 (20)
Size of lesions (25 in 14 patients)	0.5-1 cm in diameter	2 (8)
	1-2 cm in diameter	11 (44)
	>2 cm in diameter	12 (48)
Type of lesions (25 lesions in 14 patients)	Papulonodular	4 (16)
	Noduloulcerative	9 (36)
	Crusted plaque/s	12 (48)

*It may be noted that in few patients, the lesions were at more than one site

While 7 (50%) cases presented with a single lesion, 4 (28%) had 2 lesions each and 3 (21%) cases had multiple lesions. Lesions were found on the exposed areas of the body, mainly on the upper limbs and facial region. The trunk and lower limb involvement was not seen in any case. The size of the lesions varied from a few mm to 8 cm in diameter. Majority of lesions were crusted plaques (48%), followed by nodulo-ulcerated lesions (36%); papulonodular lesions comprised the rest (16%). A detailed review of all cases is presented in Table 2.

Table 2

The detailed clinic, microscopic, histologic, antigenic, and molecular characterization of cutaneous lesions

Patient detail age/sex	Duration of lesion (in months)	Number of lesions	Site of lesion	Clinical description of lesion	Result of laboratory tests
					Giemsa staining	H/P	Rapid ICT	PCR
33/male	3	2	Right arm	Papulonodular lesions	+	+	+	+
13/female	3	1	Right arm	Nodulo-ulcerated lesion	+	+	+	+
25/female	7	1	Left elbow	Nodulo-ulcerated lesion	-	-	-	-
12/male	3	1	Tip of nose	Papulonodular lesion	+	+	+	+
10/female	6	2	Left arm and left hand	Nodulo-ulcerated lesion	-	-	-	+
40/female	12	2	Right middle finger and right forearm	Crusted plaque	+	+	+	+
50/female	12	3	Left forearm	Crusted plaque with surrounding erythema	+	-	+	+
15/male	4	2	Face	Nodular, crusted plaque	+	-	-	-
60/female	2	1	Forearm left	Nodulo-ulcerated lesion	+	-	-	ND
30/male	18	1	Forearm right	Crusting plaque	+	+	+	+
12/female	12	1	Face	Nodulo-ulcerated lesion with surrounding erythema	+	-	-	-
13/female	2	1	Near medial canthus eye	Crusted plaque with surrounding erythema	+	ND	+	ND
56/male	1½	3	Left hand	Nodulo-ulcerated lesion surrounding erythema	-	-	-	+
34/female	3	2	Right hand	Crusted plaque with surrounding erythema	-	-	-	ND

H/P: Histopathological examination, Rapid ICT: Rapid Immunochromatography test, PCR: Polymerase chain reaction, +: Positive, -: Negative; ND: Not done

Giemsa stained skin smears showed the amastigote form of the parasite in 10 of 14 cases (71%). (H/P) examination of the lesions showed granulomatous inflammation comprising of moderate to marked infiltration by lymphocytes, plasma cells, neutrophils, and parasitized macrophages in 5 of 14 cases (36%). Antigen detection by rapid card test was positive in 7/14 (50%) patients. PCR could be performed on 11 of 14 cases and was positive in 8, i.e., 73% [Figure 1]. Species characterization revealed L. tropica as the causative organism. There was no statistically significant difference found between the positive rates of all four tests (P > 0.05).

Figure 1

The comparative positivity rates of the different tests used to detect Leishmania parasite in the study

DISCUSSION

Leishmaniasis is one of the top five diseases targeted by the WHO special program for research and training in tropical diseases. The theme for World Health Day 2014 was also adopted as “SMALL BITE BIG THREAT” specifically to raise awareness regarding various vector-borne diseases. CL is a dermal manifestation commonly caused by various species of Leishmania by the sandfly vector bite [Figures 2–4]. This is the most common Leishmanial syndrome seen worldwide and is re-emerging in several new areas.[123]

Figure 2

Nodulo ulcerative lesion of cutaneous leishmaniasis

Figure 4

Erythematous ulcerative plaque of cutaneous leishmaniasis

Crusted plaque type lesion of cutaneous leishmaniasis

Bikaner, a district in the northwestern part of Rajasthan, India, lies almost in the middle of Thar Desert and has a hot semi-arid climate with very little rainfall. Two-thirds of the population still resides in rural areas. Historically, most of the cases of CL in India were confined to this belt.[4] In the 1960s, with the success of malaria eradication program, there was a concurrent decline of both malaria and CL in this district. However, when spraying of DDT ceased, there was a sharp rise in number of cases of both the diseases in early 1970s. The number of cases increased from 28 in 1967 to 828 in 1971. The first evidence for existence of CL was based on clinicoepidemiologic analysis of cases in 1973 by Sharma et al. during a large-scale outbreak of the disease here.[27] During this time, >2000 people suffered from this infection. During 1981, 1% of random population of Bikaner was surveyed to determine local prevalence of CL. Among the 2860 people surveyed, 86 had chronic ulcers. However, only 18 among these were proven cases of oriental sore.[4] Ninety-eight patients were reported by Kumar et al. between 2004 and 2006, and species characterization established L. tropica as the causative organism.[11] A 10-year review of cases was done by Aara et al. and included all persons with clinically diagnosed CL during 2001–2011,[10] and a total of 1379 cases were reported. They also observed that the incidence of CL increased every year up to 2009, after which there was a decline in 2010–2011. Highest number of cases were reported in 2009.

Agrawal et al. have described pediatric CL in preschool children in this area during 2001–2012. A total of 151 patients were reported; smear positivity was seen in 70% of these cases. Parasite species identification conducted for 13 randomly selected patients by PCR identified L. tropica as the causative organism.[28] A careful review of literature has thus revealed that while the disease initially showed an upsurge, after 2009, the disease burden has consistently shown a decline.[10] In the present study, the current trends of disease have been studied, along with the available diagnostic tests.

In keeping with the earlier reported trends, the disease prevalence has shown a decline. During the 6-month study period, 14 cases presented with a total of 25 lesions. Although the incidence of disease has come down, it still needs to be understood and kept as a differential diagnosis as CL produces symptoms which are quite similar to those seen in other skin conditions such as sarcoidosis and lupus vulgaris.

The considerable decline can be explained by the change in the habitat of the vector and also regular insecticide sprays done as per government's policy. An overall improvement in sanitation and housekeeping practices probably prevents sandflies from lodging in the house. Even in rural areas, people are building and living in pukka houses, and the practice of coating the walls with mud and debris has become redundant. The cracks and crevices in houses which used to serve as potential breeding grounds are now replaced by cemented walls. Because of growing urbanization, the practice of sleeping outdoors and maintaining cattle is not very popular now. Like in earlier studies available from this area, most frequently involved groups are those of poor socioeconomic status and from rural backgrounds.[101129]

While one study has reported more number of males to be involved,[411] another reports no particular sex predilection.[10] In the current study, the prevalence was more in women. Students and homemakers of the socioeconomically poor and rural background were the most commonly affected group. Most lesions were ulcerated plaques and were present on the exposed parts of the body. This profile is similar to that reported in earlier studies.[1011] CL produces symptoms which are quite similar to those seen in other diseases such as leprosy, sarcoidosis, cutaneous tuberculosis, and lupus vulgaris and needs to be considered as a differential diagnosis.[2023]

The diagnostic modalities used in the study were microscopy [Figure 5], H/P examination, antigen testing, and confirmation with species characterization by PCR. The classic methods of demonstration of parasite by microscopy are reported to have low sensitivity and specificity. Furthermore, there is no species identification possible.[1920] With the advent of molecular techniques like PCR, issues such as low sensitivity and species identification have been answered to a great extent. However, these techniques are usually used for research purposes and are confined to specialized laboratories in low- and middle-income countries, where cases are still being diagnosed and reported.[2123] A novel rapid diagnostic immunochromatography strip test (CL Detect™ IC-RDT) has been developed and also FDA approved in 2016. This test is based on capturing the peroxidoxin antigen of Leishmania amastigotes. When Leishmania amastigote antigens are present, the mixture reacts with the affinity-purified polyclonal antibody to generate a red line at the test line region of the membrane [Figures 6 and 7]. The test has exhibited promising performance when tested in Tunisia with reports of predominant L. major infection. The reported sensitivity and specificity of the strip test is 100% and 96%, respectively.[2324] In addition, it is easy to perform, provides rapid results, and has a low cost when compared to PCR. All these properties make it a very promising potential POC test.[2430]

Figure 5

Amastigotes on Giemsa staining

Figure 6

Immunochromatography - rapid diagnostic test showing control line

Figure 7

Immunochromatography- rapid diagnostic test showing positive test

Of 14 clinically classified cases in the study, antigen strip test rapid immunochromatography test (ICT) confirmed 7 cases; thereby yielding a positive rate of 50% against 71% for microscopy, 73% for PCR, and a mere 38% for histopathology. It was also noteworthy that all positive cases identified by this test were also positive with either microscopy or PCR. There was no improvement in the number of cases diagnosed when microscopy was used alone or in conjunction with rapid ICT [Figures 1–7].

Interestingly, PCR was able to detect the presence of parasite in 2 cases which were negative by all other modalities. Both these patients responded well to therapy. However, in 2 patients, PCR was negative despite amastigote being seen on microscopy. These two patients also responded well. The lesions decreased in size and severity on follow-up examinations. The negative results in PCR can be explained by low parasite burden in the lesion as well as inaccurate and insufficient sampling from the lesion. In yet another patient, all the four diagnostic modalities used were negative. Based on the clinical diagnosis, which included the presence of similar lesions in another family member, and history of the presence of sand flies in neighborhood, treatment was initiated. This patient also responded well to treatment. A careful review of the results indicates that maximum number of cases (91%) can be diagnosed when both microscopy and PCR are combined together. Keeping these points in mind, a new diagnostic algorithm can be formulated for early and timely detection and treatment of cases.

The immunochromatography-RDT (IC-RDT) in its current form may not be very useful in the present setting if used as the only test for diagnosis. Poor performance of the test may be explained by either low parasite counts or low expression of peroxidoxin antigen in Leishmania amastigotes in Indian CL. Another drawback is that the kit can be used only in recent cases.[24] Sensitivity may be further compromised in chronic lesions when the parasite density is low. The kit has not shown encouraging results in a similar study plan conducted in Sri Lanka.[16] The authors from this study too have suggested that Giemsa-stained slit skin smear needs to be combined with PCR for better laboratory confirmation test results.[16] Similar test format with an improved sensitivity may be a valuable addition in our diagnostic armamarium, especially for POC testing.

Considering the high operational costs of PCR and relative ease involved in doing the Giemsa staining of the smear taken from suspected lesion, it may be reasonable to assume that microscopy is still the best diagnostic option available. Low cost of reagents, easy availability of microscope, requirement of minimal infrastructure, and a reasonably good sensitivity in hands of a trained microbiologist are other points which make microscopy an attractive option. The merits of microscopy have also been highlighted by several researchers.[6233029] In doubtful cases, microscopy and PCR can be combined together because when used concurrently, they were able to pick up 91% of cases [Figure 1].

Species characterization has revealed L. tropica as the causative agent which is in concordance with results reported in the earlier studies.[1127] This is especially noteworthy in light of newer foci of CL caused by L. donovani in Himachal Pradesh and Kerala.[715] The tribal population of Agasthyamala biosphere reserve forest, Western Ghats, Kerala has been described as constituting a “local endemism” by the investigators.[15] CL caused by L. donovani is also a problem and concern in a neighboring country, Sri Lanka.[1631] The genetic similarity between organisms isolated from mountainous ranges of Western Ghats and Sri Lanka is thought to be because of their constituting a “single biogeographic unit.”[15]

Limitations of study include small number of cases studied and also that PCR could not be done in all the patients. Vector studies too could not be undertaken. More widespread community-based studies are needed in future to enhance our understanding. Furthermore, medical officers working in primary healthcare centers need to be reoriented and trained to include CL in the differential diagnosis; laboratory tests were required to diagnose such cases.

CONCLUSIONS

A careful review of the results indicates that the number of cases have declined substantially over the past decade. Yet, it is important to study the disease because it forms an important differential diagnosis in various skin conditions and can lead to scarring and stigmatization. It is important to use molecular techniques for diagnosis, especially for species identification and to understand its evolving epidemiology. Despite being the oldest technique used, microscopy still is a good option for diagnosis. Combined with PCR, diagnostic accuracy and hence utility is increased multifold. In its current form, IC-RDT may not be very useful. More research is needed to increase the sensitivity of the rapid antigen detection kit.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.