Utility of smear examination, culture, and serological tests in the diagnosis of visceral leishmaniasis/post-kala-azar dermal leishmaniasis at National Centre for Disease Control, Delhi.

Introduction: A range of assays have been developed to detect specific antileishmanial antibody, such as rK 39 immunochromatographic test (ICT), KE 16 ICT, ELISA test, and indirect immunofluorescent antibody test (IFAT), which play a crucial role in serological diagnosis of visceral leishmaniasis (VL). However, limited published reports are available on the utility of serological test (IFAT test/rk 39), smear examination, and culture in the diagnosis of VL and post-kala-azar dermal leishmaniasis (PKDL) in our country. Materials and
Methods: We present utility of serological test (IFAT test/rK 39), smear examination for Leishmania donovani (LD) bodies, and culture in 2589 samples from 2294 VL/PKDL suspected patients (January 2009-December 2019) tested in Centre for Arboviral and Zoonotic diseases, National Centre for Disease Control, New Delhi, India, for laboratory diagnosis of VL/PKDL.
Results: A total of 80/553 (14.4%) cases were confirmed of VL (74/522 cases by demonstration of LD bodies in bone marrow smear examination, 5/12 in splenic smear examination 1/19 by culture) and 4/21 (19.0%) cases were confirmed of PKDL (demonstration of LD bodies in slit skin smear examination. In our study 197/1368 (14.4%) cases were diagnosed positive by IFAT, 34/646 (5.2%) cases by rk 39 ICT for VL/PKDL by demonstration of specific antileishmanial antibodies.
Conclusion: As the goal of elimination of VL as a public health problem is approaching, apart from serological tests such as rk 39 and IFAT, direct methods of detection such as (parasitic demonstration in BM smear, culture, and molecular tests) for Leishmania may play a crucial role for achieving a correct diagnosis and treatment. We also concluded that IFAT though not field-friendly, its optimal use as an adjunct test with BM smear in all stages of infections may be required. Further rk39 is a simple, reliable, noninvasive, and field-friendly test for diagnosis VL, especially in endemic areas. Copyright:

INTRODUCTION

Visceral leishmaniasis (VL) is a chronic disease caused by protozoan parasite Leishmania donovani (LD). In India, L. donovani is the only parasite that causes this disease commonly called as kala-azar (KA) which is transmitted by bite of female phlebotomine sandflies. VL patients have clinical presentations such as fever, hepatosplenomegaly, weight loss, and loss of appetite. It is usually fatal if not treated with specific chemotherapy. In India, presently, disease is endemic in Bihar, Jharkhand, West Bengal, and Uttar Pradesh with sporadic incidence reported from Himachal Pradesh, Jammu and Kashmir, Gujarat, Tamil Nadu, Madhya Pradesh, and Kerala in past.[1]

Post-kala-azar dermal leishmaniasis (PKDL), a cutaneous sequela of VL, develops in some patients and they are an important but neglected reservoir of anthroponotic leishmaniasis. The early detection and treatment of PKDL cases is important in control of VL elimination.

A range of assays have been developed to detect specific antileishmanial antibody such as rK 39 immunochromatographic test (ICT), KE 16 ICT, ELISA test, and indirect Immunofluorescent antibody test (IFAT).[23] Serological tests using rk39 in rapid ICT format have been widely used for the diagnosis of VL. However, there are limited published reports available on the role of parasitological, immunological, and molecular methods in diagnosis and management of leishmaniasis in our country.[23]

The utility of serological test (IFAT test/rk 39), smear examination, and culture in 2589 samples sent from 2294 VL/PKDL suspected patients from January 2009–December 2019 for laboratory diagnosis of VL/PKDL done at Centre for Arboviral and Zoonotic diseases, National Centre for Disease Control, New Delhi, India is presented in this study.

MATERIALS AND METHODS

Serological test (IFAT test/rk 39), smear examination, and culture in 2589 samples (522 bone marrow smears, 12 splenic smears, 21 slit skin smears for demonstration of L. donovani bodies, 19 bone marrow aspirates for culture, 1369 serum samples for IFAT, and 646 samples for rK 39 antigen test) from 2294 (2273 VL + 21 PKDL) VL/PKDL-suspected patients (January 2009–December 2019) were done in Centre for Arboviral and Zoonotic diseases, National Centre for Disease Control, New Delhi, India, for laboratory diagnosis of VL/PKDL.

The clinical case definition used for VL/PKDL-suspected cases as per the National Vector Borne Disease Control Programme guidelines was as follows.[34]

Visceral leishmaniasis

Persons with fever of more than 3 weeks’ duration and hepato/splenomegaly, not responding to a full course of anti-malarials and antibiotics.

Post-kala-azar dermal leishmaniasis

A patient from a KA-endemic area with multiple hypopigmented macules, papules, plaques, or nodules.

Culture

1–2 drops of bone marrow/splenic aspirate was inoculated under all aseptic precautions in modified Novy-MacNeal-Nicolle medium and incubated at 22–25°C as per the standard protocol.[5] A drop from culture was examined for promastigotes by making a wet smear and its examination under microscope every 4th day for 28 days before declaring it negative.

Direct microscopic examination of the bone marrow/splenic/slit skin smear smears

Smears slides were fixed using methanol and air-dried. Giemsa staining was done on fixed dried smears. Stained smears were examined under oil immersion for amastigotes form of the parasite (L.D bodies). The slides were examined in duplicate by two different observers and at least 1000 fields were examined to label them as “Negative” as per the WHO criteria (×10 eyepiece and ×100 oil immersion lens).[5]

Serological test

In-house immunofluorescent antibody test.

It is a indirect IFAT which detects the specific Ig G antibodies which was standardized in-house.

Preparation of slides coated with Leishmania donovani promastigotes

Multispots Teflon-coated slides were degreased by immersing them in acetone for 10 min, taken out and dried. Slides were coated with crude Leishmania promastigotes prepared from 4 to 5 fresh L. donovani strain culture bottles of Tobie's media from our laboratory (MHOM/IN/80/DD8)[6] and fixed with chilled acetone. The Leishmania culture from selected bottles was pooled and centrifuged at 800–1000 rpm at 4° C for 10 min and washed with phosphate-buffered saline (PBS), the same was repeated three times. The sediments were suspended in PBS followed by the addition of 2 drops of formalin and kept for 1 min, then recentrifuged for 10 min. The supernatant was discarded and sediments were examined to adjust 5–10 parasite per field (6 × 106 promastigotes/ml). After that, sediments were coated as spots on slides and incubated in BOD incubator (temp. 24°C to 27°C) for 5 days. The slides were allowed to dry and then stored at −20°C till the use. Antihuman IgG (γ-chain specific) fluorescein isothiocyanate conjugate (Sigma F6380, Saint Louis Missouri, USA) was used in working dilution of 1:100 which was being standardized using positive and negative controls. The test was performed according to the method of Shaw and Voller.[7]

Test procedure

Twofold serial dilutions of 10 μl of patients’ serum from 1:25 to 1: 1600 were transferred to each coated spot on the slides. Positive and negative controls were included in the test. After adding a patient serum to coated spots, incubation was done at room temperature for 60 min in a moist chamber. Slides were washed thrice with PBS for 3 min each time followed by the addition of 30 μl of fluorescent isothiocyanate conjugate IgG antihuman globulin (1:100 dilution) and kept for 1 h. Finally, the slides were washed with distilled water. A fluorescence microscope was used to read the mounted slides by visualizing them through blue–green filter.

Interpretation

The specific titer of serum sample giving 2+ fluorescence was noted. IFAT 50 positive represents sample with 2+ flouroscence in 1:50 dilution. IFAT 100 positive represents sample with 2+ fluorescence in 1:100 dilution. IFAT 200 positive represents sample with 2+ fluorescence in 1:200 dilution.

rK 39 Rapid test

The Kalazar Detect™ Test (Inbios Seattle, WA, USA) for VL is a rapid immunochromatographic strip (precoated with rK39 recombinant antigen obtained from Leishmania chagasi) assay for the qualitative detection of antibodies to members of L. donovani in human serum to aid in the presumptive diagnosis of VL. The test was performed and interpreted as per the manufacturer's kit instructions.

Evaluation of immunofluorescent antibody test and bone marrow smear examination for diagnosis of visceral leishmaniasis

We also further tried to evaluate performance of IFAT and bone marrow smear examination in diagnosis of VL in 350 VL suspected cases tested in our laboratory (January 2008–December 2019). The statistical analysis was done using online graph pad software quick cals.

RESULTS

A total of 80/553 (14.4%) cases were confirmed of VL (74/522 cases by demonstration of LD bodies in Bone marrow smear examination, 5/12 in splenic smear examination 1/19 by culture) and 4/21 (19.0%) cases were confirmed of PKDL (demonstration of LD bodies in slit-skin smear examination. In our study, 197/1369 (14.4%) cases were diagnosed positive by IFAT, 34/646 (5.2%) cases by rk 39 ICT for VL/PKDL by demonstration of specific antileishmanial antibodies [Table 1].

Table 1

Utility of smear examination and culture and serological tests in diagnosis of visceral leishmaniasis/post-kala-azar dermal leishmaniasis

Year	Total samples/total suspected patients	Total samples for culture	Number of samples positive for culture/total samples for culture	Total samples for smear examination	Smear positive for LD bodies/total bone marrow smear	Smear positive for LD bodies/total splenic smear	Smear positive for LD bodies/total skin biopsy smear	Total serum samples	IFAT positive/total IFAT tested	rK39 positive/total rK 39 tested
2009	522/424	2	0/2	202	27/199	1/3	Nil	318	45/318	NT
2010	350/318	2	0/2	112	18/108	0/1	0/3	236	44/236	NT
2011	342/289	6	1/6	91	10/85	2/3	2/3	245	33/245	NT
2012	253/251	4	0/4	54	7/50	1/2	0/2	195	33/195	NT
2013	202/188	Nil	-	37	8/35	0/1	0/1	165	27/165	NT
2014	225/151	4	0/4	31	2/23	Nil	0/8	190	13/190	NT
2015	162/149	1	0/1	5	0/3	Nil	0/2	156	0/14	9/142
2016	188/185	Nil	-	11	1/7	1/2	2/2	177	NT	17/177
2017	117/115	Nil	-	2	1/2	Nil	Nil	115	NT	2/115
2018	72/68	Nil	-	10	0/10	Nil	Nil	62	NT	2/62
2019	156/156	Nil	-	0	Nil	Nil	Nil	156	2/6	4/150
Total	2589/2294	19	1/19	555	74/522	5/12	4/21	2015	197/1369	34/646

NT: Not tested, LD: Leishmania donovani, IFAT: Indirect immunofluorescent antibody test

Anti-Leishmania antibodies end titer determined by IFAT and parasitological detection in 350 VL suspected cases is depicted in Table 2. With IFAT 100 as cutoff, we were able to detect 129 cases out of 350 VL suspected cases including all 72 parasitologically positive KA patients (missing no confirmed case of VL) and 57 out of 278 (20.5%) parasitologically negative cases. With IFAT 200 as cutoff, we were able to detect 59 cases out of 350, including 31 out of 72 (43.05%) parasitologically positive KA patients (missing 30 confirmed cases of VL). In our study, we found that IFAT 100 and above can be taken as optimum cutoff titer as it showed 85.3% accuracy. The sensitivity and specificity of IFAT test are 84.7% and 85.6%, respectively, in our study. Demonstration of homogeneous cytoplasmatic green fluorescence of promsatigotes in IFAT of positive sample is shown in Figure 1.

Table 2

Anti-Leishmania antibodies end titer determined by indirect immunofluorescent antibody test and Leishmania donovani bodies detection in bone marrow smears of visceral leishmaniasis suspected cases (n=350)

IFAT end point titers	LD bodies present	LD bodies absent	Total
≤50	0	221	221
100	41	29	70
≥200	31	28	59
Total	72	278	350

LD: Leishmania donovani, IFAT: Indirect immunofluorescent antibody test

Figure 1

(a) Demonstration of nonfluorescent promastigotes (negative samples) (b) homogeneous cytoplasmatic green fluorescence promastigotes in (positive samples) by immunofluorescent antibody test for diagnosis of leishmaniasis

DISCUSSION

KA is presently endemic in selected districts of Bihar, Jharkhand, and West Bengal besides the occurrence of sporadic cases reported from Uttar Pradesh and Uttarakhand.[3] Confirmed diagnosis of VL/PKDL requires demonstration of parasites in stained smears or culture of bone marrow aspirate, splenic aspirates, lymph nodes, liver, and skin biopsy obtained by invasive procedures.[8] However, the accuracy of microscopic examination is influenced by the ability of the laboratory expert, the quality of reagents, procedures, and low parasitemia. Bone marrow smear is most often sample received in laboratories for LD bodies which was also evident in our setting

Serological tests can be used in conjunction with bone marrow examination for confirmation of VL with the added advantage of detecting anti-Leishmania antibodies, especially when parasites are not found either by microscopy or by culture.[8]

Significant IFAT titer in parasitological smear-negative VL-suspected patients may require additional testing by other direct methods such as molecular testing wherever possible or must clinically respond to specific treatment for confirmation of VL. The demonstrated antibodies in parasitologically negative kala-azar patients in our study may be due to very early stages of infection or probable relapse with low parasitemia or persisting antibodies of previous infection. Various studies on IFAT have suggested 1:80–1:160 titers to be considered diagnostic for humans with VL symptoms, in different epidemiological areas.[910] The IFAT is specialized labor intensive technique which is not suitable for field purpose but has some striking advantages. Antibody titer measured by the IFAT is for the whole antigen and can be used to monitor the patient's long-term recovery.[1011] Antibodies are undetectable by IFAT 6 to 9 months after cure. If the antibodies persist in low titers, it is good indication of a probable relapse.[7]

PCR-based methods have proven to be sensitive to detect even very low parasitemia.[9] The major limitation of PCR-based methods for disease survey is availability of the procedure in low-resource facilities.[12131415]

Serological tests using rk39 in ELISA or rapid immunochromatographic format are simpler and less invasive as compared to IFAT for diagnosis of VL; however, they are not quantitative, less specific, and use rK39 recombinant antigen obtained from L. chagasi.[1315] Rk 39 cannot discriminate between current, subclinical, or past infections and is not useful for diagnosis of relapses and as a prognostic (cure) test.[16] The diagnostic potential of rk 39 rapid test for VL in our country is very good which has been reinforced in a recent study done in a nearby endemic country,[13] but the utility of other direct tests needs to be established.

With the advent of technology, highly specific and sensitive molecular-based tools such as PCR have been developed for detecting infection, diagnosis, and species differentiations, these hold considerable further promise for delivering better point-of-care diagnostic tests in elimination and postelimination setting.[1718]

CONCLUSION

Hence, we conclude that direct demonstration of parasite in smear or by culture or by PCR is required for confirmation of VL/PKDL-suspected cases. IFAT though not field-friendly, its optimal use as an adjunct test along with BM smear in all stages of infections may be required. Further, rk39 is a simple, reliable, noninvasive, and field-friendly test for diagnosis VL, especially in endemic areas. Importantly, as the goal of elimination of VL as a public health problem is approaching, apart from serological tests such as rk 39 and IFAT, direct methods of detection such as (parasitic demonstration in BM smears, molecular tests) for Leishmania may play a crucial role for achieving correct diagnosis and treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.