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ORIGINAL ARTICLE
Year : 2022  |  Volume : 12  |  Issue : 1  |  Page : 54-58  

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


1 Centre for Arboviral and Zoonotic Diseases, National Centre for Disease Control, Delhi, India
2 Centre for Arboviral and Zoonotic Diseases, National Centre for Disease Control, Delhi; BCG Vaccine Laboratory, Chennai, Tamil Nadu, India
3 Centre for Medical Entomology and Vector Management, National Centre for Disease Control, Delhi, India
4 National Centre for Disease Control, Delhi, India

Date of Submission06-Feb-2021
Date of Decision06-Feb-2021
Date of Acceptance01-Jun-2021
Date of Web Publication25-Jun-2022

Correspondence Address:
Monil Singhai
Centre for Arboviral and Zoonotic Diseases, National Centre for Disease Control, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tp.tp_7_21

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   Abstract 


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.

Keywords: Bone marrow smear, indirect immunofluorescent antibody test, rK 39 test, post–kala-azar dermal leishmaniasis, visceral leishmaniasis


How to cite this article:
Singhai M, Kakkar N, Gupta N, Bala M, Singh R, Singh SK. 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. Trop Parasitol 2022;12:54-8

How to cite this URL:
Singhai M, Kakkar N, Gupta N, Bala M, Singh R, Singh SK. 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. Trop Parasitol [serial online] 2022 [cited 2022 Nov 28];12:54-8. Available from: https://www.tropicalparasitology.org/text.asp?2022/12/1/54/348307




   Introduction Top


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).[2],[3] 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.[2],[3]

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 Top


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.[3],[4]

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 Top


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

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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)

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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

Click here to view



   Discussion Top


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.[9],[10] 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.[10],[11] 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.[12],[13],[14],[15]

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.[13],[15] 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.[17],[18]


   Conclusion Top


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.



 
   References Top

1.
Dey A, Sharma U, Singh S. First case of indigenous visceral leishmaniasis from central India. Am J Trop Med Hyg 2007;77:95-8.  Back to cited text no. 1
    
2.
Mittal V, Bhatia R, Sehgal S. Serodiagnosis of Indian kala-azar: Evaluation of IFA, ELISA and CIEP tests. J Commun Dis 1991;23:131-4.  Back to cited text no. 2
    
3.
Thakur S, Joshi J, Kaur S. Leishmaniasis diagnosis: An update on the use of parasitological, immunological and molecular methods. J Parasit Dis 2020;44:253-72.  Back to cited text no. 3
    
4.
Operational Guidelines On Kala-Azar (Visceral Leishmaniasis) Elimination In India (2015). Directorate of National Vector Borne Disease Control Programme (NVBDCP), Directorate General of Health Services, Minister of Health and Family Welfare. Available from: https://www.nvbdcp.gov.in/Doc/opertional-guideline-KA-2015.pdf.  Back to cited text no. 4
    
5.
Control of the Leishmaniasis: Report of a Meeting of the WHO Expert Committee on the Control of Leishmaniases. Geneva: WHO Technical Report Series; No. 949; 2010.http://apps.who.int/iris/bitstream/handle/10665/44412/WHO_TRS_949_eng.pdf.  Back to cited text no. 5
    
6.
Thakur S, Singh S, Pasha ST, Rawat DS, Lal S, Mittal V. Use of multilocus microsatellite typing (MLMT) for the genetic analysis of Indian isolates of Leishmania donovani. Ann Trop Med Parasitol 2009;103:461-6.  Back to cited text no. 6
    
7.
Shaw JJ, Voller A. The detection of circulating antibody to kala-azar by means of immunofluorescent techniques. Trans R Soc Trop Med Hyg 1964;58:349-52.  Back to cited text no. 7
    
8.
Machado de Assis TS, Rabello A, Werneck GL. Predictive models for the diagnostic of human visceral leishmaniasis in Brazil. PLoS Negl Trop Dis 2012;6:e1542.  Back to cited text no. 8
    
9.
Srivastava P, Dayama A, Mehrotra S, Sundar S. Diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg 2011;105:1-6.  Back to cited text no. 9
    
10.
Christodoulou V, Antoniou M, Ntais P, Messaritakis I, Ivovic V, Dedet JP, et al. Re-emergence of visceral and cutaneous leishmaniasis in the Greek Island of Crete. Vector Borne Zoonotic Dis 2012;12:214-22.  Back to cited text no. 10
    
11.
Akin M, Polat A, Balci YI, Kaya B, Karaca A, Turk M. Multiple relapses of visceral leishmaniasis in a patient treated with liposomal amphotericin. Indian J. Pediatr 2009;76:436-7.  Back to cited text no. 11
    
12.
Pereira MR, Rocha-Silva F, Graciele-Melo C, Lafuente CR, Magalhães T, Caligiorne RB. Comparison between conventional and real-time PCR assays for diagnosis of visceral leishmaniasis. Biomed Res Int 2014;2014:639310.  Back to cited text no. 12
    
13.
Banu SS, Ahmed BN, Shamsuzzaman AKM, Lee R. Evaluation of recombinant K39 antigen and various promastigote antigens in sero-diagnosis of visceral leishmaniasis in Bangladesh. Parasite Epidemiol Control 2016;1:219-28.  Back to cited text no. 13
    
14.
Mniouil M, Fellah H, Amarir F, Sadak A, Et-Touys A, Bakri Y, et al. Comparative evaluation of immunochromatographic dipstick test (ICT) rk39, soluble antigen ELISA and IFAT for the sero-diagnosis of visceral leishmaniasis in Morocco. Acta Trop 2018;182:185-9.  Back to cited text no. 14
    
15.
Burns JM Jr., Shreffler WG, Benson DR, Ghalib HW, Badaro R, Reed SG. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci USA 1993;90:775-9.  Back to cited text no. 15
    
16.
Eslami G, Hajimohammadi B, Jafari AA, Mirzaei F, Gholamrezai M, Anvari H, Khamesipour A. Molecular identification of Leishmania tropica infections in patients with cutaneous leishmaniasis from an endemic central of Iran. Trop Biomed 2014;31:592-9.  Back to cited text no. 16
    
17.
Sundar S, Singh OP. Molecular diagnosis of visceral leishmaniasis. Mol Diagn Ther 2018;22:443-57.  Back to cited text no. 17
    
18.
Suman R, Shyam S, Dinesh M, Pradeep D, Jorge A, Marleen B, et al. Eliminating visceral leishmaniasis in South Asia: The road ahead. BMJ 2019;364:k5224.  Back to cited text no. 18
    


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  [Table 1], [Table 2]



 

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