Author:
- Abstract.
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- Study population.
- Analytical sensitivity and specificity of RealAmp.
- Establishing the utility of RealAmp for the detection of leishmaniasis.
- Assessing the utility of RealAmp for diagnosis and as a test of cure in PKDL using slit aspirate sample.
- Direct sample lysis–LAMP assay of slit aspirate.
- Comparative ROC curve analysis for assessing diagnostic accuracy of RealAmp for detection of leishmaniasis.
- DISCUSSION
- Supplemental appendix, tables, and figure
- Acknowledgments:
- REFERENCES
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
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Zijlstra EE, Alves F, Rijal S, Arana B, Alvar J, 2017. Post-kala-azar dermal leishmaniasis in the Indian subcontinent: a threat to the south-east Asia region kala-azar elimination programme. PLoS Negl Trop Dis 11: e0005877.
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Zijlstra EE, 2019. Biomarkers in post kala-azar dermal leishmaniasis (PKDL). Front Cell Infect Microbiol 9: 228.
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Gedda MR, Singh B, Kumar D, Singh AK, Madhukar P, Upadhyay S, Singh OP, Sundar S, 2020. Post kala-azar dermal leishmaniasis: a threat to elimination program. PLoS Negl Trop Dis 14: e0008221.
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Rajni E, Ghiya BC, Singh S, Shankar P, Swami T, Jadon DS, Negi SR, Malik M, Khatri PK, 2019. 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. Trop Parasitol 9: 115–123.
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Abeijon C, Alves F, Monnerat S, Mbui J, Viana AG, Almeida RM, Bueno LL, Fujiwara RT, Campos-Neto A, 2020. Urine-based antigen detection assay for diagnosis of visceral leishmaniasis using monoclonal antibodies specific for six protein biomarkers of Leishmania infantum/Leishmania donovani. PLoS Negl Trop Dis 14: e0008246.
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Njiru ZK, Mikosza ASJ, Armstrong T, Enyaru JC, Ndung’u JM, Thompson ARC, 2008. Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. PLoS Negl Trop Dis 2: e147.
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Khan MGM, Bhaskar KRH, Salam MA, Akther T, Pluschke G, Mondal D, 2012. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors 5: 280.
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Chaouch M, Mhadhbi M, Adams ER, Schoone GJ, Limam S, Gharbi Z, Darghouth MA, Guizani I, BenAbderrazak S, 2013. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes. Vet Parasitolo 198: 78–84.
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Real-Time Fluorimetry Loop-Mediated Isothermal Amplification for Diagnosis of Leishmaniasis and as a Tool for Assessment of Cure for Post–Kala-Azar Dermal Leishmaniasis
Keerti Kaumudee Dixit
Keerti Kaumudee Dixit
ICMR-National Institute of Pathology (NIOP), Safdarjung Hospital Campus, New Delhi, India;
Faculty of Health and Biological Sciences, Symbiosis International (Deemed University), Pune, India;
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Faculty of Health and Biological Sciences, Symbiosis International (Deemed University), Pune, India;
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Venkatesh Ramesh
Venkatesh Ramesh
Department of Dermatology and STD, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India;
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Ratan Gupta
Ratan Gupta
Department of Paediatrics, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India;
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Narendra Singh Negi
Narendra Singh Negi
Department of Medicine, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India
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Ruchi Singh
Ruchi Singh
ICMR-National Institute of Pathology (NIOP), Safdarjung Hospital Campus, New Delhi, India;
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Poonam Salotra
Poonam Salotra
ICMR-National Institute of Pathology (NIOP), Safdarjung Hospital Campus, New Delhi, India;
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Abstract.
Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post–kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68–100) for diagnosis of VL and PKDL (95% CI: 79.61–100) and 96% (95% CI: 86.68–100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64–98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20–96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I–based (Molecular Probes, Eugene, OR) visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.
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Author Notes
Disclosure: P. S. is grateful to DST_SERB for providing JC Bose fellowship. K. K. D. received fellowship from the Indian Council of Medical Research (ICMR), New Delhi, India.
Financial support: The work was supported by the Indian Council of Medical Research (ICMR) (Grant no. 6/9–7[79]/2014/ECD II) New Delhi, India.
Authors’ addresses: Keerti Kaumudee Dixit, ICMR-National Institute of Pathology (NIOP), Safdarjung Hospital Campus, New Delhi, India, and Faculty of Health and Biological Sciences, Symbiosis International (Deemed University), Pune, India, E-mail: keerti.dixit@gmail.com. Venkatesh Ramesh, Department of Dermatology and STD, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India, and Department of Dermatology, Hamdard Institute of Medical sciences and Research, Jamia Hamdard University, New Delhi, India, E-mail: weramesh@gmail.com. Ratan Gupta, Department of Paediatrics, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India, E-mail: ratangupta100@yahoo.com. Narendra Singh Negi, Department of Medicine, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India, E-mail: drnsnegi@gmail.com. Ruchi Singh and Poonam Salotra, ICMR-National Institute of Pathology (NIOP), Safdarjung Hospital Campus, New Delhi, India, E-mails: ruchisp@gmail.com and poonamsalotra@hotmail.com.
- Abstract.
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- Study population.
- Analytical sensitivity and specificity of RealAmp.
- Establishing the utility of RealAmp for the detection of leishmaniasis.
- Assessing the utility of RealAmp for diagnosis and as a test of cure in PKDL using slit aspirate sample.
- Direct sample lysis–LAMP assay of slit aspirate.
- Comparative ROC curve analysis for assessing diagnostic accuracy of RealAmp for detection of leishmaniasis.
- DISCUSSION
- Supplemental appendix, tables, and figure
- Acknowledgments:
- REFERENCES
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
WHO, 2020. WHO Factsheet on Leishmaniasis. Geneva, Switzerland: World Health Organization. Available at: https://www.who.int/en/news-room/fact-sheets/detail/leishmaniasis. Accessed August 20, 2020.
-
2.
Zijlstra EE, Alves F, Rijal S, Arana B, Alvar J, 2017. Post-kala-azar dermal leishmaniasis in the Indian subcontinent: a threat to the south-east Asia region kala-azar elimination programme. PLoS Negl Trop Dis 11: e0005877.
-
3.
Zijlstra EE, 2019. Biomarkers in post kala-azar dermal leishmaniasis (PKDL). Front Cell Infect Microbiol 9: 228.
-
4.
Gedda MR, Singh B, Kumar D, Singh AK, Madhukar P, Upadhyay S, Singh OP, Sundar S, 2020. Post kala-azar dermal leishmaniasis: a threat to elimination program. PLoS Negl Trop Dis 14: e0008221.
-
5.
Rahman KM et al. 2010. Increasing incidence of post-kala-azar dermal leishmaniasis in a population-based study in Bangladesh. Clin Infect Dis 50: 73–76.
-
6.
Ramesh V, Kaushal H, Mishra AK, Singh R, Salotra P, 2015. Clinico-epidemiological analysis of post kala-azar dermal leishmaniasis (PKDL) cases in India over last two decades: a hospital based retrospective study. BMC Public Health 15: 1092.
-
7.
Zijlstra EE, Musa AM, Khalil EAG, El-Hassan IM, 2003. Post-kala-azar dermal leishmaniasis. Lancet Infect Dis 3: 87–98.
-
8.
Musa AM, Khalil EAG, Raheem MA, Zijlstra EE, Ibrahim ME, Elhassan IM, Mukhtar MM, El Hassan AM, 2002. The natural history of Sudanese post-kala-azar dermal leishmaniasis: clinical, immunological and prognostic. Ann Trop Med Parasitol 96: 765–772.
-
9.
Das VNR et al. 2012. Clinical epidemiologic profile of a cohort of post–kala-azar dermal leishmaniasis patients in Bihar, India. Am J Trop Med Hyg 86: 959–961.
-
10.
Desjeux P, Ghosh RS, Dhalaria P, Strub-Wourgaft N, Zijlstra EE, 2013. Report of the post kala-azar dermal leishmaniasis (PKDL) consortium meeting, New Delhi, India, 27–29 June 2012. Parasit Vectors 6: 196.
-
11.
WHO, 2016. Process of Validation of Elimination of Kala-Azar as a Public Health Problem in South-East Asia. Geneva, Switzerland: World Health Organization. Available at: https://www.who.int/leishmaniasis/resources/Validation_of_elimination_of_VL_as_a_public_health_prob_SEARO/en/. Accessed August 20, 2020.
-
12.
WHO, 2017. Accelerated Plan for Kala-Azar Elimination. Geneva, Switzerland: World Health Organization. Available at: https://www.who.int/leishmaniasis/resources/Accelerated_plan_for_Kala-azar_Elimination_2017/en/. Accessed August 20, 2020.
-
13.
National Vector Borne Disease Control Programme (NVBDCP), 2020. Kala-Azar Situation in India, National Vector Borne Disease Control Programme (NVBDCP). Available at: https://nvbdcp.gov.in/index4.php?lang=1&level=0&linkid=467&lid=3750. Accessed August 20, 2020.
-
14.
Kumar A, Saurabh S, Jamil S, Kumar V, 2020. Intensely clustered outbreak of visceral leishmaniasis (kala-azar) in a setting of seasonal migration in a village of Bihar, India. BMC Infect Dis 20: 10.
-
15.
Rajni E, Ghiya BC, Singh S, Shankar P, Swami T, Jadon DS, Negi SR, Malik M, Khatri PK, 2019. 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. Trop Parasitol 9: 115–123.
-
16.
Cunningham J et al. 2012. A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis. Clin Infect Dis 55: 1312–1319.
-
17.
Chappuis F, Rijal S, Soto A, Menten J, Boelaert M, 2006. A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis. Br Med J 333: 723.
-
18.
Abeijon C, Alves F, Monnerat S, Mbui J, Viana AG, Almeida RM, Bueno LL, Fujiwara RT, Campos-Neto A, 2020. Urine-based antigen detection assay for diagnosis of visceral leishmaniasis using monoclonal antibodies specific for six protein biomarkers of Leishmania infantum/Leishmania donovani. PLoS Negl Trop Dis 14: e0008246.
-
19.
Mitarai S et al. 2011. Evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis. Int J Tuberc Lung Dis 15: 1211–1217.
-
20.
Boehme CC et al. 2007. Operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries. J Clin Microbiol 45: 1936–1940.
-
21.
Njiru ZK, Mikosza ASJ, Armstrong T, Enyaru JC, Ndung’u JM, Thompson ARC, 2008. Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. PLoS Negl Trop Dis 2: e147.
-
22.
Hopkins H et al. 2013. Highly sensitive detection of malaria parasitemia in an endemic setting: performance of a new LAMP kit in a remote clinic in Uganda. J Infect Dis 208: 645–652.
-
23.
Polley SD et al. 2013. Clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria. J Infect Dis 208: 637–644.
-
24.
Takagi H, Itoh M, Islam MZ, Razzaque A, Ekram AS, Hashighuchi Y, Noiri E, Kimura E, 2009. Sensitive, specific, and rapid detection of Leishmania donovani DNA by loop-mediated isothermal amplification. Am J Trop Med Hyg 81: 578–582.
-
25.
Adams ER, Schoone GJ, El Safi S, Schallig HD, 2010. Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples. Am J Trop Med Hyg 82: 591–596.
-
26.
Khan MGM, Bhaskar KRH, Salam MA, Akther T, Pluschke G, Mondal D, 2012. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors 5: 280.
-
27.
Verma S, Avishek K, Sharma V, Negi NS, Ramesh V, Salotra P, 2013. Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis. Diagn Microbiol Infect Dis 75: 390–395.
-
28.
Chaouch M, Mhadhbi M, Adams ER, Schoone GJ, Limam S, Gharbi Z, Darghouth MA, Guizani I, BenAbderrazak S, 2013. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes. Vet Parasitolo 198: 78–84.
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