• 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.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 5.

    Rahman KM 2010. Increasing incidence of post-kala-azar dermal leishmaniasis in a population-based study in Bangladesh. Clin Infect Dis 50: 7376.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 7.

    Zijlstra EE, Musa AM, Khalil EAG, El-Hassan IM, 2003. Post-kala-azar dermal leishmaniasis. Lancet Infect Dis 3: 8798.

  • 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: 765772.

    • Search Google Scholar
    • Export Citation
  • 9.

    Das VNR 2012. Clinical epidemiologic profile of a cohort of post–kala-azar dermal leishmaniasis patients in Bihar, India. Am J Trop Med Hyg 86: 959961.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 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: 115123.

    • Search Google Scholar
    • Export Citation
  • 16.

    Cunningham J 2012. A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis. Clin Infect Dis 55: 13121319.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 19.

    Mitarai S 2011. Evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis. Int J Tuberc Lung Dis 15: 12111217.

    • Search Google Scholar
    • Export Citation
  • 20.

    Boehme CC 2007. Operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries. J Clin Microbiol 45: 19361940.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 22.

    Hopkins H 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: 645652.

    • Search Google Scholar
    • Export Citation
  • 23.

    Polley SD 2013. Clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria. J Infect Dis 208: 637644.

  • 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: 578582.

    • Search Google Scholar
    • Export Citation
  • 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: 591596.

    • Search Google Scholar
    • Export Citation
  • 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.

    • Search Google Scholar
    • Export Citation
  • 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: 390395.

    • Search Google Scholar
    • Export Citation
  • 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: 7884.

    • Search Google Scholar
    • Export Citation
  • 29.

    Ghasemian M, Gharavi MJ, Akhlaghi L, Mohebali M, Meamar AR, Aryan E, Oormazdi H, 2014. Development and assessment of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of human visceral leishmaniasis in Iran. Iran J Parasito 9: 50.

    • Search Google Scholar
    • Export Citation
  • 30.

    Sriworarat C, Phumee A, Mungthin M, Leelayoova S, Siriyasatien P, 2015. Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection. Parasit Vectors 8: 591.

    • Search Google Scholar
    • Export Citation
  • 31.

    Gao CH, Ding D, Wang JY, Steverding D, Wang X, Yang YT, Shi F, 2015. Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples. Parasit Vectors 8: 370.

    • Search Google Scholar
    • Export Citation
  • 32.

    Kothalawala HS, Karunaweera ND, 2016. Loop-mediated isothermal amplification assay as a sensitive diagnostic tool for Leishmania donovani infections in Sri Lanka. Ceylon Med J 61: 6870.

    • Search Google Scholar
    • Export Citation
  • 33.

    Nzelu CO 2016. A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool. Acta Trop 153: 116119.

    • Search Google Scholar
    • Export Citation
  • 34.

    Verma S, Singh R, Sharma V, Bumb RA, Negi NS, Ramesh V, Salotra P, 2017. Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection. BMC Infect Dis 17: 223.

    • Search Google Scholar
    • Export Citation
  • 35.

    Mukhtar M, Ali SS, Boshara SA, Albertini A, Monnerat S, Bessell P, Mori Y, Kubota Y, Ndung’u JM, Cruz I, 2018. Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP). PLoS Negl Trop Dis 12: e0006264.

    • Search Google Scholar
    • Export Citation
  • 36.

    Imai K 2018. Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing. Parasitol Int 67: 3437.

    • Search Google Scholar
    • Export Citation
  • 37.

    Adams ER 2018. Development and evaluation of a novel loop-mediated isothermal amplification assay for diagnosis of cutaneous and visceral leishmaniasis. J Clin Microbiol 56: e00386-18.

    • Search Google Scholar
    • Export Citation
  • 38.

    Vink MM 2018. Evaluation of point-of-care tests for cutaneous leishmaniasis diagnosis in Kabul, Afghanistan. EBioMedicine 37: 453460.

  • 39.

    Dixit KK 2018. Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL). PLoS Negl Trop Dis 12: e0006922.

    • Search Google Scholar
    • Export Citation
  • 40.

    Avelar DMD, Carvalho DM, Rabello A, 2019. Development and clinical evaluation of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of human visceral leishmaniasis in Brazil. BioMed Res Int 2019: 8240784.

    • Search Google Scholar
    • Export Citation
  • 41.

    Nzelu CO, Kato H, Peters NC, 2019. Loop-mediated isothermal amplification (LAMP): an advanced molecular point-of-care technique for the detection of Leishmania infection. PLoS Negl Trop Dis 13: e0007698.

    • Search Google Scholar
    • Export Citation
  • 42.

    Ibarra-Meneses AV, Cruz I, Chicharro C, Sánchez C, Biéler S, Broger T, Moreno J, Carrillo E, 2018. Evaluation of fluorimetry and direct visualization to interpret results of a loop-mediated isothermal amplification kit to detect Leishmania DNA. Parasit Vectors 11: 250.

    • Search Google Scholar
    • Export Citation
  • 43.

    Verma S, Kumar R, Katara GK, Singh LC, Negi NS, Ramesh V, Salotra P, 2010. Quantification of parasite load in clinical samples of leishmaniasis patients: IL-10 level correlates with parasite load in visceral leishmaniasis. PLoS One 5: e10107.

    • Search Google Scholar
    • Export Citation
  • 44.

    Hanley JA, McNeil BJ, 1982. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 143: 2936.

  • 45.

    Hanley JA, McNeil BJ, 1983. A method of comparing the areas under receiver operating characteristic curves derived from the same cases. Radiology 148: 839843.

    • Search Google Scholar
    • Export Citation
  • 46.

    Verma S, Bhandari V, Avishek K, Ramesh V, Salotra P, 2013. Reliable diagnosis of post‐kala‐azar dermal leishmaniasis (PKDL) using slit aspirate specimen to avoid invasive sampling procedures. Trop Med Int Health 18: 268275.

    • Search Google Scholar
    • Export Citation
  • 47.

    Sundar S, Singh OP, Chakravarty J, 2018. Visceral leishmaniasis elimination targets in India, strategies for preventing resurgence. Expert Rev Ant Infect Ther 16: 805812.

    • Search Google Scholar
    • Export Citation
  • 48.

    Le Rutte EA, Zijlstra EE, de Vlas SJ, 2019. Post-kala-azar dermal leishmaniasis as a reservoir for visceral leishmaniasis transmission. Trends Parasitol 35: 590592.

    • Search Google Scholar
    • Export Citation
  • 49.

    Medley GF, Hollingsworth TD, Olliaro PL, Adams ER, 2015. Health-seeking behaviour, diagnostics and transmission dynamics in the control of visceral leishmaniasis in the Indian subcontinent. Nature 528: S102S108.

    • Search Google Scholar
    • Export Citation
  • 50.

    Ramesh V, Dixit KK, Sharma N, Singh R, Salotra P, 2020. Assessing the efficacy and safety of liposomal amphotericin B and miltefosine in combination for treatment of post kala-azar dermal leishmaniasis. J Infect Dis 221: 608617.

    • Search Google Scholar
    • Export Citation
  • 51.

    Bhargava A, Ramesh V, Verma S, Salotra P, Bala M, 2018. Revisiting the role of the slit-skin smear in the diagnosis of Indian post-kala-azar dermal leishmaniasis. Indian J Dermatol Venereol Leprol 84: 690695.

    • Search Google Scholar
    • Export Citation
  • 52.

    Molina R, Ghosh D, Carrillo E, Monnerat S, Bern C, Mondal D, Alvar J, 2017. Infectivity of post-kala-azar dermal leishmaniasis patients to sand flies: revisiting a proof of concept in the context of the kala-azar elimination program in the Indian subcontinent. Clin Infect Dis 65: 150153.

    • Search Google Scholar
    • Export Citation
  • 53.

    Sengupta R, Chowdhury SJ, Moulik S, Ghosh MK, Saha B, Das NK, Chatterjee M, 2019. Active surveillance identified a neglected burden of macular cases of post kala-azar dermal leishmaniasis in West Bengal. PLoS Negl Trop Dis 13: e0007249.

    • Search Google Scholar
    • Export Citation
  • 54.

    Salotra P, Singh R, 2006. Challenges in the diagnosis of post kala-azar dermal leishmaniasis. Indian J Med Res 123: 295310.

  • 55.

    Martzy R, Kolm C, Krska R, Mach RL, Farnleitner AH, Reischer GH, 2019. Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis. Anal Bioanal Chem 411: 16951702.

    • Search Google Scholar
    • Export Citation
  • 56.

    Mikita K, Maeda T, Yoshikawa S, Ono T, Miyahira Y, Kawana A, 2014. The direct boil-LAMP method: a simple and rapid diagnostic method for cutaneous leishmaniasis. Parasitol Int 63: 785789.

    • Search Google Scholar
    • Export Citation
  • 57.

    Modak SS, Barber CA, Geva E, Abrams WR, Malamud D, Ongagna YSY, 2016. Rapid point-of-care isothermal amplification assay for the detection of malaria without nucleic acid purification. Infect Dis (Auckl) 9: 19.

    • Search Google Scholar
    • Export Citation
  • 58.

    Bentaleb EM, Abid M, El Messaoudi MD, Lakssir B, Amzazi S, Sefrioui H, Benhassou HA, 2016. Development and evaluation of an in-house single step loop-mediated isothermal amplification (SS-LAMP) assay for the detection of Mycobacterium tuberculosis complex in sputum samples from Moroccan patients. BMC Infect Dis 16: 110.

    • Search Google Scholar
    • Export Citation

 

 

 

 

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

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  • 1 ICMR-National Institute of Pathology (NIOP), Safdarjung Hospital Campus, New Delhi, India;
  • 2 Faculty of Health and Biological Sciences, Symbiosis International (Deemed University), Pune, India;
  • 3 Department of Dermatology and STD, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India;
  • 4 Department of Paediatrics, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India;
  • 5 Department of Medicine, Safdarjung Hospital, Vardhman Mahavir Medical College, New Delhi, India

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

Address correspondence to Ruchi Singh or Poonam Salotra, ICMR-National Institute of Pathology, Safdarjung Hospital Campus New Delhi, 110029, India. E-mails: ruchisp@gmail.com or poonamsalotra@hotmail.com

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.

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