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

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 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.

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. V. 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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