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Quantitative monitoring of experimental and human leishmaniasis employing amastigote-specific genes

Published online by Cambridge University Press:  10 May 2022

Madhurima Roy ,
Deblina Sarkar  and
Mitali Chatterjee Open the ORCID record for Mitali Chatterjee [Opens in a new window]
Madhurima Roy
Affiliation:
Department of Pharmacology, Institute of Postgraduate Medical Education and Research (IPGME&R), 244B, Acharya JC Bose Road, Kolkata 700020, India
Deblina Sarkar
Affiliation:
Department of Pharmacology, Institute of Postgraduate Medical Education and Research (IPGME&R), 244B, Acharya JC Bose Road, Kolkata 700020, India
Mitali Chatterjee*
Affiliation:
Department of Pharmacology, Institute of Postgraduate Medical Education and Research (IPGME&R), 244B, Acharya JC Bose Road, Kolkata 700020, India
*
Author for correspondence: Mitali Chatterjee, E-mail: ilatimc@gmail.com
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Abstract

The gold standard for diagnosis of leishmaniasis is the microscopic detection of amastigotes/Leishman Donovan (LD) bodies, but its moderate sensitivity necessitates the development of molecular approaches. This study aimed to quantify in experimental animal models and human leishmaniasis the expression of amastigote-specific virulence genes, A2 and amastin by droplet digital polymerase chain reaction (ddPCR). Total RNA was isolated from L. donovani-infected hamsters or murine peritoneal macrophages and lesional biopsies from patients with post kala-azar dermal leishmaniasis (PKDL). Following cDNA conversion, EvaGreen-based ddPCR was performed using specific primers for A2 or amastin and parasite load expressed in copies per μL. Assay was optimized and the specificity of amastigote-specific A2 and amastin was confirmed. In hepatic and splenic tissues of L. donovani-infected hamsters and peritoneal macrophages, ddPCR demonstrated a greater abundance of A2 than amastin. Treatment of L. donovani-infected peritoneal macrophages with conventional anti-leishmanials, miltefosine and amphotericin B translated into a dose-dependent reduction in copies per μL of A2 and amastin, and the extrapolated IC50 was comparable with results obtained by counting LD bodies in Giemsa-stained macrophages. Similarly, in dermal biopsies of patients with PKDL, A2 and amastin were detected. Overall, monitoring of A2 by ddPCR can be an objective measure of parasite burden and potentially adaptable into a high throughput approach necessary for drug development and monitoring disease progression when the causative species is L. donovani.

Keywords

A2amastigotesamastindroplet digital polymerase chain reaction (ddPCR)post kala-azar dermal leishmaniasis (PKDL)
Type
Research Article
Information
Parasitology , Volume 149 , Issue 8 , July 2022 , pp. 1085 - 1093
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Copyright
Copyright © The Author(s), 2022. Published by Cambridge University Press

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References

Abbasi, I, Aramin, S, Hailu, A, Shiferaw, W, Kassahun, A, Belay, S, Jaffe, C and Warburg, A (2013) Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia. BMC Infectious Diseases 13, 153.CrossRefGoogle ScholarPubMed
Akhoundi, B, Mohebali, M, Shojaee, S, Jalali, M, Kazemi, B, Bandehpour, M, Keshavarz, H, Edrissian, GH, Eslami, MB, Malekafzali, H and Kouchaki, A (2013) Rapid detection of human and canine visceral leishmaniasis: assessment of a latex agglutination test based on the A2 antigen from amastigote forms of Leishmania infantum. Experimental Parasitology 133, 307–313.CrossRefGoogle ScholarPubMed
Avishek, K, Ahuja, K, Pradhan, D, Gannavaram, S, Selvapandiyan, A, Nakhasi, HL and Salotra, P (2018) A leishmania-specific gene upregulated at the amastigote stage is crucial for parasite survival. Parasitology Research 117, 3215–3228.CrossRefGoogle ScholarPubMed
Bates, PA (2018) Revising Leishmania's life cycle. Nature Microbiology 3, 529–530.CrossRefGoogle ScholarPubMed
Belmonte, FR, Martin, JL, Frescura, K, Damas, J, Pereira, F, Tarnopolsky, MA and Kaufman, BA (2016) Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions. Scientific Reports 6, 25186.CrossRefGoogle ScholarPubMed
Bhattacharyya, T, Ayandeh, A, Falconar, AK, Sundar, S, El-Safi, S, Gripenberg, MA, Bowes, DE, Thunissen, C, Singh, OP, Kumar, R, Ahmed, O, Eisa, O, Saad, A, Silva Pereira, S, Boelaert, M, Mertens, P and Miles, MA (2014) IgG1 as a potential biomarker of post-chemotherapeutic relapse in visceral leishmaniasis, and adaptation to a rapid diagnostic test. PLoS Neglected Tropical Diseases 8, e3273.CrossRefGoogle ScholarPubMed
Cai, Y, Li, X, Lv, R, Yang, J, Li, J, He, Y and Pan, L (2014) Quantitative analysis of pork and chicken products by droplet digital PCR. Biomed Research International 2014, 810209.CrossRefGoogle ScholarPubMed
Carvalho, FA, Charest, H, Tavares, CA, Matlashewski, G, Valente, EP, Rabello, A, Gazzinelli, RT and Fernandes, AP (2002) Diagnosis of American visceral leishmaniasis in humans and dogs using the recombinant Leishmania donovani A2 antigen. Diagnostic Microbiology and Infectious Disease 43, 289–295.CrossRefGoogle ScholarPubMed
Chappuis, F, Sundar, S, Hailu, A, Ghalib, H, Rijal, S, Peeling, RW, Alvar, J and Boelaert, M (2007) Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nature Reviews. Microbiology 5, 873–882.CrossRefGoogle Scholar
Charest, H and Matlashewski, G (1994) Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene. Molecular and Cellular Biology 14, 2975–2984.Google ScholarPubMed
de Paiva, RM, Grazielle-Silva, V, Cardoso, MS, Nakagaki, BN, Mendonça-Neto, RP, Canavaci, AM, Souza Melo, N, Martinelli, PM, Fernandes, AP, daRocha, WD and Teixeira, SM (2015) Amastin knockdown in Leishmania braziliensis affects parasite-macrophage interaction and results in impaired viability of intracellular amastigotes. PLoS Pathogens 11, e1005296.CrossRefGoogle ScholarPubMed
Didwania, N, Ejazi, SA, Chhajer, R, Sabur, A, Mazumder, S, Kamran, M, Kar, R, Pandey, K, Das, V, Das, P, Rahaman, M, Goswami, RP and Ali, N (2020) Evaluation of cysteine protease C of Leishmania donovani in comparison with glycoprotein 63 and elongation factor 1α for diagnosis of human visceral leishmaniasis and for posttreatment follow-up response. Journal of Clinical Microbiology 58, e00213–20.CrossRefGoogle Scholar
Dighal, A, Mukhopadhyay, D, Sengupta, R, Moulik, S, Mukherjee, S, Roy, S, Chaudhuri, SJ, Das, NK and Chatterjee, M (2020) Iron trafficking in patients with Indian post kala-azar dermal leishmaniasis. PLoS Neglected Tropical Diseases 14, e0007991.CrossRefGoogle ScholarPubMed
Ejazi, SA, Bhattacharya, P, Bakhteyar, MA, Mumtaz, AA, Pandey, K, Das, VN, Das, P, Rahaman, M, Goswami, RP and Ali, N (2016) Noninvasive diagnosis of visceral leishmaniasis: development and evaluation of two urine-based immunoassays for detection of Leishmania donovani infection in India. PLoS Neglected Tropical Diseases 10, e0005035.CrossRefGoogle ScholarPubMed
Ejazi, SA, Ghosh, S, Saha, S, Choudhury, ST, Bhattacharyya, A, Chatterjee, M, Pandey, K, Das, VNR, Das, P, Rahaman, M, Goswami, RP, Rai, K, Khanal, B, Bhattarai, NR, Deepachandi, B, Siriwardana, YD, Karunaweera, ND, deBrito, MEF, Gomes, YM, Nakazawa, M, Costa, CHN, Adem, E, Yeshanew, A, Melkamu, R, Fikre, H, Hurissa, Z, Diro, E, Carrillo, E, Moreno, J and Ali, N (2019) A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain. Scientific Reports 9, 9932.CrossRefGoogle ScholarPubMed
Escobar, P, Matu, S, Marques, C and Croft, SL (2002) Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18–OCH(3) (edelfosine) and amphotericin B. Acta Tropica 81, 151–157.CrossRefGoogle ScholarPubMed
Floren, C, Wiedemann, I, Brenig, B, Schütz, E and Beck, J (2015) Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR). Food Chemistry 173, 1054–1058.CrossRefGoogle Scholar
Garg, R and Dube, A (2006) Animal models for vaccine studies for visceral leishmaniasis. The Indian Journal of Medical Research 123, 439–454.Google ScholarPubMed
Gedda, MR, Singh, B, Kumar, D, Singh, AK, Madhukar, P, Upadhyay, S, Singh, OP and Sundar, S (2020) Post kala-azar dermal leishmaniasis: a threat to elimination program. PLoS Neglected Tropical Diseases 14, e0008221.CrossRefGoogle ScholarPubMed
Ghosh, S, Das, NK, Mukherjee, S, Mukhopadhyay, D, Barbhuiya, JN, Hazra, A and Chatterjee, M (2015) Inadequacy of 12-week miltefosine treatment for Indian post-kala-azar dermal leishmaniasis. The American Journal of Tropical Medicine and Hygiene 93, 767–769.CrossRefGoogle ScholarPubMed
Gobert, G, Cotillard, A, Fourmestraux, C, Pruvost, L, Miguet, J and Boyer, M (2018) Droplet digital PCR improves absolute quantification of viable lactic acid bacteria in faecal samples. Journal of Microbiological Methods 148, 64–73.CrossRefGoogle ScholarPubMed
Holst-Jensen, A, Rønning, SB, Løvseth, A and Berdal, KG (2003) PCR technology for screening and quantification of genetically modified organisms (GMOs). Analytical and Bioanalytical Chemistry 375, 985–993.CrossRefGoogle Scholar
Hossain, F, Ghosh, P, Khan, M, Duthie, MS, Vallur, AC, Picone, A, Howard, RF, Reed, SG and Mondal, D (2017) Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of visceral leishmaniasis patients and the monitoring of their response to treatment. PLoS ONE 12, e0185606.CrossRefGoogle Scholar
Jackson, AP (2010) The evolution of amastin surface glycoproteins in trypanosomatid parasites. Molecular Biology and Evolution 27, 33–45.CrossRefGoogle ScholarPubMed
Johnston, DA, Blaxter, ML, Degrave, WM, Foster, J, Ivens, AC and Melville, SE (1999) Genomics and the biology of parasites. Bioessays: News and Reviews in Molecular, Cellular and Developmental Biology 21, 131–147.3.0.CO;2-I>CrossRefGoogle ScholarPubMed
Karmakar, J, Roy, S and Mandal, C (2019) Modulation of TLR4 sialylation mediated by a Sialidase Neu1 and impairment of its signaling in Leishmania donovani infected macrophages. Frontiers in Immunology 10, 2360.CrossRefGoogle ScholarPubMed
Mary, C, Faraut, F, Lascombe, L and Dumon, H (2004) Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. Journal of Clinical Microbiology 42, 5249–5255.CrossRefGoogle ScholarPubMed
Mary, C, Faraut, F, Drogoul, MP, Xeridat, B, Schleinitz, N, Cuisenier, B and Dumon, H (2006) Reference values for Leishmania infantum parasitemia in different clinical presentations: quantitative polymerase chain reaction for therapeutic monitoring and patient follow-up. The American Journal of Tropical Medicine and Hygiene 75, 858–863.CrossRefGoogle ScholarPubMed
Matlashewski, G (2001) Leishmania infection and virulence. Medical Microbiology and Immunology 190, 37–42.CrossRefGoogle ScholarPubMed
McCall, LI and Matlashewski, G (2010) Localization and induction of the A2 virulence factor in Leishmania: evidence that A2 is a stress response protein. Molecular Microbiology 77, 518–530.CrossRefGoogle ScholarPubMed
Melby, PC, Chandrasekar, B, Zhao, W and Coe, JE (2001) The hamster as a model of human visceral leishmaniasis: progressive disease and impaired generation of nitric oxide in the face of a prominent Th1-like cytokine response. Journal of Immunology 166, 1912–1920.CrossRefGoogle ScholarPubMed
Mondal, D, Bern, C, Ghosh, D, Rashid, M, Molina, R, Chowdhury, R, Nath, R, Ghosh, P, Chapman, L, Alim, A, Bilbe, G and Alvar, J (2019) Quantifying the infectiousness of post-kala-azar dermal leishmaniasis toward sand flies. Clinical Infectious Diseases 69, 251–258.CrossRefGoogle ScholarPubMed
Moreira, ND, Vitoriano-Souza, J, Roatt, BM, Vieira, PM, Ker, HG, de Oliveira Cardoso, JM, Giunchetti, RC, Carneiro, CM, de Lana, M and Reis, AB (2012) Parasite burden in hamsters infected with two different strains of leishmania (Leishmania) infantum: ‘Leishman Donovan units’ versus real-time PCR. PLoS ONE 7, e47907.CrossRefGoogle Scholar
Moulik, S, Chaudhuri, SJ, Sardar, B, Ghosh, M, Saha, B, Das, NK and Chatterjee, M (2018) Monitoring of parasite kinetics in Indian post-kala-azar dermal leishmaniasis. Clinical Infectious Diseases 66, 404–410.CrossRefGoogle ScholarPubMed
Moulik, S, Karmakar, J, Joshi, S, Dube, A, Mandal, C and Chatterjee, M (2021a) Status of IL-4 and IL-10 driven markers in experimental models of visceral leishmaniasis. Parasite Immunology 43, e12783.CrossRefGoogle Scholar
Moulik, S, Sengupta, S and Chatterjee, M (2021b) Molecular tracking of the Leishmania parasite. Frontiers in Cellular and Infection Microbiology 11, 623437.CrossRefGoogle Scholar
Mukhopadhyay, D, Das, NK, De Sarkar, S, Manna, A, Ganguly, DN, Barbhuiya, JN, Maitra, AK, Hazra, A and Chatterjee, M (2012) Evaluation of serological markers to monitor the disease status of Indian post kala-azar dermal leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 106, 668–676.CrossRefGoogle ScholarPubMed
Pinheiro, LB, Coleman, VA, Hindson, CM, Herrmann, J, Hindson, BJ, Bhat, S and Emslie, KR (2012) Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Analytical Chemistry 84, 1003–1011.CrossRefGoogle ScholarPubMed
Rafati, S, Hassani, N, Taslimi, Y, Movassagh, H, Rochette, A and Papadopoulou, B (2006) Amastin peptide-binding antibodies as biomarkers of active human visceral leishmaniasis. Clinical and Vaccine Immunology 13, 1104–1110.CrossRefGoogle ScholarPubMed
Rochette, A, McNicoll, F, Girard, J, Breton, M, Leblanc, E, Bergeron, MG and Papadopoulou, B (2005) Characterization and developmental gene regulation of a large gene family encoding amastin surface proteins in Leishmania spp. Molecular and Biochemical Parasitology 140, 205–220.CrossRefGoogle ScholarPubMed
Saha, S, Mazumdar, T, Anam, K, Ravindran, R, Bairagi, B, Saha, B, Goswami, R, Pramanik, N, Guha, SK, Kar, S, Banerjee, D and Ali, N (2005) Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis. Journal of Clinical Microbiology 43, 1269–1277.CrossRefGoogle ScholarPubMed
Saini, S and Rai, AK (2020) Hamster, a close model for visceral leishmaniasis: opportunities and challenges. Parasite Immunology 42, e12768.CrossRefGoogle ScholarPubMed
Salotra, P, Sreenivas, G, Pogue, GP, Lee, N, Nakhasi, HL, Ramesh, V and Negi, NS (2001) Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. Journal of Clinical Microbiology 39, 849–854.CrossRefGoogle ScholarPubMed
Sengupta, S and Chatterjee, M (2021) IgG3 and IL10 are effective biomarkers for monitoring therapeutic effectiveness in post kala-azar dermal leishmaniasis. PLoS Neglected Tropical Diseases 15, e0009906.CrossRefGoogle ScholarPubMed
Sengupta, R, Chaudhuri, SJ, Moulik, S, Ghosh, MK, Saha, B, Das, NK and Chatterjee, M (2019) Active surveillance identified a neglected burden of macular cases of post kala-azar dermal leishmaniasis in West Bengal. PLoS Neglected Tropical Diseases 13, e0007249.CrossRefGoogle ScholarPubMed
Sharma, P, Gurumurthy, S, Duncan, R, Nakhasi, HL and Salotra, P (2010) Comparative in vivo expression of amastigote up regulated Leishmania genes in three different forms of leishmaniasis. Parasitology International 59, 262–264.CrossRefGoogle ScholarPubMed
Sudarshan, M, Weirather, JL, Wilson, ME and Sundar, S (2011) Study of parasite kinetics with antileishmanial drugs using real-time quantitative PCR in Indian visceral leishmaniasis. The Journal of Antimicrobial Chemotherapy 66, 1751–1755.CrossRefGoogle ScholarPubMed
Sundar, S, Singh, RK, Maurya, R, Kumar, B, Chhabra, A, Singh, V and Rai, M (2006) Serological diagnosis of Indian visceral leishmaniasis: direct agglutination test versus rK39 strip test. Transactions of the Royal Society of Tropical Medicine and Hygiene 100, 533–537.CrossRefGoogle ScholarPubMed
Sunter, J and Gull, K (2017) Shape, form, function and Leishmania pathogenicity: from textbook descriptions to biological understanding. Open Biology 7, 170165.CrossRefGoogle ScholarPubMed
Verma, S, Kumar, R, Katara, GK, Singh, LC, Negi, NS, Ramesh, V and 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.CrossRefGoogle ScholarPubMed
Vermeersch, M, da Luz, RI, Toté, K, Timmermans, JP, Cos, P and Maes, L (2009) In vitro susceptibilities of Leishmania donovani promastigote and amastigote stages to antileishmanial reference drugs: practical relevance of stage-specific differences. Antimicrobial Agents and Chemotherapy 53, 3855–3859.CrossRefGoogle ScholarPubMed
Yardley, V, Croft, SL, De Doncker, S, Dujardin, JC, Koirala, S, Rijal, S, Miranda, C, Llanos-Cuentas, A and Chappuis, F (2005) The sensitivity of clinical isolates of Leishmania from Peru and Nepal to miltefosine. The American Journal of Tropical Medicine and Hygiene 73, 272–275.CrossRefGoogle ScholarPubMed
Zijlstra, EE, Alves, F, Rijal, S, Arana, B and 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 Neglected Tropical Diseases 11, e0005877.CrossRefGoogle Scholar
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