Investigation of single nucleotide polymorphisms in MRPA and AQP-1 genes of Leishmania donovani as resistance markers in visceral leishmaniasis in Kenya

Anna Kapambwe Bwalya; Robinson Mugasiali Irekwa; Amos Mbugua; Matthew Mutinda Munyao; Peter Kipkemboi Rotich; Tonny Teya Nyandwaro; Caroline Wangui Njoroge; Anne Wanjiru Mwangi; Joanne Jepkemei Yego; Shahiid Kiyaga; Samson Muuo Nzou; Anna Kapambwe Bwalya; Robinson Mugasiali Irekwa; Amos Mbugua; Matthew Mutinda Munyao; Peter Kipkemboi Rotich; Tonny Teya Nyandwaro; Caroline Wangui Njoroge; Anne Wanjiru Mwangi; Joanne Jepkemei Yego; Shahiid Kiyaga; Samson Muuo Nzou

doi:10.3934/molsci.2021011

AIMS Molecular Science

2021, Volume 8, Issue 2: 149-160. doi: 10.3934/molsci.2021011

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Research article

Investigation of single nucleotide polymorphisms in MRPA and AQP-1 genes of Leishmania donovani as resistance markers in visceral leishmaniasis in Kenya

1.
Department of Molecular Biology and Biotechnology, Pan-African University Institute of Basic Sciences, Technology and Innovation, P.O. Box 62000-00200, Nairobi, Kenya
2.
Innovation Technology Transfer Division Department, Kenya Medical Research Institute, P.O. Box 54840-00200, Nairobi, Kenya
3.
Department of Medical Laboratory Sciences, College of Health Sciences, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200, Nairobi, Kenya
4.
International Center for Insect Physiology and Ecology, P.O. Box 30772-00100, Nairobi, Kenya
5.
Centre for Microbiology Research, Kenya Medical Research Institute, P.O. Box 54840-00200, Nairobi, Kenya

Received: 08 April 2021 Accepted: 22 June 2021 Published: 02 July 2021

Visceral Leishmaniasis (VL) remains a major public health problem mainly affecting the poorest populations across Asia, Africa, Middle East, Europe, Southern and Central America. For seven-decade now, the first-line drug of choice for leishmaniasis has been pentavalent antimonials. However, the clinical value of these drugs is threatened by the emergence of drug-resistant parasites. Clinical resistance to sodium stibogluconate (pentostam) has been a challenge in the Indian subcontinent, raising concerns for the endemic countries in Africa. This study aimed to identify and describe Single Nucleotide Polymorphism (SNPs) in gene markers associated with drug resistance among the clinical samples. The study was an experimental laboratory investigation on Dry Blood Spots (DBS). DNA was extracted from 18 VL positive samples, and Internal Transcribed Spacer-1 Polymerase Chain Reaction confirmed the positivity. Two target resistance markers, aquaglyceroporin 1 (AQP-1) and the Multi-Drug Resistant Protein A (MRPA), were PCR-amplified and resulting amplicons sequenced using the Sanger sequencing platform. Multiple sequence alignments were performed using ClustalW, and the phylogenetic tree was constructed in MegaX using the Maximum Likelihood method. A total of 84 SNPs in the AQP-1 gene were identified from six clinical samples. Fifty-nine of the SNPs (70.2%) were non-synonymous, while 25 (29.8%) were synonymous. Among the non-synonymous SNPs, three (5.1%) were nonsense, and 56 (94.9%) were missense point mutations. Two missense SNPs A188T and E185A in S17608 reported to be associated with drug resistance phenotype were observed. The study describes the resistance associated with the pentostam uptake by Leishmania donovani.
- antimony sodium stibogluconate,
- visceral leishmaniasis,
- single nucleotide polymorphism,
- drug resistance,
- aquaglyceroporin
Citation: Anna Kapambwe Bwalya, Robinson Mugasiali Irekwa, Amos Mbugua, Matthew Mutinda Munyao, Peter Kipkemboi Rotich, Tonny Teya Nyandwaro, Caroline Wangui Njoroge, Anne Wanjiru Mwangi, Joanne Jepkemei Yego, Shahiid Kiyaga, Samson Muuo Nzou. Investigation of single nucleotide polymorphisms in MRPA and AQP-1 genes of Leishmania donovani as resistance markers in visceral leishmaniasis in Kenya[J]. AIMS Molecular Science, 2021, 8(2): 149-160. doi: 10.3934/molsci.2021011

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Abstract

Visceral Leishmaniasis (VL) remains a major public health problem mainly affecting the poorest populations across Asia, Africa, Middle East, Europe, Southern and Central America. For seven-decade now, the first-line drug of choice for leishmaniasis has been pentavalent antimonials. However, the clinical value of these drugs is threatened by the emergence of drug-resistant parasites. Clinical resistance to sodium stibogluconate (pentostam) has been a challenge in the Indian subcontinent, raising concerns for the endemic countries in Africa. This study aimed to identify and describe Single Nucleotide Polymorphism (SNPs) in gene markers associated with drug resistance among the clinical samples. The study was an experimental laboratory investigation on Dry Blood Spots (DBS). DNA was extracted from 18 VL positive samples, and Internal Transcribed Spacer-1 Polymerase Chain Reaction confirmed the positivity. Two target resistance markers, aquaglyceroporin 1 (AQP-1) and the Multi-Drug Resistant Protein A (MRPA), were PCR-amplified and resulting amplicons sequenced using the Sanger sequencing platform. Multiple sequence alignments were performed using ClustalW, and the phylogenetic tree was constructed in MegaX using the Maximum Likelihood method. A total of 84 SNPs in the AQP-1 gene were identified from six clinical samples. Fifty-nine of the SNPs (70.2%) were non-synonymous, while 25 (29.8%) were synonymous. Among the non-synonymous SNPs, three (5.1%) were nonsense, and 56 (94.9%) were missense point mutations. Two missense SNPs A188T and E185A in S17608 reported to be associated with drug resistance phenotype were observed. The study describes the resistance associated with the pentostam uptake by Leishmania donovani.

Abbreviations

AQP-1

Aquaglyceroporin 1

Cutaneous Leishmaniasis

DBS

Dry Blood Spot

LdAQP-1

Leishmania donovani Aquaglyceroporin 1

MCL

Mucocutaneous Leishmaniasis

MRPA

Multi-Drug Resistant Protein A

PCR

Polymerase Chain Reaction

SSG

Sodium Stibogluconate

Visceral Leishmaniasis

Acknowledgments

The African Union Commission supported this study through the Pan African University Institute for Basic Sciences, Technology, and Innovation (PAUSTI); and the molecular epidemiology project, KEMRI. In addition, the authors appreciate the infrastructural support by the Nagasaki University Institute of Tropical Medicine- Kenya Medical Research Institute project (NUITM-KEMRI). We also appreciate the efforts of Jomo Kenyatta University for Agriculture and Technology (JKUAT) and Pan African University Institute for Basic Sciences, Technology and Innovation (PAUSTI) lecturers for their invaluable input in this study.

Conflict of interest

The authors declare there was no conflict of interest.

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