African Journal of Malaria and Tropical Diseases

African Journal of Malaria and Tropical Diseases ISSN 4123-0981 Vol. 8 (2), pp. 001-006, February, 2020. © International Scholars Journals

Full Length Research Paper

Mutations within folate metabolising genes of Plasmodium falciparum in Cameroon

Wilfred Fon Mbacham¹*, Marie-Solange Bebandoue Evehe¹, Palmer Masumbe Netongo¹, Innocent Mbuli Ali.¹, Nfor Emmanuel Nfor³, Ateh Isabel Akaragwe², Patrice Nsangou Mimche ¹, Akindeh Nji⁴ and Cyrille Finyom Djoko

¹The Biotechnology Center, University of Yaounde I, Cameroon, P. O. Box 8094, Yaounde, Cameroon

²Biotechnology Unit, University of Buea , Cameroon.

³Department of Animal Biology and Physiology, University of Yaounde I, Cameroon.

⁴Bota District Hospital, Limbe South West Province, Cameroon.

Accepted 20 November, 2019

Abstract

Sulfadoxine-Pyrimethamine (S-P) still used in some parts of the country was suggested as the second line drug to amodiaquine following widespread failure of chloroquine in Cameroon in 2002. We investigated the efficacy of S-P and determined the baseline mutations on marker genes for folate metabolism (dhfr and dhps) in the forest and Guinea-Savanna ecozones of Cameroon, as a way of tracking resistance in patients aged between 0.5 and 10 years in Limbe (n=138), Nkambe (n=103), Fontem (n=100 ) and Dschang (n=93 ). Filter paper blood sample were collected prior to treatment and on clinical failure days to determine the prevalence of molecular markers of resistance and to assess the mutation rates on the folate metabolising genes by restriction fragment length polymorphism assays or dot-blot assays with 32-P labeled mutation-specific probes. Sequencing using the dideoxy- chain termination method by PCR was conducted to confirm doubtful cases. Late parasitological failure (LPF) was higher in Limbe (30.6%) compared to Nkambe 10.3% (p=0.001). The prevalence of the 437-Gly mutation though lower in Nkambe, 57.6%, than in Limbe, 60% were statistically not different (p=0.2). All genotypes with the 108N mutation also carried the 51-Ile and 59-Arg mutations. All sensitive alleles (S108) also carried the amino acids, 51-Asn and 59-Cys. S-P is no longer efficacious in Limbe and Nkambe, Cameroon for treating uncomplicated malaria in children below 10 years. Instead 437G rather than the 108 N of Plasmodium falciparum may be determinant as the marker for tracking the spread of S-P resistance in Cameroon.

Key words: Mutations, plasmodium, sulfadoxine-pyrimethamine, resistance, dhfr, dhps

African Journal of Malaria and Tropical Diseases ISSN 4123-0981 Vol. 8 (1), pp. 001-010, January, 2020. © International Scholars Journals

Full Length Research Paper

The Hsp40-Hsp70 chaperone machinery of Plasmodium falciparum

Eva-Rachele Pesce* and Gregory L. Blatch

Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa.

Accepted 14 December, 2019

Abstract

Plasmodium falciparum is the protozoan parasite responsible for the most virulent form of malaria. The majority of the asexual stages of its life cycle occur in the human erythrocyte. Since the infected erythrocytes undergo dramatic structural and functional changes upon parasite infection, malaria research has been focusing on investigating the proteins potentially involved in host cell modifications. Molecular chaperones are believed to play an important role in erythrocyte remodelling as many of these proteins are predicted to be exported into the erythrocyte cytoplasm. A family of molecular chaperones that has recently received much attention is the heat shock protein family (Hsps), and in particular members belonging to the 40 and 70 kDa heat shock proteins classes (Hsp40s and Hsp70s). This review summarises the latest in silico and in vivo data available on P. falciparum Hsp40s and Hsp70s.

Key words: Plasmodium falciparum, malaria, Hsp40s, Hsp70.

African Journal of Malaria and Tropical Diseases ISSN 4123-0981 Vol. 8 (1), pp. 001-007, January, 2020. © International Scholars Journals

Full Length Research Paper

Optimization of a protocol for extraction of Plasmodium falciparum RNA from infected whole blood samples for use in DNA microarrays

Jaffu Chilongola¹*, Sakurani Balthazary² and Erasto Mbugi³

¹Faculty of Medicine, Kilimanjaro Christian Medical College, Tumaini University, Tanzania.

²Department of, Physiology, Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, Tanzania.

³Department of Biochemistry, School of Medicine, Muhimbili University of Health and Allied  Sciences (MUHAS), P.O. Box 65001, Dar es Salaam, Tanzania.

Accepted 28 October, 2019

Abstract

This study was carried out to determine the efficiency of two reagents, RNAlater and RNAwiz, for their ability to stabilize Plasmodium falciparum RNA in infected whole blood and saponin lysed parasite pellets for use in DNA microarrays. Eight infected blood samples were stored in each of the reagents, and RNA extracted at days 0 and 56 post collection. RNA yields and quality were compared at the different time points between the two test reagents. We show that for both reagents, higher RNA yields and quality is obtained when RNA is isolated immediately after sample collection (day 0), however, results show that RNAwiz storage provides a marginally higher RNA yield compared to RNAlater storage. Our results indicate that whole blood gave slightly higher RNA yields with superior quality as compared to saponin lysed samples when such whole blood samples are stored in RNA wiz, but not in RNAlater. From our results, we recommend RNAwiz as a better reagent for use in storage of whole infected blood intended for extraction of P. falciparum RNA for DNA microarrays and other sensitive techniques.

Key words: RNAwiz, RNAlater, RNA extraction, Plasmodium falciparum, DNA microarrays.

African Journal of Malaria and Tropical Diseases ISSN 4123-0981 Vol. 8 (1), pp. 001-011, January, 2020. © International Scholars Journals

Full Length Research Paper

Limited variation of the 5’cis-control region of the transmission blocking vaccine candidate Pfs25 amid great genetic diversity of Plasmodium falciparum in Cameroon

Wilfred Fon Mbacham¹*, Patrice Nsangou Mimche¹, Palmer Masumbe Netongo¹, Evehe Bebandoue Marie-Solange¹, Akindeh Nji¹, Immaculate Amunom¹, Johanna Daily², Valerie Makoge¹, Kayla Laserson^2, Songmbe Michael Yong³, Nicoline Lomah³, Peter Enyong⁴, Vincent P. Titanji³ and Dyann F. Wirth²

¹The Biotechnology Center, University of Yaounde I, Cameroon.

²Department of Immunology and Infectious Diseases, Harvard School of Public Health, U.S.A.

³Biotechnology Unit, University of Buea, Cameroon.

⁴Center for Medical and Medicinal Plant Research, Ministry of Scientific Research, Kumba, SWP, Cameroon.

Accepted 16 September, 2019

Abstract

Genetic recombination during sexual reproduction within Plasmodium sp. contributes to parasite diversity and altered gene expression of certain surface markers. The pfs25 gene involved in the upset of gametocytogenesis is a candidate antigen in transmission blocking vaccine. This study investigated the polymorphism of Pfs25 within its 5’cis-control region in field isolates from different ecotypes in Cameroon. Symptomatic patients and asymptomatic healthy school children with a positive smear and from different ecozones were included. Parasite DNA was extracted and polymorphisms within pfs25, cg2-ω, msp-1, msp-2 and glurp genes were investigated by PCR-RFLP and DNA sequencing. Putative control elements of the 5’cis control regions of Pfs25 were identified by PCGENE software and enzymes were selected whose sequences produced or abolished restriction sites by mutations. Malaria infection was mainly caused by Plasmodium falciparum with sporadic occurrence of Plasmodium malariae and Plasmodium ovale. Analysis of the Pfs25 5’ cis-control region identified only one polymorphism (0.002%) that abolished an RsaI restriction site as part of the sequence TTTCTGTAC, located 40 bp downstream of the promoter and found at – 478 bp of the ATG. Analysis of the 5’ cis-control sequence of Pfs25 revealed minimal variation of the promoter region amid great zonal differences in parasite population. Altitudinal differences in parasite populations were not easily discernable.

Key words: Plasmodium falciparum, Pfs25, cis-control elements, genetic polymorphism.