Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparumparasites in a Brazilian endemic area

De, Gama Bianca, Zalis, Santos Fátima, Daniel-Ribeiro Cláudio, Ferreira-Da-Cruz, Ferreira-Da-Cruz Maria

Description

The goal of the present study was the characterization of Plasmodium falciparum genes associated to malaria drug resistance ( pfcrt , pfdhfr and pfdhps) , in samples from two Brazilian localities. Methods Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct DNA sequencing. Results Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from Paragominas, that showed a SP-sensitive genotype. Conclusion This study is the first to document the sensitivity of P. falciparum parasites to CQ and SP in Brazilian field samples. The importance of these findings is discussed.

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• Publié par: biomed
• Publié le: 01 janvier 2009
• Nombre de lectures: 26

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BioMed CentralMalaria Journal
Open AccessResearch
Chloroquine and sulphadoxine-pyrimethamine sensitivity of
Plasmodium falciparum parasites in a Brazilian endemic area
1 1 2Bianca Ervatti Gama , Natália K Almeida de Oliveira , Mariano G Zalis ,
3 4 1José Maria de Souza , Fátima Santos , Cláudio Tadeu Daniel-Ribeiro and
1Maria de Fátima Ferreira-da-Cruz*
1 2Address: Laboratory of Malaria Research, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro (RJ), Brazil, Laboratory of Infectiology and
3Parasitology, Hospital Universitário Clementino Fraga Filho, Federal University of Rio de Janeiro, Rio de Janeiro (RJ), Brazil, Ambulatory and
4Laboratory of Malaria Clinical Assays, Secretariat of Health Vigilance, Instituto Evandro Chagas, Belém (PA), Brazil and Laboratory of
Entomology, LACEN, Porto Velho (RO), Brazil
Email: Bianca Ervatti Gama - [e-mail]; Natália K Almeida de Oliveira - [e-mail];
Mariano G Zalis - [e-mail]; José Maria de Souza - [e-mail]; Fátima Santos - [e-mail];
Cláudio Tadeu Daniel-Ribeiro - [e-mail]; Maria de Fátima Ferreira-da-Cruz* - [e-mail]
* Corresponding author
Published: 14 July 2009 Received: 3 March 2009
Accepted: 14 July 2009
Malaria Journal 2009, 8:156 doi:10.1186/1475-2875-8-156
This article is available from: http://www.malariajournal.com/content/8/1/156
© 2009 Gama et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: The goal of the present study was the characterization of Plasmodium falciparum
genes associated to malaria drug resistance (pfcrt, pfdhfr and pfdhps), in samples from two Brazilian
localities.
Methods: Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct
DNA sequencing.
Results: Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype
associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant
genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from
Paragominas, that showed a SP-sensitive genotype.
Conclusion: This study is the first to document the sensitivity of P. falciparum parasites to CQ and
SP in Brazilian field samples. The importance of these findings is discussed.
The first documented case of CQ resistance in Brazil wasBackground
Malaria is a challenging infectious disease to many coun- in 1954; in the sixties several authors in South America
tries in the world, especially to those located in tropics (SA) reported the occurrence of in vivo falciparum malaria
and subtropical regions, and the increasing numbers of resistance to CQ and amodiaquine. The spread of 4-
drug resistant parasites worsens this situation. In Brazil, as amino-quinoleine resistant strains of P. falciparum lead
well as in other endemic countries, drug-sensitivity tests also to the use of SP in the seventies. Unfortunately, at the
revealed that Plasmodium falciparum isolates displayed end of eighteens the SP resistance it was no less than 90%
high levels of resistance to many drugs, including chloro- [5] and at the beginning of nineties the CQ failure rate was
quine (CQ) and sulphadoxine-pyrimethamine (SP) [1-4]. considered 100% [6] and, consequently, the Brazilian
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National Malaria Programme withdrew SP and CQ for fal- systems), and 10 pmol of each primer (Alpha DNA):
ciparum malaria treatment. Then, the combination qui- CRTP1 (5' CCG TTA ATA ATA AAT ACA CGC AG 3') and
CRTP2 (5' CGG ATG TTA CAA AAC TAT AGT TAC C 3') tonine plus tetracycline was introduced, followed by the
usage of the combination quinine plus doxycycline or, as amplify a 537 bp region. The reactions were settled with
second-line drug, mefloquine plus primaquine. In 2007, an initial hold (95°C/10 minutes), 45 cycles (94°C/30
a fixed combination of artemether plus lumefantrine seconds, 56°C/30 seconds and 60°C/1 minute) plus one
®(Coartem ) was introduced as first-line drug [7] and since final hold (60°C/3 minutes). Then, 5 μl of the first PCR
2008, the fixed combination artesunate plus mefloquine amplification were added to 45 μl of a second mixture
(FarManguinhos, Fiocruz) was produced, and its imple- containing the second set of primers (Alpha DNA):
mentation in Brazilian endemic areas is in progress to CRTD1 (5' TGT GCT CAT GTG TTT AAA CTT 3') and
counteract parasite resistance, according to WHO guide- CRTD2 (5' CAA AAC TAT AGT TAC CAA TTT TG 3') and
lines [8]. 32 steps (1 hold 95°C/10 min, 30 cycles 92°C/30 sec,
48°C/30 sec and 65°C/30 sec, and 1 hold 65°C/3 min)
Interestingly, the reemergence of CQ-sensitive P. falci- were performed to amplify a 134 bp fragment. PCR prod-
parum parasites as well as the downturn of P. falciparum ucts were analysed by ethidium bromide-stained agarose-
triple mutants associated to SP resistance, were reported gel (2%) electrophoresis.
after cessation of monotherapy using CQ or SP for the
treatment of P. falciparum malaria [9-13]. These findings DNA sequencing
provide a rationale for the search of drug-sensitive haplo- After DNA purification using the Wizard SV Gel and PCR
types in P. falciparum isolates in Brazilian areas where the Clean-Up System (Promega), the amplified fragments
® use of these two drugs for falciparum malaria treatment were sequenced using Big Dye Terminator Cycle Sequenc-
has been interrupted since 1990. ing Ready Reaction version 3.1 (Applied Biosystems) and
ABI PRISM DNA Analyzer 3730 (Applied Biosystems)
The goal at the present study was, therefore, the character- from the Genomic Platform/PDTIS/Fiocruz [16].
ization of pfcrt, pfdhfr and pfdhps genes that are known
molecular markers of P. falciparum resistance to CQ and Results
The pfcrt nested-PCR generated DNA fragments in all theSP [14].
65 samples, the pfdhps in 63 samples and the pfdhfr
nested-PCR in 52 samples, showing different sensitivityMethods
Study sites, blood samples and DNA extraction threshold among the PCRs.
Parasites from 65 P. falciparum samples were genotyped.
These samples were collected at the time of diagnosis from The pfdhfr gene analysis revealed the existence of seven
uncomplicated malaria patients living in two Brazilian haplotypes: six associated with drug resistance profiles
malaria-endemic areas: Porto Velho – Rondônia state (n = (ARICNVI, ACICNVI, ACICNVL, ACNRNVI, ACNCNVI
46), and Paragominas – Pará state (n = 19). After obtain- and ACIRNVI) and one, from Paragominas locality, asso-
ing the informed consent, venous blood collection was ciated with SP sensitivity (ACNCSVI). The drug resistance
done according to protocols previously approved by the pfdhfr haplotypes displayed up to three (50R + 51I and
ethics research committees of Fiocruz and of local study 108N; 51I + 108N and 164L, or 51I + 59R and 108N) out
sites. DNA was extracted from 1 ml of cryopreserved of seven SNPs herein analysed. Concerning the pfdhps
blood using QIAamp midi columns as described by the gene, three single haplotypes (SGEGA, SGEAA, SGKAA),
manufacturer (Qiagen). displaying up to three (437G + 540E and 581G) out of the
five SNPs analysed were observed and, in this case, no
Polymerase chain reactions (PCRs) and electrophoresis drug sensitive haplotype (SAKAA) was detected. In rela-
A nested-PCR technique was employed in order to tion to pfcrt gene, two single haplotypes were observed:
amplify a partial DNA sequence containing the major sin- the SVMNT CQ-resistant in 97% of the samples and the
gle-nucleotide polymorphisms (SNPs) related to drug- CVMNK CQ-sensitive in the same sample from Paragom-
resistance for each target gene as: SNPs A16V/S, C50R, inas that showed the sensitive genotype to SP (pfdhfr
N51I, C59R, S108N, V140L plus I164L for pfdhfr, S436A/ gene). All the three genes displayed at least one mixed
F/C, A437G, K540E, A581G plus A613T/S for pfdhps, and haplotype. These results are summarized in Table 1.
SNPs C72S, M74I, N75E/D plus K76T for pfcrt. The proto-
cols of pfdhfr and pfdhps nested PCRs have been described A multilocus analysis among the 47 samples showing sin-
elsewhere [15]. The amplification of the pfcrt gene frag- gle haplotypes was performed. The pfdhfr + pfdhps + pfcrt
ment was performed with 5 μl of DNA into a 45 μl mix- genes presented some usual haplotype associations as,
ture containing 2.5 mM of MgCl , 250 μM of dNTPs, 1.25 respectively, follows: ARICNVI + SGEGA + SVMNT2
® units of Amplitaq Gold DNA polymerase (Applied Bio- (42.5%), ACICNVL + SGEGA + SVMNT (27.6%), ARIC-
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Table 1: Pfcrt, pfdhfr and pfdhps haplotypes of P. falciparum parasites from Paragominas (PRG) and Porto Velho (PV), Brazilian
Amazon.
Gene Haplotypes n (Locality) % Mutated codons
pfcrt SVMNT 63 (18 PRG and 45 PV) 97 2
CVMNK 1 (PRG) 1.5 0
C/S VMNT 1 (PV) 1.5 1 or 2
pfdhfr A