Molecular analysis of two local falciparum malaria outbreaks on the French Guiana coast confirms the msp1 B-K1/varD genotype association with severe malaria
© Legrand et al; licensee BioMed Central Ltd. 2005
Received: 30 March 2005
Accepted: 21 June 2005
Published: 21 June 2005
Plasmodium falciparum outbreaks can occur in the coastal area of French Guiana, where the population is essentially non-immune. Two sporadic outbreaks were observed, including one with severe malaria cases. To characterize these outbreaks and verify previous observations of specific genotype characteristics in severe malaria in this area, all cases from each outbreak were studied.
P. falciparum genotypes for six genetic loci were determined by PCR amplification from peripheral blood parasites. The msp1/ block2 and msp2 genotypes were determined by DNA sequencing. Microsatellite and varD genotyping was based on size polymorphism and locus-specific amplification.
The outbreak including severe malaria cases was associated with a single genotype. The other mild malaria outbreak was due to at least five distinct genotypes.
Two distinct types of outbreak occured despite systematic and sustained deployement of malaria control measures, indicating a need for reinforced vigilance. The varD/B-K1 msp1 linkage and its association with severe malaria in this area was confirmed.
The annual malaria incidence in French Guiana has increased ten-fold during the last 30 years, reaching nowadays approximately 3%, with 60% due to Plasmodium falciparum and 40 % due to Plasmodium vivax. French Guiana is an area with multidrug resistant P. falciparum malaria. Transmission occurs in isolated foci located in settlement pockets within the Amazonian forest and along the rivers. The main malaria-endemic areas are located along the Maroni and Oyapock rivers, which serve as natural frontiers with Surinam and Brazil, respectively . As a result of three-monthly insecticide spraying campaigns, there are virtually no malaria cases along the coast, where 80% of the population resides. However, outbreaks, which usually are of short duration and affect a small number of patients, are occasionally reported in the coastal area.
The P. falciparum parasite population of French Guiana presents a remarkably low degree of polymorphism, with a clonal type structure . The parasite diversity is so restricted that the circulation of specific genotypes could be followed within the area, making it possible to investigate the clinical impact of specific parasite genotypes. A comparative analysis of parasite genetic characteristics in isolates collected from mild and severe malaria patients has highlighted a significant linkage disequilibrium between a particular msp1/block2 allele that was called B-K1 and a particular var gene called varD in isolates from patients with severe P. falciparum malaria .
To verify this association and better understand the possible cause of local outbreaks in the coastal area located away from the main endemic sites, two P. falciparum outbreaks were studied, one with five cases, including two cases of severe malaria and one death, the other with nine mild malaria cases. The isolates from each case were characterized for both outbreaks using a six-locus genotyping approach, including varD, msp1/block2, msp2 [2–7], as well as three microsatellite loci .
Parasite isolates and patient information
DNA extraction and genotyping
Microsatellite typing was done using the C1M4, C3M27 and C4M69 loci, located on chromosomes 1, 3 and 4, respectively. The locus-specific primers described by Su et al.  were used (see Figure 2). Amplification was done in 15 μL reaction volume containing the DNA template, 1.5 mM MgCl2, 2 μM each primer, 200 μM each dNTP, and 0.5 Units Taq polymerase (Promega, Madison, WI, USA). The samples were subjected to 1 cycle at 94°C for 7 min, followed by 35 cycles at 94°C for 30 sec, 52°C for 30 sec, 47°C for 30 sec, 72°C for 30 sec, and a final extension step at 72°C for 15 min. The PCR products were analysed by 3% Metaphor agarose gel (FMC Bioproduct, Rockland, Maine, USA) electrophoresis and stained with ethidium bromide. Fragment size was calculated using the Taxotron software (P.A.D. Grimont, Institut Pasteur, Paris).
Results and discussion
To further explore the genetic make-up of the P. falciparum isolates, three microsatellite loci were studied. A single 140-bp C1M4 product was observed for all isolates, except F79, F89 and F113, where no band was visualized (Fig. 3). A 150-bp C3M27 product was amplified from all Macouria isolates and from the F33, F40, F52, F53, F96 and F105 Matoury isolates. A distinct 130-bp C3M27 allele was detected in isolates F79, F89 and F113. For the C4M69 locus, a single 520-bp band was obtained for all Macouria isolates, a 430-bp band for Matoury isolates F33, F40, F52, F53, F96 and F105 and a 360-bp fragment for the Matoury isolates F79 and F89. No product was obtained for this locus from isolate F113 (Fig. 3). This data indicated that for all five single copy loci investigated, all isolates generated a single, monomorphic PCR fragment. We can thus reasonably consider them as being monoclonal infections. This confirms previous findings in this area, where a very high percentage of single infections was observed [2, 3], and is in line with reports from other areas of South America [5, 6].
French Guiana is a hypoendemic area, where the population has limited, if any, immunity. There are between 6,000–7,000 mild malaria cases and only between four and 20 severe malaria cases each year [1, 3]. Previous studies have highlighted specific genotype characteristics in P. falciparum isolates from severe cases in this setting, with the B-K1 msp1 allele in strong linkage disequilibrium with a particular var gene, called varD . The var genes code for the variant adhesins that mediate the cytoadhesion of parasitized erythrocytes to specific host receptors that undoubtedly contribute to severe malaria. As highlighted in Figure 3, all five Macouria isolates, including the two severe cases, had the same 5-loci genotype, suggesting that a single virulent isolate was present. These isolates were also tested fo the presence of the varD gene. A typical varD product was amplified from all five Macouria isolates. Only two Matoury isolates (isolates F33 and F52) carried the varD locus, none of which harboured the B-K1 msp1 allele (Fig. 3). Overall, the same genotype was observed in all Macouria isolates while there were at least five distinct genotypes in Matoury. The Matoury 1 and 2 genotypes differed at the varD locus. The Matoury 2 and Matoury 3 genotypes had a different msp2 allele. Furthermore, isolates F40 and F96 had a different drug susceptibility profile (data not shown). The Matoury 4 and Matoury 5 genotypes differed by the C4M69 microsatellite locus.
These data confirm the varD/B-K1 msp1 linkage and its association with severe malaria in this area. It is important to note that this association was previously detected in isolates collected in 1994-6, more than six years before the Macouria epidemic. Such a stability over time in a species with a high recombination rate  is consistent with previous data pointing to a high selfing rate in this area . The severe malaria cases studied here originated from a geographic area quite distinct from the previous cases reported where the varD/B-K1 association was observed, as illustrated in Figure 1. As observed previously , the varD/B-K1 msp1 association was not strictly specific for severe malaria, as the same genotype was also observed in the non-severe Macouria cases. Whether the patients with mild malaria received an earlier treatment compared to those with severe malaria or were less susceptible due to their genetic or immune make up is unclear. The occurrence of two severe cases at one day interval in patients infected with this strain suggests a particular inherent virulence. Whether this is due to the particular msp1 and/or varD allele present is unknown. It may reflect physical association with another locus implicated in virulence. It is worth noting that isolate F52, which harboured varD together with the A-K1 msp1 allele, was collected from a patient with very high peripheral parasitaemia (20%), also considered a sign of severity . VarD is one of approximately 60 members of the P. falciparum var genomic repertoire . The presence of a particular var gene is not synonymous with its expression. Expression of varD was demonstrated in a patient with fatal P. falciparum malaria in a previous study , but could not be studied here. The data indicate that future investigations on varD expression in severe and non-severe malaria are warranted. Work is in progress to characterize the full varD gene sequence.
The different genetic profiles of the isolates involved in the two outbreaks reveal distinct onset and dynamics scenarios. Genotyping strongly suggests that the Macouria outbreak was due to one single parasite strain, but the origin of this strain is uncertain. Transmission from E57, the first registered case, to the other cases is unlikely, due to the short delay between the clinical attack and the attack experienced by E61, E62 and E64 (12, 13 and 20 days later, respectively). E67 may possibly have subsequently transmitted the strain to E72, who experienced a malaria attack 37 days later. The diversity of the Matoury isolates indicates that the outbreak was certainly caused by several distinct strains. Matoury accommodates the largest airport in the country and consequently may serve as an occasional transmission focus for parasites originating in neighbouring endemic malaria areas. The possible cause and mode of transmission in this city at that time have not been identified.
The results point to two distinct types of outbreak in a region where malaria control measures are systematically deployed and sustained. Reinforced vigilance and rapid case notification are needed to ensure rapid deployment of vector control and personal protection measures to prevent such sporadic epidemics. The Macouria outbreak provoked two severe cases, including one death, a rare event in this area, where health facilities are well-equipped and treatment policy is regularly updated. Parasite genotyping confirmed the association of the B-K1 msp1/varD genotype with severe malaria, reinforcing the notion that some P. falciparum strains might cause more severe infections than others.
This project was supported by the French Ministry of Health (InVS agency, Paris) and the French Academy of Sciences (Prix Louis D., 2000). The authors thank Prof. B. Carme and colleagues from the Parasitology Unit of the Central Hospital, Cayenne and Drs. P. Duval and B. Maubert, from the Biomedical Unit of the Institut Pasteur de Guyane, Cayenne for providing the blood samples.
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