Michela Menegon

Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction

A molecular assay has been developed for the specific detection and genetic characterisation of Plasmodium falciparum gametocytes in the blood of malaria infected individuals. The assay is based on the reverse transcription and polymerase chain reaction (RT-PCR) amplification of the messenger RNA of gene pfg377, a sexual-stage specific transcript abundantly produced in maturing gametocytes. The gene contains four regions of repetitive sequences, of which region 3 was shown to be the most polymorphic in laboratory clones and field isolates of the parasite. Analysis of samples of malaria infected blood by RT-PCR specific for region 3 has enabled identification of multiple gametocyte-producing clones within single infections. The assay is able to detect gametocytes below the threshold of microscopic detection, and is highly specific for its gametocyte targets also in the presence of a vast excess of asexual forms.

Genetic diversity of Plasmodium vivax isolates from Azerbaijan

BackgroundPlasmodium vivax, although causing a less serious disease than Plasmodium falciparum, is the most widespread of the four human malarial species. Further to the recent recrudescence of P. vivax cases in the Newly Independent States (NIS) of central Asia, a survey on the genetic diversity and dissemination in Azerbaijan was undertaken. Azerbaijan is at the crossroads of Asia and, as such, could see a rise in the number of cases, although an effective malaria control programme has been established in the country.MethodsThirty-six P. vivax isolates from Central Azerbaijan were characterized by analysing the genetic polymorphism of the circumsporozoite protein (CSP) and the merozoite surface protein 1 (MSP-1) genes, using PCR amplifications and amplicons sequencing.ResultsAnalysis of CSP sequences showed that all the processed isolates belong to the VK 210 type, with variations in the alternation of alanine residue (A) or aspartic acid residue (D) in the repeat motif GDRA(A/D)GQPA along the sequence. As far as MSP-1 genotyping is concerned, it was found that the majority of isolates analysed belong to Belem and Sal I types. Five recombinant isolates were also identified. Combined analysis with the two genetic markers allowed the identification of 19 plasmodial sub-types.ConclusionThe results obtained in the present study indicate that there are several P. vivax clones circulating in Azerbaijan and, consequently, a careful malaria surveillance could be of paramount importance to identify, at early stage, the occurrence of possible P. vivax malaria outbreaks.

Plasmodium vivax Diversity and Population Structure across Four Continents

Plasmodium vivax is the geographically most widespread human malaria parasite. To analyze patterns of microsatellite diversity and population structure across countries of different transmission intensity, genotyping data from 11 microsatellite markers was either generated or compiled from 841 isolates from four continents collected in 1999–2008. Diversity was highest in South-East Asia (mean allelic richness 10.0–12.8), intermediate in the South Pacific (8.1–9.9) Madagascar and Sudan (7.9–8.4), and lowest in South America and Central Asia (5.5–7.2). A reduced panel of only 3 markers was sufficient to identify approx. 90% of all haplotypes in South Pacific, African and SE-Asian populations, but only 60–80% in Latin American populations, suggesting that typing of 2–6 markers, depending on the level of endemicity, is sufficient for epidemiological studies. Clustering analysis showed distinct clusters in Peru and Brazil, but little sub-structuring was observed within Africa, SE-Asia or the South Pacific. Isolates from Uzbekistan were exceptional, as a near-clonal parasite population was observed that was clearly separated from all other populations (F ST>0.2). Outside Central Asia F ST values were highest (0.11–0.16) between South American and all other populations, and lowest (0.04–0.07) between populations from South-East Asia and the South Pacific. These comparisons between P. vivax populations from four continents indicated that not only transmission intensity, but also geographical isolation affect diversity and population structure. However, the high effective population size results in slow changes of these parameters. This persistency must be taken into account when assessing the impact of control programs on the genetic structure of parasite populations.

Monitoring for multidrug‐resistant Plasmodium falciparum isolates and analysis of pyrimethamine resistance evolution in Uige province, Angola

Objectives  To assess the extent of drug resistance in Uige through molecular genetic analysis and to test whether the dhfr triple mutant alleles present in Angola are of southeast Asian origin.

A Quality Control Program within a Clinical Trial Consortium for PCR Protocols To Detect Plasmodium Species

ABSTRACT Malaria parasite infections that are only detectable by molecular methods are highly prevalent and represent a potential transmission reservoir. The methods used to detect these infections are not standardized, and their operating characteristics are often unknown. We designed a proficiency panel of Plasmodium spp. in order to compare the accuracy of parasite detection of molecular protocols used by labs in a clinical trial consortium. Ten dried blood spots (DBSs) were assembled that contained P. falciparum, P. vivax, P. malariae, and P. ovale; DBSs contained either a single species or a species mixed with P. falciparum. DBS panels were tested in 9 participating laboratories in a masked fashion. Of 90 tests, 68 (75.6%) were correct; there were 20 false-negative results and 2 false positives. The detection rate was 77.8% (49/63) for P. falciparum, 91.7% (11/12) for P. vivax, 83.3% (10/12) for P. malariae, and 70% (7/10) for P. ovale. Most false-negative P. falciparum results were from samples with an estimated ≤5 parasites per μl of blood. Between labs, accuracy ranged from 100% to 50%. In one lab, the inability to detect species in mixed-species infections prompted a redesign and improvement of the assay. Most PCR-based protocols were able to detect P. falciparum and P. vivax at higher densities, but these assays may not reliably detect parasites in samples with low P. falciparum densities. Accordingly, formal quality assurance for PCR should be employed whenever this method is used for diagnosis or surveillance. Such efforts will be important if PCR is to be widely employed to assist malaria elimination efforts.

Effects of Mefloquine Use on Plasmodium vivax Multidrug Resistance

Use of mefloquine against P. falciparum jeopardizes its future use against P. vivax.

Frequency distribution of antimalarial drug resistance alleles among Plasmodium falciparum isolates from Gezira State, Central Sudan, and Gedarif State, Eastern Sudan.

In 2004, Sudan adopted artesunate + sulfadoxine/pyrimethamine (SP) combination as the first-line drug, in response to the high level of falciparum resistance to antimalarials. In 2007, a molecular study on antimalarial resistance linked genes, pfcrt, pfmdr1, pfdhfr, pfdhps, and pfATPase6, was conducted on 198 isolates from central and eastern Sudan. We observed a high frequency of point mutations at almost all loci analyzed, mainly of pfcrt 76T (72.7%), pfdhfr 51I (75.3%), and pfdhfr 108N (72.7%) alleles. The MARK III in vitro test for chloroquine sensitivity in 45 P. falciparum isolates showed that 37.8% of the isolates were low resistant and 6.7% were fully resistant. This study represents the most recent molecular investigation on antimalarial resistance in this area after the adoption of artemisinin-based combination therapy (ACT), and underlines the importance of the analysis of SP resistance evolution to monitor the efficacy of ACT therapy in endemic areas.

Genetic Confirmation of Quinine-Resistant Plasmodium falciparum Malaria Followed by Postmalaria Neurological Syndrome in a Traveler from Mozambique

ABSTRACT A case of quinine-resistant Plasmodium falciparum malaria, followed by a postmalaria neurological syndrome and a recurrence episode, is described. Genetic characterization of the P. falciparum isolate obtained by analysis of msp1 and msp2 amplicons revealed the coexistence of two genotypes causing the first malaria episode and the presence of a unique isolate responsible for the recurrence.

Use of the Plasmodium vivax merozoite surface protein 1 gene sequence analysis in the investigation of an introduced malaria case in Italy.

Malaria due to Plasmodium vivax is globally widespread and is associated with substantial morbidity. The parasite was previously prevalent in temperate areas from which it has been eradicated, however there is a risk of re-introduction because of increased international travel and migration. Following the occurrence of an autochthonous case of P. vivax malaria in Italy after decades of malaria eradication, we applied a molecular approach to compare parasites involved in the introduced case and to determine whether a highly polymorphic gene marker could be useful to tag a P. vivax isolate geographically. To this end, the sequence encompassing the interspecies conserved blocks 5 and 6 of the gene encoding for merozoite surface protein 1 (msp-1) was determined in 16 P. vivax isolates from different regions, and analysed along with 24 pvmsp-1 sequences downloaded from published data. Results have shown that: (i). parasites from the introduced case and the putative source of infection identified following epidemiological investigation, although very similar, differed in three nucleotide substitutions, of which one non synonymous; ii). some geographical isolates looked tightly clustered (e.g. Korean and Punjab isolates), but others were less so.

Plasmodium vivax congenital malaria in an area of very low endemicity in Guatemala: implications for clinical and epidemiological surveillance in a malaria elimination context.

This is a report of the first Plasmodium vivax congenital malaria case in Guatemala and the first case in Latin America with genotypical, histological and clinical characterization. The findings show that maternal P. vivax infection still occurs in areas that are in the pathway towards malaria elimination, and can be associated with detrimental health effects for the neonate. It also highlights the need in very low transmission areas of not only maintaining, but increasing awareness of the problem and developing surveillance strategies, based on population risk, to detect the infection especially in this vulnerable group of the population.