Identification of Plasmodium falciparum,P. vivax,P. ovale and P. malariae and detection of mixed infection in patients with imported malaria in Italy

The species-specific nested-PCR previously described by Snounou and others for detecting the four parasite species that cause human malaria is evaluated in the current study testing 230 blood samples. The results are compared with those obtained by microscopy and, for 101 samples out of 230, with those previously obtained by a genus-specific PCR based method (pg-PCR) followed by species-specific Southern-blot hybridization. All blood specimens were obtained from patients (127 foreigners and 103 Italians) with a suspect clinical diagnosis of imported malaria in Italy: 76 were positive by microscopy and 83 were positive by nested-PCR. The last method also revealed 10 double infections (8 foreigners and 2 Italians) which were not identified by microscopy or by pg-PCR with species-specific Southern-blot hybridization. Fifty-four out of 83 positive samples tested by nested-PCR were submitted to genomic sequence analysis, which confirmed the presence of DNA region portion encoding the 18S rRNA corresponding to the Plasmodium species identified by nested-PCR. These results demonstrate that the nested-PCR assay surpasses microscopy and pg-PCR with species-specific Southern-blot hybridization, both in sensitivity and in diagnostic accuracy. Moreover, it is quicker because it requires no further blotting or hybridization of PCR amplification products. This method also offers a clear advantage in the detection of mixed infections, which is important not only for successful medical treatment but also for the study of malaria epidemiology. Finally, our study also highlights the value of genomic sequence analysis for validating PCR results.     

Plasmodium falciparum: distribution of msp2 genotypes among symptomatic and asymptomatic individuals from the Wosera region of Papua New Guinea

The merozoite surface protein 2 (MSP2) is a leading asexual-stage malaria vaccine candidate that has already proven to have an effect in phase I/IIb vaccine trials, where it was tested in combination with other antigens. Alleles of msp2 fall within two major allelic families, 3D7 and FC27. We analyzed the msp2 genotype in 306 asymptomatic and 63 symptomatic infections from the Wosera region of Papua New Guinea. The multiplicity of infection and the distribution of msp2 alleles was similar to that found in previous studies in the region, but there was no association found between FC27-type or 3D7-type forms of MSP2 and clinical malaria.     

Infection of Peruvian Aotus nancymai monkeys with different strains of Plasmodium falciparum, P. vivax, and P. malariae

Aotus nancymai (karyotype I) monkeys from Peru were studied for their susceptibility to infection with Plasmodium falciparum, P. vivax, and P. malariae. Three strains of P. falciparum (Santa Lucia from El Salvador, Indochina I/CDC from Thailand, and Uganda Palo Alto) were inoculated into 38 monkeys. The results indicated that this species of Aotus monkey is highly susceptible to infection. The Uganda Palo Alto and the Santa Lucia strain parasites appear to be the most useful for immunologic and chemotherapeutic studies. Five strains of P. vivax (Chesson, ONG, Vietnam Palo Alto, Salvador I, and Honduran I/CDC) were inoculated into 28 monkeys. The Vietnam Palo Alto strain produced the highest level parasitemias ranging from 23,800 to 157,000/mm3. Mosquito infections were obtained with the ONG, Chesson, and Salvador I strains. Two out of 6 attempts to transmit P. vivax via sporozoite inoculation to splenectomized monkeys were successful with prepatent periods of 39 and 57 days. Five monkeys were infected with the Uganda I/CDC strain of P. malariae. Maximum parasitemias ranged from 10 to 5,390/mm3.     

On-site diagnosis of Plasmodium falciparum, P. vivax, and P. malariae by using the Quantitative Buffy Coat system.

The Quantitative Buffy Coat (QBC) system was used for the detection and identification of malaria parasites in blood specimens from 570 residents of Oksibil, an isolated highland valley in the eastern Jayawijaya Mountains of Irian Jaya (Indonesian New Guinea). The availability of a battery-powered centrifuge and a fiberoptic Paralens enabled us to complete and interpret the assay in this remote environment. Of 322 QBC tubes examined for 2-4 min each, results of 295 (92%) concurred with findings on the matched Giemsa-stained thick smear (GTS). The 27 discrepant results included 13 QBC+/GTS- that, upon reexamination, were found to be GTS+. When using the corrected GTS results as the standard, the sensitivity and specificity of the QBC were 94% and 96%, respectively. Because electricity was available only 3 hr per day, it was decided to decrease the examination for an additional 248 QBC to a maximum of 90 sec per tube. This shortened inspection time resulted in a reduction of sensitivity to 53% but specificity was preserved at 89%. Forty-two of 45 conflicting results, QBC-/GTS+ from cases of light Plasmodium falciparum infections with < 1 trophozoite or gametocyte per field, were resolved by reexamination of the QBC in the laboratory. Tubes held at 4 C could be reexamined, without noticeable loss of fluorescence, for at least 6 wk after collection. Despite some difficulty in the identification of Plasmodium species, it was concluded that the QBC is an easy, sensitive method for the rapid diagnosis of malaria in the field and that it provides the inexperienced microscopist with an additional means for on-site identification of individuals needing treatment.     

ama1 genes of sympatric Plasmodium vivax and P. falciparum from Venezuela differ significantly in genetic diversity and recombination frequency

Background

We present the first population genetic analysis of homologous loci from two sympatric human malaria parasite populations sharing the same human hosts, using full-length sequences of ama1 genes from Plasmodium vivax and P. falciparum collected in the Venezuelan Amazon.

Methodology/Principal Findings

Significant differences between the two species were found in genetic diversity at the ama1 locus, with 18 distinct haplotypes identified among the 73 Pvama1 sequences obtained, compared to 6 unique haplotypes from 30 Pfama1 sequences, giving overall diversity estimates of h = 0.9091, and h = 0.538 respectively. Levels of recombination were also found to differ between the species, with P. falciparum exhibiting very little recombination across the 1.77kb sequence. In contrast, analysis of patterns of nucleotide substitutions provided evidence that polymorphisms in the ama1 gene of both species are maintained by balancing selection, particularly in domain I. The two distinct population structures observed are unlikely to result from different selective forces acting upon the two species, which share both human and mosquito hosts in this setting. Rather, the highly structured P. falciparum population appears to be the result of a population bottleneck, while the much less structured P. vivax population is likely to be derived from an ancient pool of diversity, as reflected in a larger estimate of effective population size for this species. Greatly reduced mosquito transmission in 1997, due to low rainfall prior to the second survey, was associated with far fewer P. falciparum infections, but an increase in P. vivax infections, probably due to hypnozoite activation.

Conclusions/Significance

The relevance of these findings to putative competitive interactions between these two important human pathogen species is discussed. These results highlight the need for future control interventions to employ strategies targeting each of the parasite species present in endemic areas.     

High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections

Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples (n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia.     

Elucidating relationships between P.falciparum prevalence and measures of genetic diversity with a combined genetic-epidemiological model of malaria

There is an abundance of malaria genetic data being collected from the field, yet using these data to understand the drivers of regional epidemiology remains a challenge. A key issue is the lack of models that relate parasite genetic diversity to epidemiological parameters. Classical models in population genetics characterize changes in genetic diversity in relation to demographic parameters, but fail to account for the unique features of the malaria life cycle. In contrast, epidemiological models, such as the Ross-Macdonald model, capture malaria transmission dynamics but do not consider genetics. Here, we have developed an integrated model encompassing both parasite evolution and regional epidemiology. We achieve this by combining the Ross-Macdonald model with an intra-host continuous-time Moran model, thus explicitly representing the evolution of individual parasite genomes in a traditional epidemiological framework. Implemented as a stochastic simulation, we use the model to explore relationships between measures of parasite genetic diversity and parasite prevalence, a widely-used metric of transmission intensity. First, we explore how varying parasite prevalence influences genetic diversity at equilibrium. We find that multiple genetic diversity statistics are correlated with prevalence, but the strength of the relationships depends on whether variation in prevalence is driven by host- or vector-related factors. Next, we assess the responsiveness of a variety of statistics to malaria control interventions, finding that those related to mixed infections respond quickly (∼months) whereas other statistics, such as nucleotide diversity, may take decades to respond. These findings provide insights into the opportunities and challenges associated with using genetic data to monitor malaria epidemiology.     

High prevalence of Plasmodium malariae and Plasmodium ovale in co-infections with Plasmodium falciparum in asymptomatic malaria parasite carriers in southwestern Nigeria

Asymptomatic malaria parasite carriers do not seek anti-malarial treatment and may constitute a silent infectious reservoir. In order to assess the level of asymptomatic and symptomatic carriage amongst adolescents in a highly endemic area, and to identify the risk factors associated with such carriage, we conducted a cross-sectional survey of 1032 adolescents (ages 10–19 years) from eight schools located in Ibadan, southwestern Nigeria in 2016. Blood films and blood spot filter paper samples were prepared for microscopy and DNA analysis. The prevalence of asymptomatic malaria was determined using microscopy, rapid diagnostic tests and PCR for 658 randomly selected samples. Of these, we found that 80% of asymptomatic schoolchildren were positive for malaria parasites by PCR, compared with 47% and 9%, determined by rapid diagnostic tests and microscopy, respectively. Malaria parasite species typing was performed using PCR targeting the mitochondrial CoxIII gene, and revealed high rates of carriage of Plasmodium malariae (53%) and Plasmodium ovale (24%). Most asymptomatic infections were co-infections of two or more species (62%), with Plasmodium falciparum + P. malariae the most common (35%), followed by P. falciparum + P. malariae + P. ovale (21%) and P. falciparum + P. ovale (6%). Single infections of P. falciparum, P. malariae and P. ovale accounted for 24%, 10% and 4% of all asymptomatic infections, respectively. To compare the species composition of asymptomatic and symptomatic infections, further sample collection was carried out in 2017 at one of the previously sampled schools, and at a nearby hospital. Whilst the species composition of the asymptomatic infections was similar to that observed in 2016, the symptomatic infections were markedly different, with single infections of P. falciparum observed in 91% of patients, P. falciparum + P. malariae in 5% and P. falciparum + P. ovale in 4%.     

Elucidating genetic relationships,diversity and population structure among the Turkish female figs

A collection of 96 female Turkish fig (Ficus carica L.) accessions was studied to elucidate genetic structure and estimate diversity and genetic similarity distribution among the female figs present in Turkish genetic resources, using 157 molecular genome markers including 129 sequence-related amplified polymorphisms, 21 random amplified polymorphic DNAs, and 7 simple-sequence repeats. The plant samples mainly included Turkish fig collections selected throughout the country over the course of a half-century. Neighbor-joining analysis revealed continuous dissimilarity range, and it was difficult to classify figs into distinct groups. The principle component analysis produced similar results. The analysis of molecular variance indicated that 95 and 93% of genetic variation were explained by within geographic origins and similar fruit rind color, respectively. Sub-structuring Bayesian analysis assigned the 96 female figs into four sub-populations, and indicated that they were highly related. The corrected allelic pairwise distances among the six geographic origins were less than 5%. This study suggests that geography- and color-based groups were not genetically distinct among the Turkish figs.     

Effect of sequential infection with Plasmodium vivax and P. falciparum in the Aotus trivirgatus monkey.

Aotus trivirgatus monkeys with prior experience with Plasmodium vivax were inoculated with P. falciparum via the bites of infected mosquitoes. The animals with prior malaria had higher parasitemias and significantly higher levels of mosquito infectivity than monkeys with no prior P. vivax experience. Monkeys with a history of P. falciparum that were inoculated with P. vivax had essentially the same parasitemias as those with no prior malaria. However, levels of mosquito infectivity were markedly increased in those monkeys with a history of P. falciparum. The results imply that the introduction of another malaria species into a malarious area may result in higher levels of mosquito infection and more rapid establishment and distribution of that species.     

Widespread occurrence of antibodies against circumsporozoite protein and against blood forms of Plasmodium vivax, P. falciparum and P. malariae in Brazilian wild monkeys

BACKGROUND: A survey of malaria antibodies was carried out over 7 years and a total of 777 serum samples from wild monkeys were collected in three distinct ecological areas of Brazil where autochthonous malaria has been reported: the 'Cerrado' (similar to savanna), the Atlantic Forest and the Atlantic Semideciduous Forest. METHODS: We carried out enzyme-linked immunosorbent assay to investigate the presence of IgG antibodies against peptides of the circumsporozoite protein (CSP) repeat region of 'classic'Plasmodium vivax, P. vivax VK247, human P. vivax-like/P. simiovale, P. brasilianum/P. malariae and P. falciparum. We also carried out immunofluorescence assay with asexual forms of P. vivax, P. malariae and P. falciparum. RESULTS: The high prevalence of antibodies against CSP in all areas indicates that the monkeys had intense contact with sporozoites from infected anophelines. The immune response against asexual forms of Plasmodium in the monkeys from the Atlantic Forest indicates the development of the infection. CONCLUSIONS: We discuss the possibility of monkeys being malaria reservoirs in non-endemic areas.     

Relative frequencies of polymorphisms of variation in Block 2 repeats and 5' recombinant types of Plasmodium falciparum msp1 alleles

The mechanisms producing the genetic polymorphism at Plasmodium falciparum merozoite surface antigen-1 locus (pfmsp1) include the insertion and deletion of the different type of dimorphic Block 2 9-nucleotide repeat units as well as the intragenic recombination. To study relative occurrence frequencies of these two distinct mechanisms, we have developed a sensitive PCR strategy to identify both 5' recombinant types and the number of Block 2 repeats from the same sample. This method can specifically detect the target 5' recombinant type (Blocks 2-6) at the sensitivity of 1-4 copies of the pfmsp1. Applying the new method to field isolates from the Solomon Islands enabled us to identify six different 5' recombinant types and variation in Block 2 repeat number in three of them, thus distinguishing 10 different alleles. Distribution of these alleles in local three villages in the study area suggests that frequencies of variation in the number of Block 2 9-bp repeats and recombination events within Blocks 2-6 are mutually independent and the frequency of repeat variation is relatively high as compared to that of recombination events at the pfmsp1 locus in P. falciparum populations from the Solomon Islands.     

"Sexual" population structure and genetics of the malaria agent P. falciparum

The population genetics and structure of P. falciparum determine the rate at which malaria evolves in response to interventions such as drugs and vaccines. This has been the source of considerable recent controversy, but here we demonstrate the organism to be essentially sexual, in an area of moderately high transmission in the Lower Shire Valley, Malawi. Seven thousand mosquitoes were collected and dissected, and genetic data were obtained on 190 oocysts from 56 infected midguts. The oocysts were genotyped at three microsatellite loci and the MSP1 locus. Selfing rate was estimated as 50% and there was significant genotypic linkage disequilibrium (LD) in the pooled oocysts. A more appropriate analysis searching for genotypic LD in outcrossed oocysts and/or haplotypic LD in the selfed oocysts found no evidence for LD, indicating that the population was effectively sexual. Inbreeding estimates at MSP1 were higher than at the microsatellites, possibly indicative of immune action against MSP1, but the effect was confounded by the probable presence of null mutations. Mating appeared to occur at random in mosquitoes and evidence regarding whether malaria clones in the same host were related (presumably through simultaneous inoculation in the same mosquito bite) was ambiguous. This is the most detailed genetic analysis yet of P. falciparum sexual stages, and shows P. falciparum to be a sexual organism whose genomes are in linkage equilibrium, which acts to slow the emergence of drug resistance and vaccine insensitivity, extending the likely useful therapeutic lifespan of drugs and vaccines.     

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.     

The probability of a sequential Plasmodium vivax infection following asymptomatic Plasmodium falciparum and P. vivax infections in Myanmar,Vietnam, Cambodia,and Laos

Increasing numbers of aging individuals with chronic co-morbidities travel to regions where falciparum malaria is endemic. Non-communicable diseases are now leading risk factors for death in such countries. Thus, the influence of chronic diseases on the outcome of falciparum malaria is an issue of major importance. Aim of the present study was to assess whether non-communicable diseases increase the risk for severe imported falciparum malaria. A retrospective observational study of all adult cases with imported falciparum malaria hospitalized between 2001 and 2015 in the tertiary care Charité University Hospital, Berlin, was performed. A total of 536 adult patients (median age 37 years; 31.3% female) were enrolled. Of these, 329 (61.4%) originated from endemic countries, 207 patients (38.6%) from non-endemic regions. Criteria for severe malaria were fulfilled in 68 (12.7%) cases. With older age, lack of previous malaria episodes, being a tourist, and delayed presentation, well-characterized risk factors were associated with severe malaria in univariate analysis. After adjustment for these potential confounders hypertension (adjusted odds ratio aOR, 3.06 95% confidence interval, CI 1.34–7.02), cardiovascular diseases (aOR, 8.20 95% CI 2.30–29.22), and dyslipidaemia (aOR, 6.08 95% CI 1.13–32.88) were individual diseases associated with severe disease in multivariable logistic regression. Hypertension proved an independent risk factor among individuals of endemic (aOR, 4.83, 95% CI 1.44–16.22) as well as of non-endemic origin (aOR, 3.60 95% CI 1.05–12.35). In imported falciparum malaria hypertension and its related diseases are risk factors for severe disease.     

Clearance of asymptomatic P. falciparum Infections Interacts with the number of clones to predict the risk of subsequent malaria in Kenyan children

Background

Protective immunity to malaria is acquired after repeated infections in endemic areas. Asymptomatic multiclonal P. falciparum infections are common and may predict host protection. Here, we have investigated the effect of clearing asymptomatic infections on the risk of clinical malaria.

Methods

Malaria episodes were continuously monitored in 405 children (1–6 years) in an area of moderate transmission, coastal Kenya. Blood samples collected on four occasions were assessed by genotyping the polymorphic P. falciparum merozoite surface protein 2 using fluorescent PCR and capillary electrophoresis. Following the second survey, asymptomatic infections were cleared with a full course of dihydroartemisinin.

Results

Children who were parasite negative by PCR had a lower risk of subsequent malaria regardless of whether treatment had been given. Children with ≥2 clones had a reduced risk of febrile malaria compared with 1 clone after clearance of asymptomatic infections, but not if asymptomatic infections were not cleared. Multiclonal infection was associated with an increased risk of re-infection after drug treatment. However, among the children who were re-infected, multiclonal infections were associated with a shift from clinical malaria to asymptomatic parasitaemia.

Conclusion

The number of clones was associated with exposure as well as blood stage immunity. These effects were distinguished by clearing asymptomatic infection with anti-malarials. Exposure to multiple P. falciparum infections is associated with protective immunity, but there appears to be an additional effect in untreated multiclonal infections that offsets this protective effect.     

Genetic polymorphism of cytochrome P450 2E1 in the Turkish population.

CYP 2E1 is involved in metabolic activation of carcinogenic N-nitrosamines, benzene, urethane and other low molecular weight compounds. CYP2E1 gene is present in the population in various polymorphic forms. We detected the RFLP of the human CYP2E1 gene with the restriction endonuclease PstI, RsaI and DraI in a group of 153 Turkish individuals. According to the results of the PstI/RsaI analysis, 96.07% of the subjects were of the c1/c1 genotype, and 3.93% were of the c1/c2 genotype. In the DraI RFLP analysis, 84.30% DD genotype, 15.03% CD genotype and 0.66% CC genotype were determined. The data obtained may be useful in epidemiological studies of the influence of CYP2E1 polymorphism on carcinogenesis.     

High genetic diversity in the remnant island population of hihi and the genetic consequences of re-introduction

The maintenance of genetic diversity is thought to be fundamental for the conservation of threatened species. It is therefore important to understand how genetic diversity is affected by the re-introduction of threatened species. We use establishment history and genetic data from the remnant and re-introduced populations of a New Zealand endemic bird, the hihi Notiomystis cincta, to understand genetic diversity loss and quantify the genetic effects of re-introduction. Our data do not support any recent bottleneck events in the remnant population. Furthermore, all genetic diversity measures indicate the remnant hihi population has retained high levels of genetic diversity relative to other New Zealand avifauna with similar histories of decline. Genetic diversity (N(A) , alleles per locus, allelic richness, F(IS) and H(S) ) did not significantly decrease in new hihi populations founded through re-introduction when compared to their source populations, except in the Kapiti Island population (allelic richness and H(S) ) which had very slow post-re-introduction population growth. The N(e) /N(c) ratio in the remnant population was high, but decreased in first-level re-introductions, which together with significant genetic differentiation between populations (F(ST) & Fisher's exact tests) suggest that extant populations are diverging as a result of founder effects and drift. Importantly, simulations of future allele loss predict that the number of alleles lost will be higher in populations with a slow population growth, fewer founding individuals and with nonrandom mating. Interestingly, this species has very high levels of extra-pair paternity which may reduce reproductive variance by allowing social and floater males to reproduce a life history trait that together with a large remnant population size may help maintain higher levels of genetic diversity than expected.