Sequence signatures involved in targeting the male-specific lethal complex to X-chromosomal genes in Drosophila melanogaster

Background

Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance. A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.

Results

Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.

Conclusions

Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.     

An AFLP-based genetic linkage map of Plasmodium chabaudi chabaudi

Background

Plasmodium chabaudi chabaudi can be considered as a rodent model of human malaria parasites in the genetic analysis of important characters such as drug resistance and immunity. Despite the availability of some genome sequence data, an extensive genetic linkage map is needed for mapping the genes involved in certain traits.

Methods

The inheritance of 672 Amplified Fragment Length Polymorphism (AFLP) markers from two parental clones (AS and AJ) of P. c. chabaudi was determined in 28 independent recombinant progeny clones. These, AFLP markers and 42 previously mapped Restriction Fragment Length Polymorphism (RFLP) markers (used as chromosomal anchors) were organized into linkage groups using Map Manager software.

Results

614 AFLP markers formed linkage groups assigned to 10 of 14 chromosomes, and 12 other linkage groups not assigned to known chromosomes. The genetic length of the genome was estimated to be about 1676 centiMorgans (cM). The mean map unit size was estimated to be 13.7 kb/cM. This was slightly less then previous estimates for the human malaria parasite, Plasmodium falciparum

Conclusion

The P. c. chabaudi genetic linkage map presented here is the most extensive and highly resolved so far available for this species. It can be used in conjunction with the genome databases of P. c chabaudi, P. falciparum and Plasmodium yoelii to identify genes underlying important phenotypes such as drug resistance and strain-specific immunity.     

Malaria prophylaxis policy for travellers from Europe to the Indian Sub Continent

Background

Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge.

Methods

The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia.

Results

Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities.

Conclusion

It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density.     

Antipyretic effect of ibuprofen in Gabonese children with uncomplicated falciparum malaria: a randomized, double-blind, placebo-controlled trial

Background

The protection afforded by human erythrocyte polymorphisms against the malaria parasite, Plasmodium falciparum, has been proposed to be due to reduced ability of the parasite to invade or develop in erythrocytes. If this were the case, variable levels of parasitaemia and rates of seroconversion to infected-erythrocyte variant surface antigens (VSA) should be seen in different host genotypes.

Methods

To test this hypothesis, P. falciparum parasitaemia and anti-VSA antibody levels were measured in a cohort of 555 asymptomatic children from an area of intense malaria transmission in Papua New Guinea. Linear mixed models were used to investigate the effect of α⁺-thalassaemia, complement receptor-1 and south-east Asian ovalocytosis, as well as glucose-6-phosphate dehydrogenase deficiency and ABO blood group on parasitaemia and age-specific seroconversion to VSA.

Results

No host polymorphism showed a significant association with both parasite prevalence/density and age-specific seroconversion to VSA.

Conclusion

Host erythrocyte polymorphisms commonly found in Papua New Guinea do not effect exposure to blood stage P. falciparum infection. This contrasts with data for sickle cell trait and highlights that the above-mentioned polymorphisms may confer protection against malaria via distinct mechanisms.     

Detection of malaria parasites by nested PCR in south-eastern, Iran: Evidence of highly mixed infections in Chahbahar district

Background

Rapid diagnosis and correct treatment of cases are the main objectives of control programs in malaria-endemic areas.

Methods and results

To evaluate these criteria and in a comparative study, blood specimens were collected from 120 volunteers seeking care at the Malaria Health Center in Chahbahar district. One hundred and seven out of 120 Giemsa-stained slides were positive for malaria parasites by microscopy. Eighty-four (70%) and 20 (16.7%) were identified as having only Plasmodium vivax and Plasmodium falciparum infections, respectively, while only 3 (2.5%) were interpreted as having mixed P. vivax-P. falciparum infections. The target DNA sequence of the 18S small sub-unit ribosomal RNA (ssrRNA) gene was amplified by Polymerase Chain Reaction (PCR) and used for the diagnosis of malaria in south-eastern Iran. One hundred twenty blood samples were submitted and the results were compared to those of routine microscopy. The sensitivity of PCR for detection of P. vivax and P. falciparum malaria was higher than that of microscopy: nested PCR detected 31 more mixed infections than microscopy and parasite positive reactions in 9 out of the 13 microscopically negative samples. The results also confirmed the presence of P. vivax and P. falciparum.

Conclusions

These results suggest that, in places where transmission of both P. vivax and P. falciparum occurs, nested PCR detection of malaria parasites can be a very useful complement to microscopical diagnosis.     

Glycerol: An unexpected major metabolite of energy metabolism by the human malaria parasite

Background

Malaria is the third most prevalent cause of infectious disease in the world. Resistance of the parasite to classical drugs makes the discovery of new and effective drugs more urgent. The oxidized derivative of hydroxy- cis terpenone (OHCT) is a synthetic molecule that is not toxic to cultured human liver cells at concentrations as high as 60 μM and inhibits activity of cytochrome P450s that metabolize many drugs.

Methods

OHCT activity against chloroquine-sensitive and -resistant strains of Plasmodium falciparum, and a P. falciparum clone that is partially resistant to artemisinin was assayed in vitro.

Results

OHCT at nanomolar concentrations was effective against all intraerythrocytic stages of P. falciparum and exhibited activity in vitro against both chloroquine-sensitive and -resistant strains of P. falciparum as well as a P. falciparum clone that is partially resistant to artemisinin. Moreover, OHCT exhibited potent activity against gametocytes, the form that is transmitted by mosquitoes and essential for the spread of malaria.

Conclusion

OHCT displays strong growth inhibitory activity against all stages of P. falciparum and no evidence of toxicity to human cells in culture. It is easily synthesized and has the potential for inhibiting metabolism of drugs used in combination therapies.     

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

Background

Artemisinin and its derivatives have been used for falciparum malaria treatment in China since late 1970s. Monotherapy and uncontrolled use of artemisinin drugs were common practices for a long period of time. In vitro tests showed that the susceptibility of Plasmodium falciparum to artemisinins was declining in China. A concern was raised about the resistance to artemisinins of falciparum malaria in the country. It has been reported that in vitro artemisinin resistance was associated with the S769N mutation in the PfATPase6 gene. The main purpose of this study was to investigate whether that mutation has occurred in field isolates from China.

Methods

Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. A nested PCR-sequencing assay was developed to analyse the genotype of the PfATPase6 S769N polymorphism in the P. falciparum field isolates.

Results

The genotyping results of six samples could not be obtained due to failure of PCR amplification, but no S769N mutation was detected in any of the 95 samples successfully analysed.

Conclusion

The results indicate that the S769N mutation in the PfATPase6 gene is not present in China, suggesting that artemisinin resistance has not yet developed, but the situation needs to be watched very attentively.     

Electrocardiographic study in Ghanaian children with uncomplicated malaria,treated with artesunate-amodiaquine or artemether-lumefantrine

Background

In order to prepare the field site for future interventions, the prevalence of asymptomatic Plasmodium falciparum infection was evaluated in a cohort of children living in Brazzaville. Plasmodium falciparum merozoite surface protein 2 gene (msp2) was used to characterize the genetic diversity and the multiplicity of infection. The prevalence of mutant P. falciparum chloroquine resistance transporter (pfcrt) allele in isolates was also determined.

Methods

Between April and June 2010, 313 children below 10 years of age enrolled in the cohort for malaria surveillance were screened for P. falciparum infection using microscopy and polymerase chain reaction (PCR). The children were selected on the basis of being asymptomatic. Plasmodium falciparum msp2 gene was genotyped by allele-specific nested PCR and the pfcrt K76T mutation was detected using nested PCR followed by restriction endonuclease digestion.

Results

The prevalence of asymptomatic P. falciparum infections was 8.6% and 16% by microscopy and by PCR respectively. Allele typing of the msp2 gene detected 55% and 45% of 3D7 and FC27 allelic families respectively. The overall multiplicity of infections (MOI) was 1.3. A positive correlation between parasite density and multiplicity of infection was found. The prevalence of the mutant pfcrt allele (T76) in the isolates was 92%.

Conclusion

This is the first molecular characterization of P. falciparum field isolates in Congolese children, four years after changing the malaria treatment policy from chloroquine (CQ) to artemisinin-based combination therapy (ACT). The low prevalence of asymptomatic infections and MOI is discussed in the light of similar studies conducted in Central Africa.     

Application of the lumped age-class technique to studying the dynamics of malaria-mosquito-human interactions

Background

There is an increase of serum levels of IgE during Plasmodium falciparum infections in individuals living in endemic areas. These IgEs either protect against malaria or increase malaria pathogenesis. To get an insight into the exact role played by IgE in the outcome of P. falciparum infection, total IgE levels and functional anti-parasite IgE response were studied in children and adults, from two different endemic areas Gabon and India, exhibiting either uncomplicated malaria, severe non cerebral malaria or cerebral malaria, in comparison with control individuals.

Methodology and results

Blood samples were collected from controls and P. falciparum -infected patients before treatment on the day of hospitalization (day 0) in India and, in addition, on days 7 and 30 after treatment in Gabon. Total IgE levels were determined by ELISA and functional P. falciparum -specific IgE were estimated using a mast cell line RBL-2H3 transfected with a human Fcε RI α-chain that triggers degranulation upon human IgE cross-linking. Mann Whitney and Kruskall Wallis tests were used to compare groups and the Spearman test was used for correlations. Total IgE levels were confirmed to increase upon infection and differ with level of transmission and age but were not directly related to the disease phenotype. All studied groups exhibited functional parasite-specific IgEs able to induce mast cell degranulation in vitro in the presence of P. falciparum antigens. Plasma IgE levels correlated with those of IL-10 in uncomplicated malaria patients from Gabon. In Indian patients, plasma IFN-γ, TNF and IL-10 levels were significantly correlated with IgE concentrations in all groups.

Conclusion

Circulating levels of total IgE do not appear to correlate with protection or pathology, or with anti-inflammatory cytokine pattern bias during malaria. On the contrary, the P. falciparum -specific IgE response seems to contribute to the control of parasites, since functional activity was higher in asymptomatic and uncomplicated malaria patients than in severe or cerebral malaria groups.     

Increase of malaria attacks among children presenting concomitant infection by Schistosoma mansoni in Senegal

Background

Parasites incur periodic mutations which must ultimately be eliminated to maintain their genetic integrity.

Methods

It is hypothesised that these mutations are eliminated not by the conventional mechanisms of competition between parasites in different hosts but primarily by competition between parasites within the same infection.

Results

This process is enhanced by the production of a large number of parasites within individual infections, and this may significantly contribute to parasitic virulence.

Conclusions

Several features of the most virulent human malaria parasite Plasmodium falciparum can usefully be re-interpreted in this light and lend support to this interpretation. More generally, it constitutes a novel explanation for the evolution of virulence in a wider range of microparasites.     

Prevalence and risk factors for Plasmodium falciparum malaria in pregnant women of eastern Sudan

Background

Pregnant women are more susceptible to malaria, which is associated with serious adverse effects on pregnancy. The presentation of malaria during pregnancy varies according to the level of transmission in the area. Our study aimed to demonstrate the prevalence and risk factors for malaria (age, parity and gestational age) among pregnant women of eastern Sudan, which is characterized by unstable malaria transmission.

Methods

The prevalence and possible risk factors for Plasmodium falciparum malaria were investigated in 744 pregnant Sudanese women attending the antenatal clinic of New Haifa Teaching Hospital, eastern Sudan, during October 2003-April 2004.

Results

A total 102 (13.7%) had P. falciparum malaria, 18(17.6%) of these were severe cases (jaundice and severe anaemia). Univariate and multivariate analysis showed that, age and parity were not associated with malaria. Women who attended the antenatal clinic in the third trimester were at highest risk for malaria (OR = 1.58, 95% CI = 1.02–2.4; P < 0.05). Women with malaria had significantly lower mean haemoglobin (9.4 g/dl, 95% CI 9.1–9.7 versus 10.7, CI 10.6–10.8, P < 0.05). A significantly lower haemoglobin was observed in those with severe falciparum malaria compared to non-severe form (8.3 g/dl, 95% CI 7.6–9.1 versus 9.4, 95% CI 9.1–9.7, P = < 0.05).

Conclusion

The results suggest that P. falciparum malaria is common in pregnant women attending antenatal care and that anaemia is an important complication. Preventive measures (chemoprophylaxis and insecticide-treated bednets) may be beneficial in this area for all women irrespective of age or parity.     

A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR

Background

The erythrocyte binding antigen-175 (EBA-175) on Plasmodium falciparum merozoites mediates sialic acid dependent binding to glycophorin A on host erythrocytes and, therefore, plays a crucial role in cell invasion. Dimorphic allele segments have been found in its encoding gene with a 342 bp segment present in FCR-3 strains (F-segment) and a 423 bp segment in CAMP strains (C-segment). Possible associations of the dimorphism with severe malaria have been analysed in a case-control study in northern Ghana.

Methods

Blood samples of 289 children with severe malaria and 289 matched parasitaemic but asymptomatic controls were screened for eba- 175 F- and C-segments by nested polymerase chain reaction.

Results

In children with severe malaria, prevalences of F-, C- and mixed F-/C-segments were 70%, 19%, and 11%, respectively. The C-segment was found more frequently in severe malaria cases whereas mixed infections were more common in controls. Infection with strains harbouring the C-segment significantly increased the risk of fatal outcome.

Conclusion

The results show that the C-segment is associated with fatal outcome in children with severe malaria in northern Ghana, suggesting that it may contribute to the virulence of the parasite.     

Daily temperature profiles in and around Western Kenyan larval habitats of Anopheles gambiae as related to egg mortality

Background

Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins.

Methods

4.1R structural domains (30, 16, 10 and 22 kDa domain) and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R.

Results

Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R.

Conclusion

The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle.     

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Background

The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results

Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions

We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.     

Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes

Background

The 10.9× genomic sequence of Candida albicans, the most important human fungal pathogen, was published in 2004. Assembly 19 consisted of 412 supercontigs, of which 266 were a haploid set, since this fungus is diploid and contains an extensive degree of heterozygosity but lacks a complete sexual cycle. However, sequences of specific chromosomes were not determined.

Results

Supercontigs from Assembly 19 (183, representing 98.4% of the sequence) were assigned to individual chromosomes purified by pulse-field gel electrophoresis and hybridized to DNA microarrays. Nine Assembly 19 supercontigs were found to contain markers from two different chromosomes. Assembly 21 contains the sequence of each of the eight chromosomes and was determined using a synteny analysis with preliminary versions of the Candida dubliniensis genome assembly, bioinformatics, a sequence tagged site (STS) map of overlapping fosmid clones, and an optical map. The orientation and order of the contigs on each chromosome, repeat regions too large to be covered by a sequence run, such as the ribosomal DNA cluster and the major repeat sequence, and telomere placement were determined using the STS map. Sequence gaps were closed by PCR and sequencing of the products. The overall assembly was compared to an optical map; this identified some misassembled contigs and gave a size estimate for each chromosome.

Conclusion

Assembly 21 reveals an ancient chromosome fusion, a number of small internal duplications followed by inversions, and a subtelomeric arrangement, including a new gene family, the TLO genes. Correlations of position with relatedness of gene families imply a novel method of dispersion. The sequence of the individual chromosomes of C. albicans raises interesting biological questions about gene family creation and dispersion, subtelomere organization, and chromosome evolution.