Jürgen Kun

Merozoite surface antigen 1 and 2 genotypes and rosetting of Plasmodium falciparum in severe and mild malaria in Lambaréné, Gabon

We present a case-control study to investigate the distribution of Plasmodium falciparum genotypes in patients with severe and mild malaria. We compared clinical and parasitological data with the parasites genotype and rosetting. The study group consisted of 100 children suffering severe malaria, defined as severe anaemia and hyperparasitaemia. These children were matched by age, sex and provenance with 100 children with mild malaria. For characterization of the parasites we used the polymerase chain reaction to determine merozoite surface antigen (MSA) 1 and 2 genotypes and the phenomenon of rosette formation. We found a significant association between rosette formation and disease severity, and a significant association of severe anaemia with the presence of the MSA-1 allele K1. Infections with 2 genotypes in the severely affected group were significantly associated with severe anaemia and the presence of MSA-1 allele K1. Comparison with the findings of other groups led to the conclusion that the occurrence of P. falciparum genotypes seems to differ geographically.

Nitric oxide synthase 2Lambaréné (G-954C), increased nitric oxide production, and protection against Malaria

A point mutation in the promoter of the nitric oxide synthase 2 gene (NOS2), termed NOS2(Lambaréné) (NOS2-G954C), protects heterozygous carriers against severe malaria as effectively as the sickle cell trait. In a prospective longitudinal study, 841 individual infections of initially 200 children (151 wild-type vs. 49 NOS2(Lambaréné) carriers) were monitored for 4 years, to assess the rates of malarial attacks in the 2 groups; carriers of the NOS2(Lambaréné) polymorphism were significantly less likely to experience malarial attacks than were others (P=.002). The distribution of the NOS2(Lambaréné) polymorphism was investigated in malaria-endemic areas. It was found to be present with the highest frequency in Africa and at a lower frequency in Asia. Ex vivo studies showed that cells isolated from people with this polymorphism have a 7-fold higher baseline NOS activity, compared with the levels detected in cells from subjects with the wild-type gene (P=.003).

Artesunate-Clindamycin versus Quinine-Clindamycin in the Treatment ofPlasmodium falciparum Malaria: A Randomized Controlled Trial

BACKGROUNDnArtemisinin-based drug combinations are the mainstay in the fight against drug-resistant malaria in Africa. Currently available antimalarial drug combinations that include artemisinins are pharmacokinetically unmatched and are therefore potentially increasing the risk of selection of resistant mutants in areas in which the rate of transmission of malaria is high. We tested the potential value of artemisinin-based combination therapy with a short elimination half-life for the treatment of uncomplicated Plasmodium falciparum malaria in sub-Saharan Africa.nnnMETHODSnWe conducted an open-label, randomized, controlled clinical trial to evaluate the efficacy and tolerability of oral artesunate-clindamycin therapy given twice daily for 3 days (artesunate, 2 mg/kg, and clindamycin, 7 mg/kg, per dose), compared with a standard quinine-clindamycin regimen given twice daily for 3 days (quinine, 15 mg/kg, and clindamycin, 7 mg/kg, per dose), for the treatment of uncomplicated falciparum malaria in 100 Gabonese children aged 3-12 years. The primary end point of the study was the polymerase chain reaction-corrected cure rate for the per-protocol population.nnnRESULTSnThe activity of artesunate-clindamycin was comparable to that of quinine-clindamycin in the per-protocol analysis of cure rates at day 28 of follow-up (87% versus 94%). No serious adverse events were reported, and tolerability was good and was similar in both groups. Times to clearance of fever and clearance of parasites were significantly shorter in the artesunate-clindamycin group.nnnCONCLUSIONSnArtesunate-clindamycin and other matching artemisinin-based combinations with a short plasma half-life merit further attention for use in regions in which the rate of transmission of malaria is high.

Rapid detection of Pfcrt and Pfmdr1 mutations in Plasmodium falciparum isolates by FRET and in vivo response to chloroquine among children from Osogbo, Nigeria.

BackgroundChloroquine (CQ) has been in use in Africa for a long time. Because of misuse, this drug has now lost its efficacy due to the emergence of resistance strains in most parts of Africa. Recently, it was shown that after chloroquine has been withdrawn from the market, chloroquine-sensitive Plasmodium falciparum re-emerged and chloroquine could again be used successfully as an antimalarial. Surveillance of parasite populations is, therefore, important to decide whether chloroquine could be re-introduced.MethodsTo estimate the prevalence of the most pivotal polymorphisms, including Pfcrt K76T, Pfmdr1 N86Y and Pfmdr1 Y184F mutations, and their contributions to the outcome of CQ treatment, isolates from Osogbo Western Nigeria were tested using the Fluorescence Resonance Energy Transfer (FRET) method on a real-time PCR instrument.Results116 children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of CQ and followed-up for 28 days. Blood samples were collected on filter paper at enrollment and during follow-up for identification of parasite carrying the chloroquine resistant transporter (pfcrt) and P. falciparum-multi drug resistance (pfmdr1) gene mutations. Parasitological assessment of response to treatment showed that 62% of the patients were cured and 38% failed the CQ treatment. The presence of single mutant pfcrt (T76) alleles (P = 0.003) and in combination with mutant pfmdr1 Y86 (P = 0.028) was significantly associated with in vivo CQR. No other mutation on its own or in combinations was significantly associated with treatment outcome. Mutant pfcrt was more prevalent in both pre- and post-treatment isolates. No association was observed between age or initial level of parasitaemia and chloroquine treatment outcome.ConclusionThe result established the usefulness and accuracy of real time PCR in pfcrt and pfmdr1 mutation detection and also give further evidence to the reliability of the pfcrt T76 point mutation as a molecular marker for CQ resistance.

Plasmodium vivax malaria in Duffy-negative individuals from Ethiopia.

BACKGROUNDnThis study was conducted to estimate the prevalence of Plasmodium vivax and polymorphisms in the Duffy antigen receptor for chemokines (DARC) gene in patients with suspected malaria from eastern (Harar) and southwestern (Jimma) Ethiopia.nnnMETHODSnPlasmodium presence and species was assessed by microscopy in 1304 and 627 febrile patients in Harar and Jimma, respectively, during October-November 2009. All microscopy-positive samples were confirmed by PCR. DARC gene polymorphisms were identified by DNA sequencing.nnnRESULTSnPlasmodium vivax was the dominant species in Harar (74/98, 76%) and P. falciparum was more common in Jimma (70/107, 65%). We found 17/98 (17%) and 24/107 (22%) homozygous Duffy-negative patients in Harar and Jimma, respectively. Unexpectedly, three Duffy-negative patients from Harar had P. vivax malaria.nnnCONCLUSIONnThis study documents the emergence of P. vivax malaria in Duffy-negative individuals in Ethiopia. The Duffy-negative blood group does not appear to provide absolute protection against P. vivax infection in this region.

Phylogenetic nomenclature and evolution of mannose-binding lectin (MBL2) haplotypes

BackgroundPolymorphisms of the mannose-binding lectin gene (MBL2) affect the concentration and functional efficiency of the protein. We recently used haplotype-specific sequencing to identify 23 MBL2 haplotypes, associated with enhanced susceptibility to several diseases.ResultsIn this work, we applied the same method in 288 and 470 chromosomes from Gabonese and European adults, respectively, and found three new haplotypes in the last group. We propose a phylogenetic nomenclature to standardize MBL2 studies and found two major phylogenetic branches due to six strongly linked polymorphisms associated with high MBL production. They presented high Fst values and were imbedded in regions with high nucleotide diversity and significant Tajimas D values. Compared to others using small sample sizes and unphased genotypic data, we found differences in haplotyping, frequency estimation, Fu and Lis D* and Fst results.ConclusionUsing extensive testing for selective neutrality, we confirmed that stochastic evolutionary factors have had a major role in shaping this polymorphic gene worldwide.

Comparison of PCR-based detection of Plasmodium falciparum infections based on single and multicopy genes

PCR-based assays are the most sensitive and specific methods to detect malaria parasites.This study compared the diagnostic accuracy of three PCR-based assays that do not only differ in their sequence target, but also in the number of copies of their target region, for the detection of Plasmodium falciparum in 401 individuals living in a malaria-endemic area in Nigeria. Compared to a composite reference generated from results of all the 3 PCR assays, the stevor gene amplification had a sensitivity of 100% (Kappa = 1; 95% CI = 1.000–1.000), 83% (Kappa = 0.718; 95% CI = 0.648–0.788) by SSUrRNA gene PCR and 71% (Kappa = 0.552; 95% CI = 0.478–0.627) by the msa-2 gene amplification.Results from this study indicate that the stevor gene amplification is the most sensitive technique for the detection of P. falciparum. This assay may be an important reference standard, especially when a confirmatory technique with high sensitivity and specificity is needed for ruling out P. falciparum infection.

Haplotype specific-sequencing reveals MBL2 association with asymptomatic Plasmodium falciparum infection

BackgroundMannose binding lectin (MBL) has an important role in the activation of the complement system and opsonization of pathogenic microorganisms. Frequent polymorphisms found in the MBL2 gene affect the concentration and functionality of the protein and are associated with enhanced susceptibility to severe malaria in African children. Most MBL2 typing strategies were designed to the analysis of selected variants, the significance of whole haplotypes is poorly known. In this work, a new typing strategy was developed and validated in an association analysis of MBL2 with adult asymptomatic infection.MethodsMBL2 allele-specific fragments of 144 healthy Gabonese adults were amplified by using haplotype-specific sequencing (HSS), a new strategy that combines sequence-specific PCR and sequence-based typing. The Gabonese were investigated for the presence of Plasmodium falciparum parasitaemia by the amplification of parasite genes, immunochromatographic antigen detection and microscopic analysis. HSS results were also compared with a previously used real-time PCR (RT-PCR) method in 72 Euro-Brazilians.ResultsFourteen polymorphisms were identified beside the commonly investigated promoter (H, L; X, Y; P, Q) and exon 1 (A, O; O = B, C or D) variants. The MBL2*LYPA/LYPA genotype was associated with the absence of asymptomatic infection (P = 0.017), whereas the MBL2*LYQC haplotype and YA/YO + YO/YO genotypes were associated with positive parasite counts in asymptomatic adults (P = 0.033 and 0.018, respectively). The associations were specific to LYPA (identical to the reference sequence Y16577) and LYQC (Y16578) and would not have been revealed by standard genotyping, as there was no association with LYPA and LYQC haplotypes carrying new polymorphisms defined by sequence-based typing. In contrast, HSS and RT-PCR produced very similar results in the less diverse European-derived population.ConclusionIn this work, a new typing strategy for a highly polymorphic gene was developed and validated focusing on the asymptomatic status of P. falciparum- infected adults. In populations with high nucleotide diversity, it allowed for the identification of associations with fine-scaled haplotypes that would not have been found using common typing techniques. In this preliminary study, MBL2 haplotypes or SNPs linked to them were found associated with susceptibility to infection and parasitaemia control of asymptomatic adults.

Clonal expansion accounts for an excess of antimicrobial resistance in Staphylococcus aureus colonising HIV-positive individuals in Lagos, Nigeria

Nasal colonisation with Staphylococcus aureus is a risk factor for invasive infection in human immunodeficiency virus (HIV)-positive individuals. This study aimed to characterise colonising S. aureus from regions with a high HIV prevalence. Single nasal swabs were taken from a total of 374 HIV-positive and 370 healthy individuals. Overall, 202 S. aureus carriers were detected. Compared with healthy individuals, HIV-positive subjects were more likely to be S. aureus nasal carriers (33% vs. 21%; P=0.0001). Isolates from HIV-positive individuals were more often resistant to meticillin (16% vs. 8%; P=0.13), chloramphenicol (47% vs. 16%; P<0.0001), sulfamethoxazole/trimethoprim (SXT) (90% vs. 55%; P<0.0001) and ciprofloxacin (18% vs. 0%; P<0.0001). Strains belonging to the spa clonal complexes 3772/ST25 and 064/ST8 were significantly more often isolated from HIV-positive individuals and exhibited greater resistance to ciprofloxacin, SXT and chloramphenicol (spa-CC 3772) or to meticillin (spa-CC 064), respectively. Panton-Valentine leukocidin gene content was high overall and was equally distributed between isolates from HIV-positive and healthy individuals (33% vs. 30%). Genotypic characteristics of colonising isolates were similar to those reported to cause invasive infection in Nigeria. The HIV pandemic contributes to the evolution of antimicrobial resistance in S. aureus. Measures to contain antimicrobial resistance of S. aureus in Nigeria must target risk groups such as HIV-positive individuals.

NCF1 gene and pseudogene pattern: association with parasitic infection and autoimmunity.

BackgroundNeutrophil cytosolic factor 1, p47phox (NCF1) is a component of the leukocyte NADPH oxidase complex mediating formation of reactive oxygen intermediates (ROI) which play an important role in host defense and autoimmunity. An individual genomic pattern of ncf1 and its two types of pseudogenes (reflected by the ΔGT/GTGT ratio) may influence the individual capacity to produce ROI.MethodsNCF1ΔGT/GTGT ratios were correlated with clinical parameters and ROI production during Plasmodium falciparum malaria and with susceptibility to the autoimmune disease multiple sclerosis (MS).ResultsAmong Gabonese children with severe malaria, ROI production from peripheral blood tended to be higher in individuals with a ΔGT/GTGT ratio ≤ 1:1. ΔGT/GTGT ratios were not associated with susceptibility to MS, but to age-of-onset among MS patients.ConclusionThe genomic pattern of NCF1 and its pseudogenes might influence ROI production but only marginally influence susceptibility to and outcome of malaria and MS.