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1.  Host iron status and iron supplementation mediate susceptibility to erythrocytic stage Plasmodium falciparum 
Nature communications  2014;5:4446.
Iron deficiency and malaria have similar global distributions, and frequently co-exist in pregnant women and young children. Where both conditions are prevalent, iron supplementation is complicated by observations that iron deficiency anaemia protects against falciparum malaria, and that iron supplements increase susceptibility to clinically significant malaria, but the mechanisms remain obscure. Here, using an in vitro parasite culture system with erythrocytes from iron-deficient and replete human donors, we demonstrate that Plasmodium falciparum infects iron-deficient erythrocytes less efficiently. In addition, owing to merozoite preference for young erythrocytes, iron supplementation of iron-deficient individuals reverses the protective effects of iron deficiency. Our results provide experimental validation of field observations reporting protective effects of iron deficiency and harmful effects of iron administration on human malaria susceptibility. Because recovery from anaemia requires transient reticulocytosis, our findings imply that in malarious regions iron supplementation should be accompanied by effective measures to prevent falciparum malaria.
doi:10.1038/ncomms5446
PMCID: PMC4249681  PMID: 25059846
2.  Pooled Deep Sequencing of Plasmodium falciparum Isolates: An Efficient and Scalable Tool to Quantify Prevailing Malaria Drug-Resistance Genotypes 
The Journal of Infectious Diseases  2013;208(12):1998-2006.
Molecular surveillance for drug-resistant malaria parasites requires reliable, timely, and scalable methods. These data may be efficiently produced by genotyping parasite populations using second-generation sequencing (SGS). We designed and validated a SGS protocol to quantify mutant allele frequencies in the Plasmodium falciparum genes dhfr and dhps in mixed isolates. We applied this new protocol to field isolates from children and compared it to standard genotyping using Sanger sequencing. The SGS protocol accurately quantified dhfr and dhps allele frequencies in a mixture of parasite strains. Using SGS of DNA that was extracted and then pooled from individual isolates, we estimated mutant allele frequencies that were closely correlated to those estimated by Sanger sequencing (correlations, >0.98). The SGS protocol obviated most molecular steps in conventional methods and is cost saving for parasite populations >50. This SGS genotyping method efficiently and reproducibly estimates parasite allele frequencies within populations of P. falciparum for molecular epidemiologic studies.
doi:10.1093/infdis/jit392
PMCID: PMC3836461  PMID: 23908494
Plasmodium falciparum; drug resistance; molecular surveillance; tropical diseases
3.  A Quality Control Program within a Clinical Trial Consortium for PCR Protocols To Detect Plasmodium Species 
Journal of Clinical Microbiology  2014;52(6):2144-2149.
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.
doi:10.1128/JCM.00565-14
PMCID: PMC4042732  PMID: 24740073
4.  Multilocus Microsatellite Genotyping Array for Investigation of Genetic Epidemiology of Pneumocystis jirovecii 
Journal of Clinical Microbiology  2014;52(5):1391-1399.
Pneumocystis jirovecii is a symbiotic respiratory fungus that causes pneumonia (PcP) in immunosuppressed patients. Because P. jirovecii cannot be reliably cultured in vitro, it has proven difficult to study and gaps in our understanding of the organism persist. The release of a draft genome for the organism opens the door for the development of new genotyping approaches for studying its molecular epidemiology and global population structure. We identified and validated 8 putatively neutral microsatellite markers and 1 microsatellite marker linked to the dihydropteroate synthase gene (dhps), the enzymatic target of sulfa drugs used for PcP prevention and treatment. Using these tools, we analyzed P. jirovecii isolates from HIV-infected patients from three geographically distant populations: Uganda, the United States, and Spain. Among the 8 neutral markers, we observed high levels of allelic heterozygosity (average He, 0.586 to 0.842). Consistent with past reports, we observed limited global population structuring, with only the Ugandan isolates showing minor differentiation from the other two populations. In Ugandan isolates that harbored mutations in dhps, the microsatellite locus linked to dhps demonstrated a depressed He, consistent with positive directional selection for sulfa resistance mutations. Using a subset of these microsatellites, analyses of individual and paired samples from infections in San Francisco, CA, showed reliable typeability within a single infection and high discriminatory power between infections. These features suggest that this novel microsatellite typing approach will be an effective tool for molecular-epidemiological investigations into P. jirovecii population structure, transmission, and drug resistance.
doi:10.1128/JCM.02531-13
PMCID: PMC3993678  PMID: 24523468
5.  Nontypeable Haemophilus influenzae inhibits autolysis and fratricide of Streptococcus pneumoniae in vitro 
Streptococcus pneumoniae (SP) and nontypeable Haemophilus influenzae (NTHi) are common commensals of the human airway and major bacterial pathogens of otitis media (OM) and other upper airway infections. The interaction between them may play an important role in the pathogenesis of polymicrobial infections. Although previous studies suggested NTHi could promote pneumococcal survival and biofilm formation, how NTHi affects pneumococcal activities has not been defined. Our data in the present studies indicated that the outcome of the interaction between SP and NTHi was in a cell-density-dependent manner and the enhancement of pneumococcal survival happened at the later stages of culturing. Using quantitative PCR, we found that the expression of pneumococcal genes regulating autolysis and fratricide, lytA and cbpD, were significantly down-regulated in co-culture with NTHi. We further observed that influence of NTHi was not on direct cell-to-cell contact, but that this contact may contribute to the interaction between these two microorganisms. These results suggest that pneumococcal survival and biofilm formation can be enhanced by down-regulating pneumococcal cell wall hydrolase production thereby inhibiting pneumococcal autolysis and fratricide in the presence of NTHi.
doi:10.1016/j.micinf.2013.11.006
PMCID: PMC4080417  PMID: 24269704
Streptococcus pneumoniae; nontypeable Haemophilus influenzae; survival; inhibit; autolysis; fratricide
6.  Independent Lineages of Highly Sulfadoxine-Resistant Plasmodium falciparum Haplotypes, Eastern Africa 
Emerging Infectious Diseases  2014;20(7):1140-1148.
Parasites with increased resistance to sulfadoxine might undermine malaria control measures.
Sulfadoxine-resistant Plasmodium falciparum undermines malaria prevention with sulfadoxine/pyrimethamine. Parasites with a highly resistant mutant dihydropteroate synthase (dhps) haplotype have recently emerged in eastern Africa; they negated preventive benefits of sulfadoxine/pyrimethamine, and might exacerbate placental malaria. We explored emerging lineages of dhps mutant haplotypes in Malawi, the Democratic Republic of the Congo, and Tanzania by using analyses of genetic microsatellites flanking the dhps locus. In Malawi, a triple-mutant dhps SGEG (mutant amino acids are underlined) haplotype emerged in 2010 that was closely related to pre-existing double-mutant SGEA haplotypes, suggesting local origination in Malawi. When we compared mutant strains with parasites from the Democratic Republic of the Congo and Tanzania by multiple independent analyses, we found that SGEG parasites were partitioned into separate lineages by country. These findings support a model of local origination of SGEG dhps haplotypes, rather than geographic diffusion, and have implications for investigations of emergence and effects of parasite drug resistance.
doi:10.3201/eid2007.131720
PMCID: PMC4073871  PMID: 24960247
malaria; Plasmodium falciparum; parasites; sulfadoxine; drug resistance; lineages; genetics; haplotypes; population; eastern Africa
7.  Efficacy of sulphadoxine-pyrimethamine for intermittent preventive treatment of malaria in pregnancy, Mansa, Zambia 
Malaria Journal  2014;13:227.
Background
Intermittent preventive treatment of malaria in pregnancy (IPTp) with sulphadoxine-pyrimethamine (SP) decreases adverse effects of malaria during pregnancy. Zambia implemented its IPTp-SP programme in 2003. Emergence of SP-resistant Plasmodium falciparum threatens this strategy. The quintuple mutant haplotype (substitutions in N51I, C59R, S108N in dhfr and A437G and K540E in dhps genes), is associated with SP treatment failure in non-pregnant patients with malaria. This study examined efficacy of IPTp-SP and presence of the quintuple mutant among pregnant women in Mansa, Zambia.
Methods
In Mansa, an area with high malaria transmission, HIV-negative pregnant women presenting to two antenatal clinics for the 1st dose of IPTp-SP with asymptomatic parasitaemia were enrolled and microscopy for parasitaemia was done weekly for five weeks. Outcomes were parasitological failure and adequate parasitological response (no parasitaemia during follow-up). Polymerase chain reaction assays were employed to distinguish recrudescence from reinfection, and identify molecular markers of SP resistance. Survival analysis included those who had reinfection and incomplete follow-up (missed at least one follow-up).
Results
Of the 109 women included in the study, 58 (53%) completed all follow-up, 34 (31%) had incomplete follow-up, and 17 (16%) were lost to follow-up after day 0. Of those who had complete follow-up, 15 (26%, 95% confidence interval [CI] [16–38]) had parasitological failure. For the 92 women included in the survival analysis, median age was 20 years (interquartile range [IQR] 18–22), median gestational age was 22 weeks (IQR range 20–24), and 57% were primigravid. There was no difference in time to failure in primigravid versus multigravid women. Of the 84 women with complete haplotype data for the aforementioned loci of the dhfr and dhps genes, 53 (63%, 95% CI [50–70]) had quintuple mutants (two with an additional mutation in A581G of dhps). Among women with complete follow-up and quintuple mutants, 22% had parasitological failure versus 0% without (p = 0.44).
Conclusions
While underpowered, this study found 26% failure rates of SP given the moderate prevalence of the quintuple mutant haplotype. Despite the presence of resistance, SP retained some efficacy in clearing parasites in pregnant women, and may remain a viable option for IPTp in Zambia.
doi:10.1186/1475-2875-13-227
PMCID: PMC4053579  PMID: 24909578
Malaria; Pregnancy; Intermittent-preventive treatment; Sulphadoxine-pyrimethamine
8.  Genetic Evidence of Importation of Drug-Resistant Plasmodium falciparum to Guatemala from the Democratic Republic of the Congo 
Emerging Infectious Diseases  2014;20(6):932-940.
Molecular markers and population genetics were effective tracking tools.
Imported malaria threatens control and elimination efforts in countries that have low rates of transmission. In 2010, an outbreak of Plasmodium falciparum malaria was reported among United Nations peacekeeping soldiers from Guatemala who had recently returned from the Democratic Republic of the Congo (DRC). Epidemiologic evidence suggested that the soldiers were infected in the DRC, but local transmission could not be ruled out in all cases. We used population genetic analyses of neutral microsatellites to determine the outbreak source. Genetic relatedness was compared among parasites found in samples from the soldiers and parasite populations collected in the DRC and Guatemala; parasites identified in the soldiers were more closely related to those from the DRC. A phylogenetic clustering analysis confirms this identification with >99.9% confidence. Thus, results support the hypothesis that the soldiers likely imported malaria from the DRC. This study demonstrates the utility of molecular genotyping in outbreak investigations.
doi:10.3201/eid2006.131204
PMCID: PMC4036788  PMID: 24856348
Malaria; Plasmodium falciparum; protozoan; parasite; Anopheles mosquito; microsatellites; molecular genotyping; drug-resistant; chloroquine phosphate; antimalarial; Guatemala; Democratic Republic of the Congo
9.  Dihydropteroate Synthase Mutations in Pneumocystis Pneumonia: Impact of Applying Different Definitions of Prophylaxis, Mortality Endpoints and Mutant in a Single Cohort 
Medical mycology  2013;51(6):568-575.
Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations are well-reported. Although sulfa prophylaxis generally is associated with DHPS mutant infection, whether mutant infection is associated with poorer clinical outcomes is less clear. The differing definitions of sulfa prophylaxis and the different mortality endpoints used in these studies may be one explanation for the conflicting study results. Applying different definitions of prophylaxis, mortality endpoints and DHPS mutant to 301 HIV-infected patients with Pneumocystis pneumonia, we demonstrate that prophylaxis, irrespective of definition, increased the risk of infection with pure mutant (any prophylaxis: AOR 4.00, 95% CI: 1.83–8.76, p<0.001) but not mixed genotypes (any prophylaxis: AOR 0.78, 95% CI: 0.26–2.36, p=0.65). However, infection with mutant DHPS, irrespective of definition, was not associated with increased mortality (all-cause or PCP death) at the three time-intervals examined (all p>0.05). Future studies should standardize key variables associated with DHPS mutant infection as well as examine DHPS mutant subtypes (pure mutant vs. mixed infections) – perhaps even individual DHPS mutant genotypes – so that data can be pooled to better address this issue.
doi:10.3109/13693786.2013.770604
PMCID: PMC4008324  PMID: 23470037
Pneumocystis jirovecii; PCP; dihydropteroate synthase; DHPS; DHPS mutant
11.  Increased prevalence of dhfr and dhps mutants at delivery in Malawian pregnant women receiving intermittent preventive treatment for malaria 
Summary
In the context of an Intermittent preventive treatment (IPTp) trial for pregnant women in Malawi, P. falci-parum samples from 85 women at enrollment and 35 women at delivery were genotyped for mutations associated with sulfadoxine-pyrimethamine resistance. The prevalence of the highly resistant haplotype with mutations at codons 51 and 108 of dihydrofolate reductase (dhfr) and codons 437 and 540 of dihydropteroate synthase (dhps) increased from 81% at enrollment to 100% at delivery (p=0.01). Pregnant women who were smear-positive at enrollment were more likely to have P. falciparum parasitemia at delivery. These results lend support to concerns that IPTp use may lead to increased drug resistance in pregnant women during pregnancy and emphasize the importance of screening pregnant women for malaria parasites in areas with prevalent SP resistance even when they are already on IPTp.
doi:10.1111/tmi.12028
PMCID: PMC3553254  PMID: 23198734
malaria; Intermittent preventive treatment; pregnancy; dhfr; dhps; Malawi
12.  Parasite clearance following treatment with sulphadoxine-pyrimethamine for intermittent preventive treatment in Burkina-Faso and Mali: 42-day in vivo follow-up study 
Malaria Journal  2014;13:41.
Background
Intermittent Preventive Treatment in pregnancy (IPTp) with sulphadoxine-pyrimethamine (SP) is widely used for the control of malaria in pregnancy in Africa. The emergence of resistance to SP is a concern requiring monitoring the effectiveness of SP for IPTp.
Methods
This was an in-vivo efficacy study to determine the parasitological treatment response and the duration of post-treatment prophylaxis among asymptomatic pregnant women receiving SP as part of IPTp in Mali and Burkina-Faso. The primary outcome was the PCR-unadjusted % of patients with parasites recurrence by day 42 defined as a positive diagnostic test by malaria smear at any visit between days 4 and 42. Treatment failure was based on the standard World Health Organization criteria. The therapeutic response was estimated using the Kaplan-Meier curve.
Results
A total of 580 women were enrolled in Mali (N=268) and Burkina-Faso (N=312) and followed weekly for 42 days. Among these, 94.3% completed the follow-up. The PCR-unadjusted cumulative risk of recurrence by day 42 was 4.9% overall, and 3.2% and 6.5% in Mali and Burkina Faso respectively (Hazard Ratio [HR] =2.14, 95%, CI [0.93-4.90]; P=0.070), and higher among the primi– and secundigravida (6.4%) than multigravida (2.2%, HR=3.01 [1.04-8.69]; P=0.042). The PCR-adjusted failure risk was 1.1% overall (Mali 0.8%, Burkina-Faso 1.4%). The frequencies (95% CI) of the dhfr double and triple mutant and dhps 437 and 540 alleles mutant genotype at enrolment were 24.2% (23.7-25.0), 4.7% (4.4-5.0), and 21.4% (20.8-22.0) and 0.37% (0.29-0.44) in Mali, and 7.1% (6.5-7.7), 44.9% (43.8-46.0) and 75.3% (74.5-76.2) and 0% in Burkina-Faso, respectively. There were no dhfr 164L or dhps 581G mutations.
Conclusion
SP remains effective at clearing existing infections when provided as IPTp to asymptomatic pregnant women in Mali and Burkina. Continued monitoring of IPTp-SP effectiveness, including of the impact on birth parameters in this region is essential.
doi:10.1186/1475-2875-13-41
PMCID: PMC3914849  PMID: 24484467
Malaria; Pregnancy; Intermittent; Sulphadoxine-pyrimethamine; Resistance; Mali; Burkina-Faso
13.  Dielectric relaxation of high-k oxides 
Nanoscale Research Letters  2013;8(1):456.
Frequency dispersion of high-k dielectrics was observed and classified into two parts: extrinsic cause and intrinsic cause. Frequency dependence of dielectric constant (dielectric relaxation), that is the intrinsic frequency dispersion, could not be characterized before considering the effects of extrinsic frequency dispersion. Several mathematical models were discussed to describe the dielectric relaxation of high-k dielectrics. For the physical mechanism, dielectric relaxation was found to be related to the degree of polarization, which depended on the structure of the high-k material. It was attributed to the enhancement of the correlations among polar nanodomain. The effect of grain size for the high-k materials' structure mainly originated from higher surface stress in smaller grain due to its higher concentration of grain boundary.
doi:10.1186/1556-276X-8-456
PMCID: PMC4228250  PMID: 24180696
Frequency dispersion; High-k; Grain size; Dielectric relaxation
14.  Peanut Allergen Threshold Study (PATS): validation of eliciting doses using a novel single-dose challenge protocol 
Background
The eliciting dose (ED) for a peanut allergic reaction in 5% of the peanut allergic population, the ED05, is 1.5 mg of peanut protein. This ED05 was derived from oral food challenges (OFC) that use graded, incremental doses administered at fixed time intervals. Individual patients’ threshold doses were used to generate population dose-distribution curves using probability distributions from which the ED05 was then determined. It is important to clinically validate that this dose is predictive of the allergenic response in a further unselected group of peanut-allergic individuals.
Methods/Aims
This is a multi-centre study involving three national level referral and teaching centres. (Cork University Hospital, Ireland, Royal Children’s Hospital Melbourne, Australia and Massachusetts General Hospital, Boston, U.S.A.) The study is now in process and will continue to run until all centres have recruited 125 participates in each respective centre.
A total of 375 participants, aged 1–18 years will be recruited during routine Allergy appointments in the centres. The aim is to assess the precision of the predicted ED05 using a single dose (6 mg peanut = 1.5 mg of peanut protein) in the form of a cookie. Validated Food Allergy related Quality of Life Questionnaires-(FAQLQ) will be self-administered prior to OFC and 1 month after challenge to assess the impact of a single dose OFC on FAQL. Serological and cell based in vitro studies will be performed.
Conclusion
The validation of the ED05 threshold for allergic reactions in peanut allergic subjects has potential value for public health measures. The single dose OFC, based upon the statistical dose-distribution analysis of past challenge trials, promises an efficient approach to identify the most highly sensitive patients within any given food-allergic population.
doi:10.1186/1710-1492-9-35
PMCID: PMC3850217  PMID: 24028324
Eliciting dose (ED); Food Allergy related Quality of Life Questionnaires-(FAQLQ); Single dose; Peanut thresholds; Oral Food Challenges (OFC); Voluntary Incidental Trace Allergen Labelling (VITAL); Peanut Allergen Threshold Study (PATS)
15.  Does public subsidy of the cost of malaria chemoprophylaxis reduce imported malaria? A comparative policy analysis 
Malaria Journal  2013;12:238.
Background
Chemoprophylaxis is recommended for at-risk travellers visiting malaria endemic regions. The majority of travellers with imported malaria have not used this, and travellers visiting friends and relatives have the largest burden of malaria and the lowest compliance to chemoprophylaxis. In 1995, the UK’s Department of Health (DH) implemented a policy to make travellers fully responsible for the cost when purchasing chemoprophylaxis. This policy was not implemented in three Primary Care Trusts (PCTs) in London due to concern about the potential increase of imported malaria in their residents, and they maintained the public subsidy. An impact evaluation of the policy change was undertaken to determine if the continued subsidy reduced the incidence of imported malaria in one of the boroughs where the subsidy was maintained when compared to a borough where no subsidy was provided.
Methods
Between 2007 and 2010 prescriptions for malaria chemoprophylaxis were collected from pharmacy records and PCTs, and all cases of imported malaria reported from the tertiary hospital in each of the two boroughs were compared.
Results
The dispensed chemoprophylaxis prescriptions were nearly 8.8 times higher in Lambeth (where subsidized drugs were provided), than in Hackney. A Poisson model revealed significantly fewer reports of imported malaria per capita were made in Lambeth compared to Hackney (p = 0.042).
Conclusions
The difference in malaria reports between the boroughs only just reached statistical significance, despite the considerable difference in chemoprophylaxis prescribing between the boroughs. Some travellers may not consider using chemoprophylaxis, irrespective of the cost. Regular evaluations of the recent policy changes in areas where malaria is subsidized will be important.
doi:10.1186/1475-2875-12-238
PMCID: PMC3723845  PMID: 23848986
Imported malaria; Chemoprophylaxis; Subsidized costs; Evaluation
16.  Antenatal Receipt of Sulfadoxine-Pyrimethamine Does Not Exacerbate Pregnancy-Associated Malaria Despite the Expansion of Drug-Resistant Plasmodium falciparum: Clinical Outcomes From the QuEERPAM Study 
Antenatal preventive administration of sulfadoxine-pyrimethamine did not potentiate pregnancy-associated malaria morbidity despite expansion and fixation of drug-resistant malaria parasites. Sulfadoxine-pyrimethamine may be a safe component of malaria prevention programs without causing adverse effects on birth outcomes.
Background. Antenatal intermittent preventive therapy with 2 doses of sulfadoxine-pyrimethamine (IPTp-SP) is the mainstay of efforts in sub-Saharan Africa to prevent pregnancy-associated malaria (PAM). Recent studies report that drug resistance may cause IPTp-SP to exacerbate PAM morbidity, raising fears that current policies will cause harm as resistance spreads.
Methods. We conducted a serial, cross-sectional analysis of the relationships between IPTp-SP receipt, SP-resistant Plasmodium falciparum, and PAM morbidity in delivering women during a period of 9 years at a single site in Malawi. PAM morbidity was assessed by parasite densities, placental histology, and birth outcomes.
Results. The prevalence of parasites with highly SP-resistant haplotypes increased from 17% to 100% (P < .001), and the proportion of women receiving full IPTp (≥2 doses) increased from 25% to 82% (P < .001). Women who received full IPTp with SP had lower peripheral (P = .018) and placental (P < .001) parasite densities than women who received suboptimal IPTp (<2 doses). This effect was not significantly modified by the presence of highly SP-resistant haplotypes. After adjustment for covariates, the receipt of SP in the presence of SP-resistant P. falciparum did not exacerbate any parasitologic, histologic, or clinical measures of PAM morbidity.
Conclusions. In this longitudinal study of malaria at delivery, the receipt of SP as IPTp did not potentiate PAM morbidity despite the increasing prevalence and fixation of SP-resistant P. falciparum haplotypes. Even when there is substantial resistance, SP may be used in modified IPTp regimens as a component of comprehensive antenatal care.
doi:10.1093/cid/cis301
PMCID: PMC3520448  PMID: 22441649
17.  Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin 
BioMed Research International  2013;2013:756789.
Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing.
doi:10.1155/2013/756789
PMCID: PMC3708386  PMID: 23878817
18.  Impact of haemoglobinopathies on the clinical epidemiology of malaria: a systematic review and meta-analysis 
The Lancet Infectious Diseases  2012;12(6):457-468.
SUMMARY
Background
Haemoglobinopathies variously reduce the risk of developing malaria syndromes. Quantifying these relationships may strengthen the foundation for translational studies of malaria pathogenesis and immunity.
Methods
The databases of MEDLINE and Embase (1950 – September 9, 2011) were searched to identify studies that estimated the risk of malaria in patients with and without haemoglobinopathies. Additional studies were identified from reference lists. Included outcomes were Plasmodium falciparum-related outcomes of severe malaria, uncomplicated malaria, asymptomatic parasitaemia, or pregnancy-associated malaria, and P. vivax malaria. Two independent reviewers identified studies, assessed their quality, and extracted data; data were meta-analyzed when outcomes were reported in more than one study.
Findings
Of 62 identified studies, 44 reported on HbAS, 19 on HbAC and HbCC, and 18 on α-thalassaemia. Case-control studies showed a decreased risk of severe malaria for HbAS (summary Odds Ratio [OR] 0.09; 95% confidence interval [CI] 0.06 – 0.12), HbCC (summary OR 0.27; 95% CI 0.11 – 0.63), homozygous α-thalassaemia (summary OR 0.63; 95% CI 0.48 – 0.83), HbAC (summary OR 0.83; 95% CI 0.74 – 0.92), and heterozygous α-thalassaemia (summary OR 0.83; 95% CI 0.74 – 0.92). Only HbAS was consistently associated with protection from uncomplicated malaria (summary Incidence Rate Ratio 0.69; 95% CI 0.61 – 0.79); none demonstrated protection from asymptomatic parasitaemia. There was a paucity of clinical studies investigating β-thalassaemia, HbE, P. vivax malaria, and pregnancy-associated malaria.
Interpretation
Protection from severe malaria syndromes is significant for HbAS, HbCC, HbAC, and homozygous and heterozygous α-thalassaemia, but these haemoglobinopathies differ substantially in the degrees of protection. Protection from uncomplicated malaria and asymptomatic parasitaemia is mild or absent. By attenuating the severity of malaria, haemoglobinopathies serve as a model for investigating the mechanisms of malaria pathogenesis and immunity.
doi:10.1016/S1473-3099(12)70055-5
PMCID: PMC3404513  PMID: 22445352
19.  Hemoglobinopathies: Slicing the Gordian Knot of Plasmodium falciparum Malaria Pathogenesis 
PLoS Pathogens  2013;9(5):e1003327.
Plasmodium falciparum malaria kills over 500,000 children every year and has been a scourge of humans for millennia. Owing to the co-evolution of humans and P. falciparum parasites, the human genome is imprinted with polymorphisms that not only confer innate resistance to falciparum malaria, but also cause hemoglobinopathies. These genetic traits—including hemoglobin S (HbS), hemoglobin C (HbC), and α-thalassemia—are the most common monogenic human disorders and can confer remarkable degrees of protection from severe, life-threatening falciparum malaria in African children: the risk is reduced 70% by homozygous HbC and 90% by heterozygous HbS (sickle-cell trait). Importantly, this protection is principally present for severe disease and largely absent for P. falciparum infection, suggesting that these hemoglobinopathies specifically neutralize the parasite's in vivo mechanisms of pathogenesis. These hemoglobin variants thus represent a “natural experiment” to identify the cellular and molecular mechanisms by which P. falciparum produces clinical morbidity, which remain partially obscured due to the complexity of interactions between this parasite and its human host. Multiple lines of evidence support a restriction of parasite growth by various hemoglobinopathies, and recent data suggest this phenomenon may result from host microRNA interference with parasite metabolism. Multiple hemoglobinopathies mitigate the pathogenic potential of parasites by interfering with the export of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of the host red blood cell. Few studies have investigated their effects upon the activation of the innate and adaptive immune systems, although recent murine studies suggest a role for heme oxygenase-1 in protection. Ultimately, the identification of mechanisms of protection and pathogenesis can inform future therapeutics and preventive measures. Hemoglobinopathies slice the “Gordian knot” of host and parasite interactions to confer malaria protection, and offer a translational model to identify the most critical mechanisms of P. falciparum pathogenesis.
doi:10.1371/journal.ppat.1003327
PMCID: PMC3656091  PMID: 23696730
20.  Adaptive evolution and fixation of drug-resistant Plasmodium falciparum genotypes in pregnancy-associated malaria: 9-year results from the QuEERPAM study 
Infection, Genetics and Evolution  2011;12(2):282-290.
Sulfadoxine-pyrimethamine (SP) has been widely deployed in Africa for malaria control and molecular evidence of parasite drug-resistance is prevalent. However, the temporal effects on the selection of Plasmodium falciparum are not well understood. We conducted a retrospective serial cross-sectional study between 1997 and 2006 to investigate changes in drug-resistant malaria among pregnant women delivering at a single hospital in Blantyre, Malawi. P. falciparum parasites were genotyped for parasite clone multiplicity and drug-resistance mutations, and the strength of selection upon mutant genotypes was quantified. Five mutations in the dihydrofolate reductase and dihydropteroate synthase genes began at moderate frequencies and achieved fixation by 2005; the frequency of the highly-SP-resistant “quintuple mutant” haplotype increased from 19% to 100%. The selective advantage of alleles and haplotypes were quantified with selection coefficients: Selection was positive on all mutant alleles and haplotypes associated with SP resistance, and the relative fitness of the quintuple mutant haplotype was 0.139 (95% C.I. 0.067 – 0.211), indicating a substantial positive selective advantage. Mutations that confer higher levels of resistance to SP did not emerge. SP-resistant haplotypes were rapidly selected for and fixed in P. falciparum populations infecting pregnant women while SP was widely deployed in Malawi. These results underscore the pressing need for new preventive measures for pregnancy-associated malaria and provide a real-world model of the selection landscape malaria parasites.
doi:10.1016/j.meegid.2011.11.006
PMCID: PMC3293939  PMID: 22119749
malaria; pregnancy-associated malaria; drug resistance; parasite evolution
21.  Low prevalence of Pneumocystis jirovecii lung colonization in Ugandan HIV-infected patients hospitalized with non-Pneumocystis pneumonia 
Pneumocystis jirovecii is an important opportunistic infection in HIV-infected patients. In the developed world, P. jirovecii epidemiology is marked by frequent colonization in immunosuppressed patients, but data on the prevalence of colonization is very limited in sub-Saharan Africa, where the majority of persons living with HIV reside. Our objective was to describe the epidemiology of P. jirovecii colonization among HIV-positive patients in a cross-sectional, hospital-based study of patients admitted with suspected pneumonia in Kampala, Uganda. P. jirovecii was detectable in bronchoalveolar lavage fluid from 7 of 124 (6%) consecutive patients with non-Pneumocystis pneumonia. Colonization was not associated with patient demographic or clinical information. This prevalence is substantially lower than in published studies in the developed world, and suggests that there is a limited reservoir of organisms for clinical infections in this Ugandan population. These findings may partially explain the low incidence of Pneumocystis pneumonia in Uganda and other sub-Saharan African countries.
doi:10.1016/j.diagmicrobio.2011.10.009
PMCID: PMC3308345  PMID: 22153850
Pneumocystis jirovecii; colonization; pneumonia; AIDS
22.  Plasmodium falciparum sulfadoxine resistance is geographically and genetically clustered within the DR Congo 
Scientific Reports  2013;3:1165.
Understanding the spatial clustering of Plasmodium falciparum populations can assist efforts to contain drug-resistant parasites and maintain the efficacy of future drugs. We sequenced single nucleotide polymorphisms (SNPs) in the dihydropteroate synthase gene (dhps) associated with sulfadoxine resistance and 5 microsatellite loci flanking dhps in order to investigate the genetic backgrounds, genetic relatedness, and geographic clustering of falciparum parasites in the Democratic Republic of the Congo (DRC). Resistant haplotypes were clustered into subpopulations: one in the northeast DRC, and the other in the balance of the DRC. Network and clonal lineage analyses of the flanking microsatellites indicate that geographically-distinct mutant dhps haplotypes derive from separate lineages. The DRC is therefore a watershed for haplotypes associated with sulfadoxine resistance. Given the importance of central Africa as a corridor for the spread of antimalarial resistance, the identification of the mechanisms of this transit can inform future policies to contain drug-resistant parasite strains.
doi:10.1038/srep01165
PMCID: PMC3558697  PMID: 23372922
24.  Quantification of the Burden and Consequences of Pregnancy-Associated Malaria in the Democratic Republic of the Congo 
The Journal of Infectious Diseases  2011;204(11):1762-1771.
Background. Pregnancy-associated malaria (PAM) produces poor birth outcomes, but its prevalence is commonly estimated in convenience samples.
Methods. We assessed the prevalence of malaria using real-time polymerase chain reaction (PCR) and estimated the consequences of infection on birth outcomes, using specimens from a nationally representative sample of 4570 women of childbearing age (WOCBA) responding to the 2007 Demographic and Health Survey in Democratic Republic of the Congo (DRC).
Results. Overall, 31.2% (95% confidence interval [CI], 29.2–33.1) of WOCBA were parasitemic, which was significantly more common in pregnant (37.2% [31.0–43.5]) than nonpregnant women (30.4% [CI, 28.4–32.5], prevalence ratio [PR] 1.22 [1.02–1.47]). Plasmodium falciparum was highest among pregnant women (36.6% vs 28.8%, PR 1.27 [1.05–1.53]). By contrast, P malariae was less common in pregnant (0.6%) compared with nonpregnant women (2.7%, PR 0.23 [0.09–0.56]). Extrapolation of the prevalence estimate to the population at risk of malaria in DRC suggests 1.015 million births are affected by P falciparum infection annually, and that adherence to preventive measures could prevent up to 549 000 episodes of pregnancy-associated malaria and 47 000 low-birth-weight births.
Conclusions. Pregnancy-associated malaria and its consequences are highly prevalent in the DRC. Increasing the uptake of malaria preventive measures represents a significant opportunity to improve birth outcomes and neonatal health.
doi:10.1093/infdis/jir625
PMCID: PMC3635529  PMID: 21990422
25.  Low Prevalence of Pneumocystis pneumonia (PCP) but High Prevalence of Pneumocystis dihydropteroate synthase (dhps) Gene Mutations in HIV-Infected Persons in Uganda 
PLoS ONE  2012;7(11):e49991.
Pneumocystis jirovecii pneumonia (PCP) is an important opportunistic infection in patients infected with HIV, but its burden is incompletely characterized in those areas of sub-Saharan Africa where HIV is prevalent. We explored the prevalence of both PCP in HIV-infected adults admitted with pneumonia to a tertiary-care hospital in Uganda and of putative P. jirovecii drug resistance by mutations in fungal dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr). In 129 consecutive patients with sputum smears negative for mycobacteria, 5 (3.9%) were diagnosed with PCP by microscopic examination of Giemsa-stained bronchoalveolar lavage fluid. Concordance was 100% between Giemsa stain and PCR (dhps and dhfr). PCP was more prevalent in patients newly-diagnosed with HIV (11.4%) than in patients with known HIV (1.1%; p = 0.007). Mortality at 2 months after discharge was 29% overall: 28% among PCP-negative patients, and 60% (3 of 5) among PCP-positive patients. In these 5 fungal isolates and an additional 8 from consecutive cases of PCP, all strains harbored mutant dhps haplotypes; all 13 isolates harbored the P57S mutation in dhps, and 3 (23%) also harbored the T55A mutation. No non-synonymous dhfr mutations were detected. PCP is an important cause of pneumonia in patients newly-diagnosed with HIV in Uganda, is associated with high mortality, and putative molecular evidence of drug resistance is prevalent. Given the reliability of field diagnosis in our cohort, future studies in sub-Saharan Africa can investigate the clinical impact of these genotypes.
doi:10.1371/journal.pone.0049991
PMCID: PMC3500344  PMID: 23166805

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