Plasmodium falciparum. An important role in immune regulation is assigned to the apoptosis-signaling cell surface receptor CD95 , encoded by the gene FAS. Here, a candidate-gene association study including variant discovery at the FAS gene locus was carried out in a case-control group comprising 1,195 pediatric cases of severe falciparum malaria and 769 unaffected controls from a region highly endemic for malaria in Ghana, West Africa. We found the A allele of c.−436C>A (rs9658676) located in the promoter region of FAS to be significantly associated with protection from severe childhood malaria  and confirmed this finding in a replication group of 1,412 additional severe malaria cases and 2,659 community controls from the same geographic area. The combined analysis resulted in an odds ratio of 0.71 . The association applied to c.−436AA homozygotes  and to a lesser extent to c.−436AC heterozygotes , and also to all phenotypic subgroups studied, including severe malaria anemia, cerebral malaria, and other malaria complications. Quantitative FACS analyses assessing CD95 surface expression of peripheral blood mononuclear cells of naïve donors showed a significantly higher proportion of CD69+CD95+ cells among persons homozygous for the protective A allele compared to AC heterozygotes and CC homozygotes, indicating a functional role of the associated CD95 variant, possibly in supporting lymphocyte apoptosis.Human genetics and immune responses are considered to critically influence the outcome of malaria infections including life-threatening syndromes caused by  Plasmodium falciparum is a major health burden, causing approximately one million fatalities annually, predominantly among young children in Sub-Saharan Africa. The occurrence of severe malaria may depend on a complex interplay of transmission dynamics and the development of a protective immune response but also on heritable differences in the susceptibility to the disease. In two large studies including a total of 2,607 affected children and 3,428 apparently healthy individuals from Ghana, West Africa, we investigated genetic variants of the FAS gene, which encodes CD95, a molecule critically involved in the programmed cell death of lymphocytes. We found that a single nucleotide variant in the FAS promoter was associated with a 29%–reduced risk of developing severe malaria. In individuals carrying two copies of the protective allele, a higher proportion of activated lymphocytes was found to express CD95. These findings indicate that a predisposition to an increased expression of CD95 may help to protect from severe malaria, possibly by rendering activated T-lymphocytes more susceptible to programmed cell death.Severe malaria caused by infection with the protozoan parasite  Plasmodium falciparum worldwide causes approximately one million fatalities annually, mostly among children in Sub-Saharan Africa Severe malaria caused by infection with the protozoan parasite FAS  at chromosome 10q24.1 encodes the cell surface receptor CD95 , known as the prototypic death receptor The gene in vitro, and serum samples of patients with P. falciparum malaria show elevated levels of soluble CD95L as compared to healthy subjects P. falciparum extract caused a marked increase in the expression of functional CD95 P. falciparum, in which its precise function has yet to be defined.Indications for an implication of CD95 during severe malaria episodes have been gained from several studies. For instance, lymphocytes from malaria patients were found to express markers of apoptosis and are susceptible to activation-induced cell death (AICD) FAS genetic variants on malaria susceptibility by conducting a candidate-gene association study, which involved a variant screen through re-sequencing and genotyping of selected variants in the FAS gene in a sample set including 1195 severe malaria cases and 769 apparently healthy controls recruited in Ghana, West Africa. In addition, the impact of variant c.−436C>A was further investigated in a replication study from the same geographical area, including 1412 children with severe malaria and 2659 community controls. With regard to the association of variant c.−436C>A (rs9658676) with protection from severe malaria in our study we characterized the allele-dependent CD95 surface expression of PBMCs by quantitative fluorescence-activated cell sorting (FACS).In the present study, we sought to elucidate the impact of FAS gene including exonic and regulatory regions in 46 individuals from our study group. Among the 16 single nucleotide polymorphisms (SNPs) detected by re-sequencing, two polymorphisms, c.−671G>A (rs1800682) in the 5′-flanking region and c.46G>A (rs3218619) in exon 1 had previously been shown to exhibit a substantial effect on the expression and function of the receptor At total of 19 variants were analyzed in the initial case-control study. Genotypes for 14 of these polymorphisms were derived using the Affymetrix Genome-Wide Human SNP Array 6.0. Additional five variants were selected for genotyping after they were identified by re-sequencing the −4. When analysing genotypes in the first case-control sample (n = 1964) a significant result was obtained for the promoter variant c.−436C>A in the trend test (p = 1.3×10−3). After adjustment for multiple testing, the association of c.−436C>A in the logistic regression analysis remained significant for the additive model of inheritance  and reached borderline significance for the dominant model  (Genotype frequencies did not deviate from Hardy-Weinberg Equilibrium (HWE) (p>0.01) except for c.334+46C>T, which in the case group showed a deviation with p = 6.2×10 = 0.05) . The A a−5). Combining the results of the two case-control groups yielded a significance level of p = 1.8×10−7  c.−436AA homozygous individuals appear to be protected to a greater extent than heterozygous individuals . The data are compatible with an additive inheritance model , whereas both the dominant and recessive genetic models were rejected .−3; ncases = 569) to a maximum of 0.73 when including cases with complications such as hyperlactatemia, prostration, and hyperparasitemia collectively . The results pointed towards a protective effect of the A allele in all sub-phenotypes of severe malaria studied. When analyzing log-transformed parasite densities in the combined case groups (n = 2353), no significant differences between the three genotypic groups were detected (p = 0.67).With the aim of specifying the association of marker c.−436C>A with regard to severe malaria pathology, a stratified analysis for clinically distinct subgroups was carried out. After combining the results from both study groups ORs for the additive mode of inheritance were found to be similar for the clinical subgroups of cerebral malaria, severe anemia, and collectively other complications independent of sample size differences . CombineIn order to evaluate a possible association of c.−436C>A with mild malaria, a quantitative transmission disequilibrium test (qTDT) was performed with the numbers of uncomplicated malaria episodes experienced by 390 Ghanaian siblings over a period of 31 weeks empirical being 0.04. This was seen only under a recessive model of inheritance. As the significant findings obtained with c.−436C>A were limited to the assumption of an additive inheritance mode, the association of c.334+46C>T being of borderline significance was not pursued further.Of the additional 18 variants studies , only c.FAS locus displayed remarkably low pairwise linkage disequilibria between markers, particularly in the promoter region. The variant c.−436C>A shares a maximum r2-value of 0.20 with c.46G>A, located 12.6 kb apart in exon 2 of FAS  for CD95+ cells were similar among the three genotypic groups for both, the entire PBMC fractions as well as the differentiated subpopulations  or homozygous c.−436CC genotypes   . No diffFAS gene was associated with protection from severe malaria in Ghanaian children. No differences were found as to the major clinical forms of the disease. Statistical analyses resulted in an OR of 0.71 for severe malaria collectively, and ranged between 0.68 and 0.73 for distinct clinical phenotypes comprising severe malaria anemia, cerebral malaria, and other forms of severe malaria, which in our study included hyperlactatemia, prostration, and hyperparasitemia.Collectively, our results show that the variant c.−436C>A in the promoter region of the http://www.gene-regulation.com/pub/databases.html). We did not obtain any evidence for additional variants which might be the causal ones by re-sequencing of the genomic region and searching for other variants which are linked to c.−436C>A and might have a stronger association with the phenotypes studied. However, re-sequencing was limited to 46 individuals and the linkage analyses were restricted to a sequence segment of 1 kb and to variants with an MAF of greater than 5%. Therefore, further association signals could have remained undiscovered and additional efforts are needed to convincingly identify the causal variant. c.334+46C>T, an intronic FAS variant which was associated with marginal significance, was not linked to −436C>A and followed a discordant model of inheritance.It is important to note, however, that the data presented solely allow the conclusion that c.−436C>A is a marker for a haplotype containing one or more genetic variants which reduce the risk of acquiring severe malaria. They do not show that c.−436C>A itself exerts this function. c.−436C>A is not predicted to directly affect a transcription factor binding site  cells, and platelets Speculating about a possible mechanism underlying the observed association, it is worth mentioning that in our study the c.−436C>A variants were not associated with differences in parasite counts, neither among our patients during a severe malaria episode nor in a longitudinal observation of 390 Ghanaian children monitored by weekly examinations over 31 weeks. In this cohort, c.−436C>A was not associated with differences in the frequency of mild malaria episodes either, although the latter finding has to be interpreted with caution because of a moderate power to detect significant differences. Nevertheless, the mechanism underlying the c.−436C>A association presumably affects the pathogenesis of the severe forms of malaria. It appears most likely to assume that an increased expression of CD95 associated with c.−436AA and possibly c.−436CA facilitates the programmed cell death of lymphocytes upon immune activation. Thereby the protective FAS allele could alleviate immunopathology. As various studies have shown, immunopathology contributes substantially to the pathogenesis of severe malaria episodes FAS was identified to be among the genes with at least one SNP associated with protozoan diversity in 52 human populations distributed worldwide.With allele frequencies of 0.12 in the present study population, 0.16 in the Yoruba from Nigeria, and 0.09 in African Americans, the A allele has exclusively been found in African populations or those with African ancestry and appears to be absent in Asian and European populations. It is possible to envisage a benefit for individuals carrying the A allele in regions endemic for malaria, which therefore might undergo a positive selection process maintaining the allele in a population. Supportive evidence for this evolutionary aspect comes from a genome-wide search for variants subjected to protozoa-driven selective pressure P. falciparum infections. It is possible that its beneficial effect during severe malaria is counterbalanced by an adverse effect of the same allele in other infections. For instance, in patients with AIDS, a disease with too much apoptosis, the depletion of CD4+ T helper cells has been shown to be mediated by CD95 + T helper cells in these patients.Although the protective A allele may have an advantageous effect, with a MAF of 0.12 it appears to be maintained at a relatively low frequency in a population constantly exposed to FAS promoter region, in particular concerning the relevance of the polymorphism c.−436C>A in gene regulation with respect to P. falciparum infections. Further analysis of the CD95/CD95L signaling as part of the immune response to P. falciparum may yield further insights into the pathogenesis of life-threatening childhood malaria.In summary, our study provides a rationale for a more detailed functional characterization of the The studies were approved by the Committee for Research, Publications and Ethics of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.Informed consent was obtained from parents or guardians of all participants for both case-control study groups, and from both parents of families after all procedures were explained in the local language.The present study was carried out in an area highly endemic for falciparum malaria in the Ashanti Region of Ghana, West Africa.FAS gene locus. In addition, five selected variants based on sequencing results (see below) were genotyped in the initial study group.The first case-control sample set comprised 1195 severe malaria cases and 769 controls and had previously been subjected to genome-wide SNP genotyping utilizing the Affymetrix Genome-Wide Human SNP Array 6.0  (manuscript in preparation). In the herein presented candidate-gene study data for SNPs included in the genome-wide chip were retrieved for the The second, independent sample set derived from the same geographical area included 1412 cases and 2659 controls and was used for the replication of significant results for variant c.−436C>A (rs9658676) observed in the first study.See P. falciparum parasites and either of the following clinical findings was diagnosed: (i) level of consciousness according to the Blantyre Coma Score (BCS) <3; (ii) hemoglobin concentration <5 g/dl; (iii) lactate concentration >5 mmol/L As previously described, all severe malaria patients were enrolled at the Komfo Anokye Teaching Hospital, Kumasi, between 2001 and 2005 in parallel with the “Severe Malaria in African Children” study P. falciparum density in mild childhood malaria, genotypes from an additional study with a total of 739 individuals comprising 390 siblings from 147 families were ascertained. DNA samples from parents and children used here were recruited and prepared as part of a genome-wide linkage analysis conducted in the Asante Akim North District, Ashanti region, Ghana P. falciparum. For each individual, the number of mild malaria episodes during the 31 week observation period was counted, whereby multiple episodes within 3 weeks were counted as one, as they were considered as recrudescences. Among the 390 siblings, a total of 504 malaria episodes were counted during the entire follow-up. Assuming a leukocyte count of 8,000 per µl blood parasite counts were recorded per 200 leukocytes (if >10 parasites/200 leukocytes) or 500 leukocytes (if ≤10 parasites/200 leukocytes). With a median of 32 parasites per µl blood, parasite densities ranged from 0–317,360/µl blood. Due the fact that the median point prevalence of malaria parasites was 54%, the 75th percentile of parasite densities was considered a representative value of parasite density for each individual.In order to determine the impact of the variant c.−436C>A on FAS gene were detected by re-sequencing the 5′-UTR, the exons, including intron/exon boundaries, and the 3′-UTR of 46 genomic DNA samples drawn from study participants. In order to capture common alleles in the population, 23 DNA samples were selected from the control group. In addition, 23 genomic DNA samples of severe malaria cases were re-sequenced to cover information about possibly selected alleles which confer excess risk for severe malaria. With 23 samples, the probability of observing a variant with a MAF≥0.05 in a study group is 90% even if HWE does not hold Variants located in the coding regions of the http://frodo.wi.mit.edu/primer3/) against the reference sequence . Sequences of oligonucleotides and PCR conditions are listed in Genomic DNA samples were amplified by PCR using primers that captured a 1000 bp region before the transcription start, the exonic sequences, including 30 bp of their intronic flanking regions, and 600 bp of the 3′-UTR. Oligonucleotides were designed using the Primer3 web-interface . Selection criteria were an estimated MAF of ≥0.05 in at least one of the two groups, cases or controls, in addition to one of the following attributes. (i) The polymorphism leads to a non-synonymous amino-acid exchange in the receptor. (ii) The polymorphism is known to have functional relevance based on literature entries. (iii) The difference in estimated MAF from the re-sequencing between cases and controls is >10%. In agreement with these conditions a set of 5 SNPs, rs1800682, rs9658676, rs3218619, rs3218621, and rs3218614 were genotyped in the initial sample set of 1195 severe malaria cases and 769 controls.In order to extract DNA for genotyping, blood samples were drawn from all participants. 0.5–1 ml blood was collected into citrate, and, before subjected to DNA extraction, the granulocyte fraction obtained from density gradient centrifugation was preserved in 4-M urea. DNA was extracted according to the suppliers' instructions . Prior to genotyping whole-genome amplification (WGA) using 10 ng DNA of each sample was conducted . SNPs were analyzed by allele-specific hybridization in a melting curve analysis based on fluorescence resonance energy transfer (FRET) in a LightTyper device . Oligonucleotides and PCR conditions can be found in the FAS gene locus or surrounding regions of 10 kb adjacent to start and end of the gene. These were then merged with genotypes from the LightTyper platform. Genotypes were called using Birdseed, and standard quality control procedures comprised the exclusion of (i) individuals with SNP-call rates below 0.96, (ii) related individuals with an IBD>12.5% or (iii) individuals with heterozygosity rates <26% or >31%. The cluster plots of individual SNPs were verified by visual inspection. SNPs with call rates <96% were excluded. As part of the standard quality control for the five polymorphisms genotyped on the LightTyper platform, individuals with more than 50% missing genotypes were excluded, and SNPs were allowed to have a maximum of 4% missing genotypes.For individuals part of the genome-wide association (GWA) study genotypes were retrieved for SNPs that are included in the Affymetrix SNP chip and are located at the FAS gene locus  spanning approximately 35 kb was analyzed using primarily PLINK v1.07 empirical)<0.05 were considered significant. After a significant result was obtained, genotypes for variant c.−436C>A were retrieved in the replication set. We calculated results in the replication sample set by logistic regression accordingly and combined the results using the inverse variance weighted fixed effects model.Hence, a genotypic data set comprising a total of 19 SNPs at the 2-test for heterogeneity of ORs based on stratification into ethnic groups was carried out in order to detect possible population stratification.For each SNP a partitioning χThe most plausible genetic model for variant c.−436C>A was selected using the approach of Bagos 2 values Linkage disequilibrium in the gene region was assessed using Haploview v4.1 for visualization of pairwise rth percentile of parasite density using the orthogonal model implemented in QTDT v2.5.1 th percentiles of parasite density.For variant c.−436C>A, the family-based qTDTs were done for the number of mild malaria attacks and the 75PBMCs of 72 naïve adult African individuals from the Ashanti region in Ghana were investigated for their level of CD95 surface expression in relation to the c.−436C>A genotype. Prior to specific antibody-labeling and FACS of cells genotypes for c.−436C>A for all 72 individuals were gained as described above. In order to isolate the PBMC fraction from peripheral blood of donors a 2.5 ml of citrate blood diluted with 2.5 ml RPMI was subjected to Ficoll gradient centrifugation at 4°C. The lymphocyte layer was collected and washed twice with RPMI before PBMCs were stored in freezing medium, RPMI/10% fetal calf serum (FCS)/10%DMSO/1×Penicillin/Strepto-mycin at −80°C for 12 hours and subsequently in liquid nitrogen for long-term storage.1 isotype, P.H. Krammer, Heidelberg, Germany) as well as with mAbs against CD4, CD19, CD8, and CD69  for differentiation of lymphocyte subsets. Biotin-conjugated mouse IgG1 antibody (BD Pharmingen) was used as an isotype control. FACS Canto II flow cytometer and FACS-Diva software  were used to perform analyzes of the cells. As cell counts varied significantly with the donors, a minimum of 10,000 analyzed cells per assay was set for a sample to be included in the analysis.After thawing cell fractions were washed with RPMI medium and re-suspended in 2 ml of pre-chilled blocking solution (1×PBS/10% FCS/10% mouse serum). PBMCs were stained with biotinylated mAb against CD95  using the Mann-Whitney test at the nominal 5% test-level together with corresponding Hodges-Lehmann 95% confidence intervals.The surface expression level of CD95 was assessed in two different ways, first by directly measuring FI of cells expressing CD95 and second by defining the percentage of CD95Figure S1FAS gene locus. Haplotypes tested for association in a sliding window comprising three SNPs.Score statistic global p-values for additive effect of sub-haplotypes at the (TIFF)Click here for additional data file.Table S1FAS from genomic DNA of 23 controls and 23 severe malaria cases identified by re-sequencing. aPosition in transcript NM_000043.3. bAlleles are matched to the forward strand.Variants in regulatory and coding regions of (DOC)Click here for additional data file.Table S2FAS locus. ap-values adjusted for gender, age, and ethnicity.Haplotype-specific p-values from score association tests with severe falciparum malaria based on inferred haplotypes with frequencies >5% at the (DOC)Click here for additional data file.Table S3FAS from genomic DNA. Each reaction mixture contained 10 ng of genomic DNA, 1×PCR Buffer BD, 1 U FIREPol DNA polymerase I , 200 µM of each dNTP, 1 µM of each PCR Primer, above stated concentration of MgCl2, and water to a final volume of 20 µl. PCR conditions were as follows: 94°C for 3 min, 40 cycles of 95°C for 1 min, specific annealing temperature for 1 min, 72°C for 1 min followed 72°C for 10 min.Oligonucleotides and PCR conditions for re-sequencing of regulatory and coding regions of (DOC)Click here for additional data file.Table S4FAS variants. Each reaction mixture contained 2 µl of a 1∶200 dilution of whole-genome wide amplified genomic DNA, 1×PCR Buffer BD, 1× Solution S, 1 U FIREPol DNA polymerase I (Solis BioDyne. Estonia), 200 µM of each dNTP, 1 µM or 0.2 µM of each PCR primer, 0.2 µM of each fluorescence-labeled primer, above stated MgCl2. Concentration, and water to a final volume of 10 µl. PCR conditions were as follows: 95°C for 3 min. 45 cycles of 95°C for 1 min. 55°C for 1 min. 72°C for 1 min, followed by 72°C for 10 min.Oligonucleotides and PCR conditions for genotyping selected (DOC)Click here for additional data file.


During a period of 11 years (2000–2010), the frequency of hyperparasitaemia (HP) (≥4% infected erythrocytes) during bouts of malaria due to Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae in patients referred to Cayenne General Hospital (CGH) in French Guiana and the frequency of their admission to the Intensive Care Unit (ICU) were evaluated.High circulating parasite load is one of the WHO criteria for severe A mean of 1,150 malaria cases were referred to the Parasitology Laboratory of CGH each year over the last decade. During this period, malaria diagnostic (microscopy) and parasitaemia evaluation have remained unchanged: determination of the parasitized erythrocytes percentage with asexual forms on thin blood smears for all cases of parasitaemia exceeding 0.1%. Patients admitted to the ICU can be counted by origin of the request for malaria testing. All the data collected retrospectively were anonymized in a standardized case report form and in database.P. vivax: 56.2%, P. falciparum: 39.5%, co-infection with both species: 3.4%, P. malariae: 0.9%. HP was observed in 262 cases, at a frequency of 4.9% for P. falciparum and only 0.041% for P. vivax, with no recorded cases for P. malariae. The need for intensive care was correlated with P. falciparum parasite load: 12.3% of cases for parasitaemia of 4-9%, 21.2% for parasitaemia 10-19%, 50% for parasitaemia 20-29% and 77.8% for parasitaemia ≥30% (n=9). The patient with the highest parasitaemia  presented a major concomitant lupus flare-up treated with corticoids. He survived without obvious sequelae.Between 2000 and 2010, 12,254 bouts of malaria were confirmed at the Parasitology Laboratory of CHG: P. falciparum and two orders of magnitude less frequent for P. vivax. HP is a severity criterion for falciparum malaria in this endemic area. However, two of the patients with HP ≥30% were not admitted to the ICU and sequel-free cure in malaria patients with 75% parasitaemia is, therefore, possible.In French Guiana during bouts of malaria, HP was observed at a frequency of ~ 5% for  Plasmodium vivax and Plasmodium falciparum predominate, whereas Plasmodium malariae is much rarer. Despite the unfavorable epidemiological conditions , the financial and health equipment resources deployed in French Guiana have ensured that severe forms remain rare and mortality low  = 0.8%. This is lower by a factor > 25 than that for cases of HP (21.9%).The frequency of HP was higher in ICU patients. Over the last 11 years, the total number of bouts of falciparum malaria in individual patients. There are several possible explanations. Circulating parasite load evaluation does not take into account the presence of mature stages in erythrocytes sequestered in deep capillaries  and does not faithfully reflect the true parasite load. Furthermore, the proportion of circulating elements  may vary considerably over time and with the parasite stage reached in bouts involving “synchronous” strains [This finding should not mask the absence of a clear link between PC based on peripheral blood screening and the severity of  strains . Note th strains , but unfmsp-1 allele (B-K1) with a specific var gene (var-D) was over-represented among patients with severe versus mild disease [Other gravity criteria linked to receptivity and host immune defenses and to the virulence of strains other than their capacity to multiply must also be considered. In French Guiana the association of a specific  disease .P. falciparum and two orders of magnitude less frequent for P. vivax. Clearly high PC is a severity criterion for falciparum malaria in this endemic area. However, two of the patients with PC ≥30% were not admitted to the ICU and sequel-free cure in malaria patients with 75% parasitaemia is, therefore, possible.In French Guiana during bouts of malaria, HP has been observed at a frequency of ~5% for CGH: Cayenne General Hospital; PC: Parasitic count; HP: Hyperparasitaemia; ICU: Intensive Care Unit.The author declare that they have no competing interests.BC completed the design of the study, performed data analysis and interpretation and prepared the manuscript. MD participated in interpretation of data and manuscript revision. All authors read and approved the final manuscript.


To be effective, national malaria guidelines must be properly followed. This study evaluated nurses' practices in the management of uncomplicated malaria cases at a District Hospital. Its objective was to identify the reasons for discrepancies between official guidelines and usual practices.This study took place at Oussouye hospital, south-western Senegal. Blood smears were available for biological diagnosis in patients aged more than five years while the Integrated Management of Childhood Illness recommended treating fevers presumptively in children under five. First line anti-malarial was Amodiaquine plus sulphadoxine-pyrimethamine (AQ+SP) bi-therapy. Hospital records of children under 13 years of age seen between 2004 and 2005 were reviewed.Among children treated with anti-malarials, 74%  received AQ+SP. However, only 22%  of febrile children and 19%  of children diagnosed with malaria got a blood smear. Moreover, an anti-malarial was prescribed for 80% (377/474) of children with a negative blood smear.The transition from chloroquine to AQ+SP was well followed. Nonetheless, blood smear use was very low and many over-prescriptions were reported. Reasons for discrepancies between guidelines and practices can be classified in three main categories: ambiguous guidelines, health system's dysfunctions and nurses' own considerations. Aside from the strengthening of the public health system, in order to guarantee practices complying with guidelines, training content should be more adapted to nurses' own considerations. In Zambia in 1997, a blood smear was requested for only 46% of patients with fever or previous fever, and among those with a negative result, 35% were treated with an anti-malarial  and in Tl [et al  reportedOnly a few studies, however, researched the reasons for these non-recommended practices ,7,8. ThiThis study took place from July to September 2005 at the District Hospital of Oussouye, south-western Senegal. In this area, malaria is meso-endemic with a seasonal recrudescence of cases during the rainy season, from June to October. The hospital was managed by a physician and was the referral centre for twelve dispensaries within the district. Two out-patient consultation rooms managed by nurses were available, one for children and one for adults. Consultations with a physician were also offered in a third room. The hospital had a laboratory facility managed by a skilled technician and equipped for blood smears. These services were open from Monday to Friday, 8:30 to 13:00.At the time of this study and since 2003, national malaria guidelines recommended AQ+SP bi-therapy for treating uncomplicated malaria . In addiA total of 5, 441 consultations involving children younger than 13 years old diagnosed with malaria or treated with an anti-malarial were retrospectively extracted from 2004 and 2005 hospital records. For each of these consultations, following data were recorded: out-patient consultation room, date, age, sex, axillary temperature, blood smear result if requested, diagnosis and prescribed anti-malarial. History of previous fever was not consigned in consultation records.Nine point five percent  of extracted consultations were excluded from the analysis: 235 consultations involving infants less than two months old, the IMCI recommending to treat fevers with antibiotics in this age group, and 282 consultations with missing data . A total of 4, 924 consultations were, therefore, analysed: 2, 371 in 2004 and 2, 553 in 2005.Blood smear use was determined according to the presence of fever (axillary temperature ≥ 37.5°C) and diagnosis made by nurses, malaria or other illness. Two aspects of anti-malarial prescription practices were analysed: The type of anti-malarials prescribed and the type of patients treated, febrile or not, with a positive or negative blood smear, and diagnosed with malaria or another illness. These analyses were performed in two age groups: under-five and from five to 13 years old. Statistical analyses were performed using STATA10. Pearson's chi square tests were used to compare two percentages (α = 0.05).Figure Only 22%  of febrile children were referred to the laboratory facility for a blood smear. Their mean axillary temperature was 38.3 ± 0.7°C (n = 404) versus 38.2 ± 0.8°C  in febrile but non-referred children. Moreover, only 19%  of children diagnosed with malaria got a blood smear. Among them, only 30% (127/429) had a positive result.By age, the use of blood smears was similar. Twenty three percent  of febrile children under five and 20% (144/735) of febrile children from five to 13 years old were referred for a blood smear (p = 0.09). And, when they were diagnosed with malaria, 22%  and 18%  respectively were referred for a blood smear (p = 0.02).Nurses also requested a blood smear in 11%  of non-febrile children and 6%  of children diagnosed with a non-malarial illness.The transition to AQ+SP was well followed, 74%  of children treated with an anti-malarial received this bi-therapy  of febrile children and 65%  of non-febrile children  of children diagnosed suffering from malaria and 22%  of children diagnosed suffering from a non-malarial illness received an anti-malarial. Among those treated while diagnosed with a non-malaria illness, 51% (291/571) were febrile. The main diagnosis recorded were respiratory infections, for 76% (452/591), or gastro-intestinal infections, for 21% (123/591).In children under five and in those from five to 13 years old respectively, the percentages of treated children were 60% (516/865) and 71% (539/762) in the absence of fever (p < 0.0001); 77% (235/305) and 84% (142/169) when blood smears were negative (p = 0.07); 27%  and 13% (119/950) when a non-malarial illness was diagnosed (p < 0.0001). Overall, 54%  of children under five and 61%  of those aged from five to 13 received an anti-malarial (p < 0.0001).In Senegal, artemisinin-based combination therapy (ACT) and rapid diagnostic tests (RDT) have been implemented in 2006 after a transitory stage recommended AQ+SP bi-therapy . This stThe transition to AQ+SP was well followed at Oussouye Hospital. This result can be attributed to the favourable context of prescription in place in a District Hospital. Indeed, nurses were trained and regularly supervised by the hospital physician. Moreover, stocks of CQ were removed and those of AQ+SP never experienced shortage.These good practices contrast with those reported in 2004, in Thies, north-western Senegal, where nurses prescribed AQ or SP mono-therapy for 15% to 78% of presumptive malaria cases . SimilarIn addition, to justify non-recommended prescriptions, nurses from Thies  and KenyIn Oussouye, informal interviews with nurses showed similar concerns and the extent to which guidelines describe standard clinical cases while nurses are confronted with various individual situations, which can lead to non-recommended prescriptions. In the paediatric consultation room, nurses preferred to prescribe AQ alone when SP had been taken in the previous month to adhere to the one-month minimum interval recommended by the Intermittent Preventive Therapy during pregnancy. When patients had started to self-medicate with CQ, AQ or AS, nurses could carry on with this treatment, conscious of some households' low incomes. In young children, they could choose to prescribe AS suppositories because of their convenience. In the adult consultation room, in cases of high fever, one particular nurse preferred to prescribe AS because of its effectiveness. Lastly, all nurses explained that pressure from parents can sometimes affect their prescribing. Parents wanted IM QN because it was perceived as being more effective or AQ drinkable suspension to store at home for treating fevers. Others refused AQ because of its adverse effects such as asthenia, stomach aches, nausea or itching.To conclude, it seems there are two kinds of potential impediments to the compliance with guidelines, first linked to the running of the health facility or its level in the health pyramid, and second linked to medical staff's handling of situations that they encounter daily with their patients.At Oussouye Hospital, compliance with the transition to AQ+SP has to be balanced. Indeed, while a laboratory facility was available, a very small proportion of patients got a blood smear. In addition, three types of over-prescription were identified. Sixty five percent  of non-febrile children were treated. However, as no history of previous fever was consigned in consultation records, these over-prescriptions are probably over-estimated. Secondly, 22%  of children diagnosed with a non-malaria illness were treated. These over-prescriptions explain why 57%  of children received an anti-malarial, while only 45%  were diagnosed with malaria. Lastly, and more surprisingly, 80% (377/474) of children received an anti-malarial despite their negative blood smear. These over-prescriptions explain why 84% (504/601) of children referred to the laboratory facility for a blood smear received an anti-malarial, while the positive blood smear percentage was only 21% (127/601).In a context of RDT deployment, similar results were reported. In Zambia in 2006, about one year after their introduction, only 23% of patients with fever or previous fever were tested. Moreover, 36% of patients with a negative RDT were treated with an anti-malarial . In Tanza precaution" if no other illness was diagnosed, probably affected nurses' prescribing. This type of ambiguous guideline is officially found in other sub-Saharan countries [et al [Considering observations made at Oussouye Hospital and informal interviews with its medical staff, three spheres of influence seem to lead to these discrepancies in guidelines. Firstly, an ambiguous guideline enacted by the hospital physician, recommending to treat fevers with a negative blood smear as "ountries ,18. In Zs [et al  also poiFurthermore, the multiplicity of guidelines, with IMCI, could also contribute to over-prescriptions. Indeed, this strategy recommends presumptive treatments of fevers in children under five to reduce malaria mortality. Nurses could, therefore, have applied this principle to older children, considering that there is no age limit for the risk of severe malaria.Alongside guidelines, dysfunctions at the laboratory facility also resulted in practices not complying with guidelines. Both nurses and laboratory technician pointed out the high workload required by blood smears, especially results reading time, and technical or material problems such as lack of supplies, microscope failures or power cuts. As a result, nurses confessed they were most of the time discouraged to request blood smears. Moreover, the laboratory technician explained that he was sometimes able to read blood smears only after the end of consultations leading nurses to prescribe without knowledge of their negative results. These dysfunctions have been reported in other sub-Saharan countries ,7,19. AtWhile RDT do not require either microscope or electricity and hence avoid material problems, their usage still requires time, in particular at least 15 minutes before the results can be read. At dispensaries where the mean consultation time is often very short, in particular five minutes or less during the rainy season in Kenya or in Tanzania ,20, it set al [Lastly, at Oussouye Hospital, nurses' own considerations seem to have played an important role in explaining both the low blood smear use and over-prescriptions. To explain low blood smear use, nurses blamed a lack of healthcare's organisation. The long period of time to obtain results led to an accumulation of patients at the laboratory and consequently delayed the end of consultations. Patients returning from laboratory also disrupted the clinic as they sometimes pushed in front of other waiting patients creating confusion and dissatisfaction. In addition, due to restricted laboratory opening times, nurses were not able to refer patients for a blood smear after 1 pm. In Zambia, Barat Another concern of Oussouye nurses was that patients refused to have a blood smear due to its cost (300 CFA or 0.46 Euro) and time involved. These refusals must be interpreted thoroughly. Indeed, patients build their own diagnosis and therapeutic approach through what they learn from different sources of information, in particular practices observed during consultations . Therefonon-febrile malaria" suspected in patients with headaches, chills, asthenia or vomiting, and "hidden malaria" in which the blood smear is negative because parasites are not yet in the blood. Furthermore, nurses mentioned once again some situations encountered with their patients, such as parents' demands for an anti-malarial or previous self-medication with an anti-malarial or an antipyretic which can explain the absence of fever or the negativity of blood smear and, therefore, do not exclude an anti-malarial prescription. In Tanzania, Chandler et al [To justify over-prescriptions, nurses' own considerations encompassed their lack of trust in the reliability of negative blood smears due to laboratory dysfunctions and their perceptions of malaria. Indeed, beyond standard symptoms of malaria, all nurses described atypical malaria cases, such as "er et al  intervieer et al ,3,23. Beer et al . In suchAlthough RDT are considered reliable, nurses could also doubt their negative results. In Tanzania, within routine practice, their sensitivity was evaluated between 19% and 86% . In thiset al [To summarise, over-prescriptions seem to be explained by practical, technical or socio-cultural considerations more than by clinical considerations. Nonetheless, as an underlying concern, interviews with Oussouye nurses also revealed their fear of severe malaria in non-treated patients. In Tanzania, Chandler et al  revealedThis study showed how difficult it is to integrate guidelines into nurses' daily practices. Reasons for discrepancies between official guidelines and usual practices can be classified in three main categories: the ambiguity and multiplicity of guidelines, health system dysfunctions and nurses' own considerations.One objective of the "Roll Back Malaria" partnership for 2015 is to treat correctly 80% of patients with malaria. To achieve this objective, the WHO has advocated, since 2001, the use of ACT for treating uncomplicated malaria  and, sinThe authors declare that they have no competing interests.SS designed the study, collected data, carried out the analysis, interpretation of data and prepared the manuscript. RL and MC contributed in the conception of the study and collection of data. JYLH contributed in the analysis and interpretation of data. All authors have participated in drafting and revising the paper and approved the manuscript.


Dengue is a major public health problem in tropical and subtropical countries. Exploring the relationships between virological features of infection with patient immune status and outcome may help to identify predictors of disease severity and enable rational therapeutic strategies.Clinical features, antibody responses and virological markers were characterized in Vietnamese adults participating in a randomised controlled treatment trial of chloroquine.P = 0.001). The clearance of DENV viremia and NS1 antigenemia occurs earlier and faster in patients with secondary dengue (P<0.0001). The maximum daily rate of viremia clearance was significantly higher in patients with secondary infections than primary (P<0.00001).Of the 248 patients with laboratory-confirmed dengue and defined serological and clinical classifications 29 (11.7%) had primary DF, 150 (60.5%) had secondary DF, 4 (1.6%) had primary DHF and 65 (26.2%) had secondary DHF. DENV-1 was the commonest serotype (57.3%), then DENV-2 (20.6%), DENV-3 (15.7%) and DENV-4 (2.8%). DHF was associated with secondary infection . DENV-1 infections resulted in significantly higher viremia levels than DENV-2 infections. Early viremia levels were higher in DENV-1 patients with DHF than with DF, even if the peak viremia level was often not observed because it occurred prior to enrolment. Peak viremias were significantly less often observed during secondary infections than primary for all disease severity grades . Little is known about the relationships between the evolution over time of virus levels in the blood, the clinical outcome and the previous infection(s) history—a better understanding of these features could help in anti-viral drug development. To analyze these relationships, we studied well characterized patients who participated in a clinical trial. The majority of these patients were infected by DENV-1 serotype and had higher levels of virus than those infected by DENV-2 and sometimes DENV-3 serotypes. We also found that patients with more severe symptoms had higher levels of virus in the first days of their illness. We found as well that the virus was cleared faster and earlier from the blood of patients previously infected. These findings are of major importance for further anti-viral drug testing. Dengue viruses (DENVs) are members of the Flavivirus genus and are the most important arboviral pathogens of humans. The four DENVs are antigenically-related and have single-stranded, positive-sense RNA genomes that share 60–70% sequence identity between each others The clinical outcome from DENV infection ranges from the asymptomatic to an acute, often debilitating illness called dengue fever (DF), to the severe and potentially life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The cardinal feature of DHF/DSS is a capillary permeability syndrome characterised by plasma leaking from the vasculature into interstitial spaces. Thrombocytopenia, a coagulopathy and a hemorrhagic diathesis are also common findings. DSS manifests when capillary permeability is severe enough to result in an inadequate intravascular volume that then leads to poor tissue perfusion. DSS is managed, and possibly prevented, by careful restoration and maintenance of the intravascular volume by use of parenteral fluids. Viral strain and host immune status have been suggested as major risk factors for DHF/DSS. In particular, two sequential infections, with the second infection caused by a DENV serotype different from the first, is a risk factor for severe disease in children and adults The literature describing the overall relationship between plasma/serum viral burden, disease severity and immune status generally supports the hypothesis that there is a positive correlation between markers of viral burden in the first 2–3 days of fever and the severity of clinical outcomes. For example, during the febrile and early convalescent periods, Taiwanese adults with secondary DENV-3 infections and DHF had higher plasma levels of viral RNA than did patients with DF In adults, where the risk of clinically apparent disease occurring in primary infection is possibly greater The dynamics of virus clearance might also be relevant to clinical outcome. In Thai children, Vaughn and others have shown that the slope of the descending portion of the viremia curve was steeper for patients with secondary infection versus those with primary infection and viremia decreased more quickly for patients with DHF than for patients with DF at defervescence A better understanding of the relationship between biomarkers of virus infection, the immune response and disease evolution is critical for the rational use of intervention therapies in dengue, e.g. anti-viral drugs or immune-modulating therapies. To this end, this study describes the kinetics of viremia and NS1 in an intensively investigated cohort of Vietnamese adults with dengue and less than 72 hrs of fever enrolled in a randomized placebo-controlled trial (RCT).A double blind RCT of chloroquine (CQ) in 307 adults hospitalized for suspected DENV infection was conducted at the Hospital for Tropical Diseases  between May 2007 and July 2008. Information on recruitment, inclusion criteria, randomization, treatment and investigations have been published previously A diagnosis of laboratory confirmed dengue was reached using serological, antigen detection and molecular methods The interpretation of primary and secondary serological responses was based on the magnitude of IgG ELISA units in early convalescent plasma samples taking into account the illness day. The cut-off in IgG ELISA units for distinguishing primary from secondary dengue by illness day was calibrated using a panel of acute and early convalescent sera from Vietnamese dengue patients that were assayed at the Centre for Vaccine Development, Mahidol University, Bangkok, Thailand using a reference IgM and IgG antibody capture ELISA described previously Clinical history and examination findings were recorded daily into case record forms. An ultrasound was performed in all patients within 24 hrs of defervescence. Venous blood samples were collected at hospital admission, then twice daily (around 9am and 3pm) for a minimum of 5 days after hospital admission and again 10–14 days after discharge from the hospital. A complete blood count, including hematocrit (Hct) and platelet measurements, was performed daily for all patients. Hct measurements were performed more frequently if clinically indicated. The extent of hemoconcentration during symptomatic illness was determined by comparing the maximum Hct recorded during hospitalization with either the value recorded at follow-up when available (191/248 i.e. 77% of the patients) or against a sex- and age-matched population value. Plasma was stored frozen in multiple aliquots at −80°C until use in the real-time RT-PCR and NS1 ELISA. The day of fever onset was self-reported by the patient and was designated illness day 1.DF and DHF were diagnosed according to 1997 World Health Organization (WHO) classification criteria and was applied to each case after review of study notes P<0.05 and were two-sided unless otherwise indicated. Uncertainty was expressed by 95% confidence intervals. The Kruskal-Wallis rank sum test was used for continuous variables and the Fisher's exact test for categorical variables. For the viremia kinetics analysis, when the RT-PCR signal was below the assay limit of detection , a value equal to concentration of the last dilution of standard that gave a specific signal divided by 10 was assigned. The maximum viremia level was defined as the highest plasma viremia level measured during illness. The maximum viremia level was considered to be a peak viremia level only in cases in which viremia rose after the enrolment specimen. To compare kinetics of viremia between patients with different serological status, disease severity and serotype, the means of log-transformed viremia measurements made on the same illness day were used as a summary measure of the viremia on that day. To estimate the maximum daily rate of DENV clearance, the slope of the viremia curve was calculated for each illness day as the change in the means of log-transformed viremia measurements made on the same illness day. Only the maximum decreasing daily rate of each patient was used for analysis. Survival analysis using the Kaplan-Meier method and log-rank test was used for all time-to-event outcomes. Time to resolution of viremia or NS1 antigenemia was defined as the time from the start of symptoms until the first of two consecutive plasma samples below the RT-PCR limit of detection or NS1 ELISA negative. The fever clearance time (FCT) was defined as the time from the start of symptoms to the start of the first 48 hours period during which axillary temperature remained below 37.5°C.The data used in this analysis was taken from a randomised controlled treatment trial of dengue. Since the intervention (CQ) had no measurable impact on virological or immunological outcomes, for the purposes of this analysis we did not distinguish between patients in the CQ or placebo arms of the study. All statistical analysis was performed and figures designed using the software R (version 2.10.1). Significance was assigned at P = 0.04, Odds ratio (OR) = 3.13, 95% CI 1.04–12.75). DHF resulting from secondary infection was more commonly associated with DENV-2 (21/45 (46.7%)) than for other serotypes (DENV-1: 33/124 (26.6%), DENV-3: 10/33 (30.3%) and DENV-4: 1/7 (14.3%)  . The characteristics of the study population are summarized in 14–4.96) .Median viremia levels by illness day for DENV-1, -2 and -3 are shown in DENV-1 was the commonest serotype detected in this patient population and therefore there was sufficient data to enable direct comparisons of viremia kinetics across serological states and clinical severity whilst controlling for the infecting serotype . These dP = 0.001, OR = 3.64, 95% CI 1.55–8.74). However, there were no significant differences in the duration of illness prior to enrolment between patients in different categories of serological status or disease severity , suggesting this snapshot of viremia levels is unbiased by differences in duration of illness at study enrolment.A limitation of these analyses is that in the majority of patients with secondary infections the viremia was already declining at the time of enrolment i.e. we did not observe an obvious peak viremia . OverallP = 0.008) and in secondary DHF than in primary DHF (P = 0.04) but there were no significant differences between primary DF and primary DHF (P = 0.73) and between secondary DF and secondary DHF (P = 0.13) for the peak viremia time (P = 0.0006) but, possibly because of small sample size, not in secondary DHF compared to primary DHF (P = 0.31). There was no significant difference between secondary DF and secondary DHF (p = 0.48) but there was a non-significant trend towards later viremia peaks in primary DF than in primary DHF (P = 0.052).Peak viremia levels were identified in 72 patients. Peak viremia occurred significantly earlier in secondary DF than in primary DF (mia time . AmongstP = 0.28) but there was a non-significant trend towards higher peak viremia during secondary infections than primary infections (primary vs secondary P = 0.096 and primary DF vs secondary DF P = 0.086). If considering the highest viremia levels (as distinct from peak viremia levels) in DENV-1 patients, these were significantly higher in DHF than in DF . Because most DHF cases were associated with secondary infections, for which peak viremia had already past by the time of enrolment, this difference is probably underestimated. These results suggest secondary infections are generally associated with earlier peak viremia but do not provide any conclusive evidence of higher peak viremia levels in DHF and/or secondary infections.There were sufficient observations of the magnitude of peak viremia in DENV-1 infections to look for associations with clinical outcome in this subgroup . Peaks oP<0.00001) (primary DF vs secondary DF P = 0.00004 and primary DHF vs secondary DHF P = 0.025). The results were very similar when considering only DENV-1 patients for analysis (data not shown). These data suggest secondary infection is associated with steeper declines in viremia.In the 239 patients with detectable viremia, the median of maximum daily rates of DENV clearance (estimated as the slope of the steepest descending daily portion of the viremia curve) was 2.2 log10 per day in primary DF, 2.8 log10 per day in secondary DF, 2.1 log10 per day in primary DHF and 3.0 log10 per day in secondary DHF. The maximum daily rate of clearance was significantly higher in patients with secondary infections (median of the maximum daily loss 2.9 logs per day) versus those who experienced primary infections , in primary DF than in secondary DF  and in primary DHF than in secondary DHF   time to resolution of viremia was significantly longer in primary infections than in secondary infections (hazard ratio (HR) = 2.88, 95% CI 1.79–4.63, log rank test = 0.015) . Median P = 0.000009), in primary DF than in secondary DF  and in primary DHF than in secondary DHF  (Amongst DENV-1 infected patients only (n = 142), times to resolution of viremia were also significantly longer in primary infections than in secondary infections  . Median P = 0.00007), in primary DF than in secondary DF , in primary DHF than in secondary DHF  but also in secondary DF than in secondary DHF  . Consistent with the viremia findings, times to resolution of NS1 antigenemia were significantly longer in primary infections than in secondary infections  . InteresP = 0.00008), in DENV-1 primary DF than in DENV-1 secondary DF , in DENV-1 primary DHF than in DENV-1 secondary DHF  and in DENV-1 secondary DF than in DENV-1 secondary DHF  (Amongst DENV-1 infected patients (n = 142), 134 were NS1 positive at the time of study enrolment. Times to resolution of NS1 antigenemia were significantly longer in DENV-1 primary infections than in DENV-1 secondary infections  . StrikinCollectively, these results suggest that DENV infection is cleared earlier and faster in secondary infections than in primary infections.P = 0.037) but there was no significant difference between primary DF and secondary DF , and between primary DHF and secondary DHF  (There were 240 patients febrile at enrolment (plus 2 afebrile patients who developed fever soon after). Overall, FCT were significantly longer in primary infections than in secondary infections (log rank test  = 0.23) . Median P<0.01). The data also suggest that leucopenia lasts longer in primary infections than in secondary infections.For descriptive purposes, the evolution over time of DENV-1 viremia in the context of white blood cell and platelet counts and percentage hemoconcentration was plotted  and summThe interplay between DEN virus infection and host immune status is postulated to play a central role in the pathophysiology of severe dengue. In this current study, we observed important features of this dynamic. First, early viremia levels were higher in patients with DHF, even if the peak viremia level was often not observed because it occurred prior to enrolment. Second, DENV-1 infections manifested as higher and longer-lived viremias, suggesting serotype dependent differences in infection kinetics. Third, the clearance of DENV viremia and NS1 antigenemia occurs earlier and faster in patients with secondary dengue and is also consistent with a faster time to defervescence.Our findings are in agreement with previous studies that found higher viremias associated with more severe disease The duration of NS1 antigenemia was shorter in patients with secondary infections and this is consistent with previous studies that have suggested reduced sensitivity of NS1-based diagnostic tests in patients with secondary infections Secondary dengue is associated with faster resolution of viremia infection and shorter duration of fever. Interestingly, the daily rates of virus clearance observed in our study were very compatible with those found by Vaughn et al in Thai children Assuming blood viremia is a reasonable surrogate of the whole-body virus burden, then the rapid decline of viremia 48–72 hrs after illness onset, especially in secondary infections that carry higher risk for severe outcomes, has implications for rational design of therapeutic pharmacological interventions. An efficacious anti-viral will need favourable pharmacokinetic properties and potency if it is to impact on the viral burden in a rapid and clinically significant manner. Pharmacological targeting (e.g. with corticosteroids) of the host immune response, which accounts for the rapid decline of viremia but which also likely contributes to the capillary permeability syndrome, may equally need to be administered early on in illness e.g. in a prophylactic way, to prevent clinical complications such as DSS. The rapid decline in viremia in secondary infections also highlights the importance of early diagnosis, since early diagnosis will provide the greatest opportunity for an intervention , to have an impact. Point of care NS1 diagnostics are available but more can be done to improve their sensitivity Current animal models of DENV infection are able to provide for in vivo measurements of anti-viral activity There are several limitations to our study. Our results are derived from hospitalized patients who are not necessarily representative of patients being seen at the primary health care level. Interestingly however, the same themes identified in this study in hospitalized Vietnamese adults were also observed in Vietnamese children presenting to primary health care level clinics in Ho Chi Minh City Our study emphasizes the importance of the period before and just after the onset of fever. This and other studies have established that early viremia levels are associated with disease severity, although they are very clearly not the only determinant of outcome. Very little is known of the virological events in the hours preceding and shortly after fever onset, mainly because this is very difficult to investigate without a good experimental model. An interesting insight was provided by clinical trials of DENV-1, -3 and -4 monovalent live attenuated vaccines in the 1980s Collectively, our findings reveal important patterns of the viremia and NS1 antigenemia kinetics according to the patient immune response, disease severity and virus serotype, and may help for the rational design of clinical trials of therapeutic interventions, especially antivirals.Table S1The characteristics of the study population by serotype.(DOC)Click here for additional data file.Table S2Levels of viremia among the study population by illness day and serotype.(DOC)Click here for additional data file.Table S3Peak viremia analysis.(DOC)Click here for additional data file.Table S4Maximum of hemoconcentration, platelet and WBC nadirs and their occurrence times by disease severity. All these markers were determined in serial plasma samples from DENV-1 infected patients with primary DF (n = 15), primary DHF (n = 3), secondary DF (n = 91) and secondary DHF (n = 33).(DOC)Click here for additional data file.


The accuracy of the World Health Organization method of estimating malaria parasite density from thick blood smears by assuming a white blood cell (WBC) count of 8,000/μL has been questioned in several studies. Since epidemiological investigations, anti-malarial efficacy trials and routine laboratory reporting in Papua New Guinea (PNG) have all relied on this approach, its validity was assessed as part of a trial of artemisinin-based combination therapy, which included blood smear microscopy and automated measurement of leucocyte densities on Days 0, 3 and 7.Plasmodium falciparum monoinfection, 14.9% with Plasmodium vivax monoinfection, and 4.8% with mixed P. falciparum/P. vivax infection. All responded to allocated therapy and none had a malaria-positive slide on Day 3. Consistent with a median baseline WBC density of 7.3 (6.5-7.8) × 109/L, there was no significant difference in baseline parasite density between the two methods regardless of Plasmodium species. Bland Altman plots showed that, for both species, the mean difference between paired parasite densities calculated from assumed and measured WBC densities was close to zero. At parasite densities <10,000/μL by measured WBC, almost all between-method differences were within the 95% limits of agreement. Above this range, there was increasing scatter but no systematic bias.168 children with uncomplicated malaria (median (inter-quartile range) age 44 (39–47) months) were enrolled, 80.3% with Diagnostic thresholds and parasite clearance assessment in most PNG children with uncomplicated malaria are relatively robust, but accurate estimates of a higher parasitaemia, as a prognostic index, requires formal WBC measurement. Plasmodium[Although the effect of malaria infection and its treatment on erythrocyte dynamics in humans is well characterized ,2, therelasmodium-6. An imlasmodium and adullasmodium.Plasmodium species [The microscopy method recommended by the World Health Organization (WHO) to estimate asexual parasite density is to multiply the parasite count for a given thick film WBC count  by an assumed peripheral blood WBC count of 8,000/μL . However species -18. The Plasmodium falciparum  and/or Plasmodium vivax (> 250/ μL) on a peripheral blood smear, and iii) no clinical or laboratory evidence of severe malaria or other infection according to WHO severity criteria [Children aged 0.5 to 5 years with uncomplicated malaria were recruited at Mugil and Alexishafen Health Centres in Madang Province on the north coast of mainland PNG between April 2011 and June 2012 during a randomized comparative efficacy trial of the artemisinin-based combination therapies artemether-lumefantrine and artemisinin-naphthoquine  . Inclusi9/L, leucopaenia as < 4.0 × 109/L and thrombocytopaenia as a platelet density < 100 × 109/L. White blood cell differentials were estimated as percentages with normal lymphocyte, monocyte and granulocyte ranges of 20.5-51.1%, 1.7-9.3% and 42.2-75.2%, respectively.On Days 0, 3 and 7, venous blood samples were collected into EDTA-containing Microtainer® tubes  and full blood counts were performed on the same day using a multichannel analyser . Leucocytosis was defined as a WBC density of ≥ 10.0 × 10Of the three blood slides per patient prepared as part of each blood collection, one was stained with 10% v/v Giemsa over 10 minutes and read on site under 100 × magnification by a skilled microscopist to determine patient eligibility. The remaining slides were transported to a reference microscopy laboratory where they were stained with 4% v/v Giemsa stain over 30 minutes and subsequently read independently by two skilled microscopists, who were blinded to the initial result. Parasitaemia was quantified using the numbers of parasites identified in fields containing a total of 200 WBC in high-density smears or 500 WBC in low density smears. Negative slides were those in which no parasites were observed after examination of 200 fields under 100 × magnification. Slides with discrepancies of more than a factor of three regarding density, speciation and/or parasite positivity/negativity were adjudicated by a senior microscopist. Parasite densities were calculated as i) assumed = (parasites counted ÷ number of WBC counted) × 8,000 and ii) confirmed = (parasites counted ÷ number of WBC counted) × .P < 0.05. Bland Altman plots were constructed with parasite densities calculated from measured WBC densities used as the gold standard [Data were analysed using non-parametric methods with a two-tailed significance level of standard .P. vivax monoinfection, 8 (4.8%) had mixed P. falciparum/P. vivax infection, while the remaining 80.3% had a P. falciparum monoinfection. All patients responded to their allocated therapies and none of the children had a malaria-positive slide on Day 3 of follow-up. There were also significant falls in axillary temperature, pulse rate and respiratory rate during this time  age was 44 (39–47) months, 49% were males and 55% had a palpable spleen at the time of enrolment. Twenty-five (14.9%) had P < 0.0001).Most of the children were anaemic at presentation with a transient further fall in haemoglobin to Day 3 before recovery by Day 7 (see Table P < 0.001) but there was no significant difference between Days 0 and 3 (P = 0.81). Excluding those with mixed-species infections, children with falciparum malaria had lower median leucocyte counts at all time-points compared to those with vivax malaria, but the differences were not statistically significant . This is an important and reassuring finding for epidemiological and intervention studies, as well as routine laboratories, in PNG. Although leucocyte densities should ideally be determined in tandem with each thick film in individual patients [The present study shows that patients , automatPNG: Papua New Guinea; WBC: White blood cell; WHO: World Health Organization.The authors declare that they have no competing interests.All authors contributed to the design of this study, collection of data, analysis of data and/or the interpretation of the results, and to the writing of the manuscript. All authors edited and approved the final version of the manuscript.


Plasmodium) drug and a slow acting blood schizontocidal agent highly effective for the prevention of malaria. When used in conjunction with a fast acting schizontocidal agent, it is also highly effective for malaria treatment. Doxycycline is especially useful as a prophylaxis in areas with chloroquine and multidrug-resistant Plasmodium falciparum malaria. Although not recommended for pregnant women and children < 8 years of age, severe adverse events are rarely reported for doxycycline. This report examines the evidence behind current recommendations for the use of doxycycline for malaria and summarizes the available literature on its safety and tolerability.Doxycycline, a synthetically derived tetracycline, is a partially efficacious causal prophylactic (liver stage of  Plasmodium falciparum in short-term travelers (< 4 months) to areas with chloroquine and/or pyrimethamine-sulfadoxine-resistant strains. Although not FDA approved, doxycycline is recommended for prophylaxis of other species of malariaP. falciparum; with both quinine sulfate and primaquine for uncomplicated chloroquine-resistant Plasmodium vivax; and with parenteral quinidine for severe malaria.2The only U.S. Food and Drug Administration (FDA)-approved indication for the use of doxycycline for malaria is for prophylaxis of Available in multiple dosage forms and strengths, doxycycline tablets or capsules of 100 mg are most often used for malaria prophylaxis or treatment. Tablets or capsules should be swallowed with an adequate amount of fluid and should be taken with food. Intravenous (IV) formulation is available for patients unable to take oral medications for treatment. For IV, avoid rapid administration.Prophylaxis: 100 mg base once daily starting 1–2 days before travel, then daily during travel, and daily for 4 weeks after leaving the malarious area.P. vivax or Plasmodium ovale.Treatment: 100 mg twice a day for 7 days; must be used in conjunction with a fast acting schizontocide. Primaquine is also required if treating For children ≥ 8 years of age.Prophylaxis: 2.2 mg/kg (up to adult dose of 100 mg) daily starting 1–2 days before travel, daily during travel, and daily for 4 weeks after leaving the malarious area.P. vivax or P. ovale.Treatment: 2.2 mg/kg (up to adult dose of 100 mg) twice a day for 7 days; must be used in conjunction with a fast acting schizontocide. Primaquine is also required if treating P. falciparum and 98% for primary P. vivax infection.Prophylaxis: Protective efficacy of doxycycline has been shown in three randomized placebo-controlled trials to be between 92% and 96% for Treatment: When used in combination with a fast acting schizontocide, treatment efficacy of doxycycline has been shown to be 96–100% in three open-label trials.Limited data suggest that milk and other dairy products may limit the bioavailability of doxycycline. It is recommended to separate doxycycline and ingestion of dairy products by 2–3 hours.Most common mild/moderate adverse drug reactions (ADRs): nausea, vomiting, abdominal pain, photosensitivity, and vaginitis. Women prone to candidal vaginitis should consider carrying a self-treatment course of an anti-fungal. Severe ADRs are uncommon and include esophagitis and esophageal ulcerations. Doxycycline monohydrate has been observed to have less gastrointestinal side effects than doxycycline hyclate.Doxycycline is contraindicated in those who have known hypersensitivity to tetracyclines.Decreased absorption of doxycycline can occur if taken concurrently with medications with divalent or trivalent cations such as antacids, laxatives, and oral iron preparations. Although the current FDA label states “concurrent use of tetracycline may render oral contraceptives less effective,” recent studies have failed to show a significant association between oral contraceptive failure and tetracyclines. Tetracyclines may potentiate the effect of oral anticoagulants, therefore patients on oral anticoagulants should have prothrombin times monitored closely and dose adjusted as needed. Concurrent use of tetracyclines and methoxyflurane anesthesia is not recommended. Barbiturates, carbamazepine, and phenytoin decrease the half-life of doxycycline.P. falciparum when no other treatment options are available. Not recommended for children < 8 years of age.Pregnancy category D: Do not use in pregnancy except for the treatment of life-threatening multidrug-resistant Excreted in low concentrations in breast milk; noted to be compatible with breastfeeding by the American Academy of Pediatrics.Streptomyces sp. bacteria.Doxycycline is a broad-spectrum bacteriostatic agent synthetically derived from naturally occurring tetracyclines produced by Doxycycline can be used by travelers to all malaria-endemic areas for malaria prophylaxis. When used in conjunction with other medications, doxycycline can also be used to treat malaria.P. falciparum. For prophylaxis, doxycycline is taken once daily beginning 1–2 days before travel, while in malarious areas, and for 4 weeks after leaving. Daily dosage for children ≥ 8 years of age is 2.2 mg/kg (not to exceed the adult dose of 100 mg) and for adults 100 mg.Doxycycline can be used for the prevention of malaria in travelers to malaria-endemic areas and is a good option for areas with chloroquine or multidrug-resistant P. falciparum or P. vivax. When used for severe malaria doxycycline should be used with intravenous quinidine. The treatment dose of doxcycyline in adults is 100 mg twice a day and for children 2.2 mg/kg twice a day up to a maximum of 100 mg twice daily. For those unable to tolerate oral medications, doxycycline is available in a parenteral form and can be made in concentrations between 0.1 and 10 mg/mL. If pain occurs while receiving doxycycline intravenously, it is recommended to use more dilute concentrations (no lower than 0.1 mg/mL) infused at rates no higher than 1 mg/minP. vivax or P. ovale, in addition to the aforementioned drugs, primaquine must also be given to prevent relapses (see Pharmacodynamics section).Doxycycline should always be used in conjunction with another drug for treatment of malaria. Quinine with doxycycline can be used for uncomplicated malaria caused by chloroquine-resistant P. falciparum in the 1960s spurred efforts to examine alternative medications such as tetracyclines with antimalarial activity. Early studies examined the treatment and prophylactic efficacy of tetracyclines such as doxycycline, tetracycline, and minocycline against chloroquine-resistant P. falciparum. Since these earlier studies, the preponderance of literature on efficacy of tetracyclines as antimalarials has focused on doxycycline.The emergence of chloroquine-resistant P. falciparum infections.P. vivax infection,P. falciparum in 67 non-immune Indonesian soldiers.P. falciparum malaria during the follow-up period. Of these 5, one developed malaria while on prophylaxis and 4 developed symptoms after discontinuation of prophylaxis during the 3 months of observation upon returning to a malaria-free country.12Overall, studies have showed that doxycycline is a slow-acting blood schizontocidal agent, had partial causal effects and no sporontocidal or gametocytocidal effects, and is effective in the treatment and prevention of malaria .P. falciparum, but is insufficient for causal prophylaxis. Doxycycline would therefore have to be continued for a sufficient amount of time post exposure for successful suppression of parasites that emerge from the liver. Studies have observed thatOther studies have also shown doxycycline to have inadequate causal activity, or activity against the liver stage of the malaria parasite, which has implications for doxycycline's post-exposure course.Plasmodium also means that it does not kill P. vivax hypnozoites. Although very effective for preventing primary P. vivax infections, doxycycline is not effective at preventing relapses.Doxycycline's limited activity in the liver stages of N = 67) was compared with full-dose doxycycline . One open-label, randomized, controlled trial found that full-dose doxycycline was significantly more effective in prevention of P. vivax, however the study was not powered to detect a difference between the two doses of doxycycline in preventing P. falciparum infections.P. falciparum and primary P. vivax infection.17The dose of doxycycline required to achieve successful malaria prophylaxis was also examined. The efficacy of half-dose doxycycline  between 3.8 and 6.0 days and fever clearance times between 3.6 and 4.0 days in patients treated with only tetracycline or doxycycline.P. falciparum, cure rates between 75% and 100% have been reported.P. falciparum infections.P. falciparum malaria.Doxycycline has been shown to be a slower acting blood schizonticide during the course of an acute An oral dose of 100–200 mg of doxycycline is almost completely absorbed in the duodenum and stomach and is detectable in the blood 15–30 minutes after administration. Following a 200 mg oral dose, peak concentrations of ∼2.6 μg/mL are reached at ∼2 hours, but this may vary as gastrointestinal (GI) absorption rates can differ among individuals.37According to the FDA, the absorption of doxycycline “is not markedly influenced by simultaneous ingestion of food and milk,” despite the reduced absorption observed with other tetracyclines. However, more recent studies on this subject, while limited, have shown that the absorption of doxycyline is affected to some degree by food and milk, but the clinical consequence of this is not well understood. Food has been shown to decrease serum levels of doxycycline from 4.4 to 4.0 μg/mL, but this has not been shown to be clinically significant.Unlike other tetracyclines, excretion of doxycycline occurs primarily by the gastrointestinal tract and to a much lesser extent by the kidneys.43N = 14 males and 14 females).44There are limited to no data on sex, age, body weight, or race differences in the pharmacokinetics of doxycycline. One study found no differences between males and females  with a median gametocyte clearance time of 62 hours.Although it is known that doxycyline is a blood schizontocide,in vitro susceptibility testing for doxycycline. Traditionally, culture-derived or field P. falciparum isolates are incubated with varying concentrations of drug and inhibition of replication is measured by several methods to yield a 50% inhibitory concentration (IC50) at 48, 72, or 96 hours. The assay conditions and the drug mechanism of action can markedly affect results.50 cutoff level of 9.6 μM for a 48-hour assay and noted that after both quinine and doxycycline were recommended as first-line malaria treatment in 1995, there was an increase of mean IC50s from 9.6 μM in 1996–1999 to 13.1 μM in 2005.P. falciparum samples of French patients who traveled to various African countries between 1997 and 2006 using a 42-hour assay and found an overall mean IC50 of 9.3 μM with country-specific means ranging between 6.2 and 11.1 μM.50s above the suggested 9.6 μM threshold. However, these findings are not consistent with the relative dearth of reporting of doxycycline failure (when taken appropriately either as prophylaxis or treatment) in the literature. Another study further identified possible molecular markers for decreased in vitro susceptibility to doxycycline defined as an IC50 of 35 μM.55There is no global consensus on the threshold of susceptibility for use in P. falciparum to doxycycline is not described but breakthroughs in prophylaxis have been associated with inadequate doses,P. falciparum parasitemia and a serum or plasma level of doxycycline or a predictive IC50 in a well-qualified in vitro susceptibility assay remains to be determined. More studies are needed to examine emergence of doxycycline resistance and establish a clinically relevant susceptibility threshold.Resistance of Adherence to daily doxycycline throughout the period of exposure to malaria and for 4 weeks following exposure is vital in achieving full effectiveness of doxycycline as a malaria prophylactic agent. The effectiveness of doxycycline among persons in whom adherence was not enforced was compared with persons in whom compliance was enforced. Effectiveness was only 89% (78–96%) for all types of malaria compared with 99% (94–100%) in soldiers in whom adherence was enforced.N = 52) was lower than that for weekly mefloquine  among United States troops in Somalia; however, it was not discussed whether administration of one or both medications was supervised.Most studies suggest that adherence to daily doxycyline is more challenging than adherence to a weekly medication. One study reported that full adherence to doxycycline daily . Most available information on AEs in doxycycline are from studies examining doxycycline at prophylactic doses (100 mg/day), and there is limited information on AEs in doxycycline at treatment doses. At prophylactic doses of doxycycline, reports of mild and moderate AEs vary widely among studies from 0.6% to 84% of the study population.Reports of AEs of doxycycline at treatment doses (100 mg twice a day) are very limited because studies often examine malaria treatment with doxycycline in combination with another drug such as quinine, and it is difficult to determine if symptoms are related to doxycycline alone, malaria, the accompanying drug, or because of a drug-drug interaction. One randomized, open-label study comparing treatment of malaria with doxycycline and quinine versus artemether-lumefantrine found that at least one AE was reported in 93.5% of patients treated with quinine-doxycycline and 89.2% of those treated with artemether-lumefantrine; statistically similar proportions.28Gastrointestinal symptoms are usually mild to moderate and include nausea (4–33%) and abdominal pain 12–33%).–33%.12,221Doxycycline and other tetracyclines are believed to be the most common cause of drug-induced esophageal disorders.63The effect of various doxycycline salts and formulations such as oral suspensions, tablets, capsules, and enteric-coated tablets are likely quite significant in their ability to cause GI adverse events. Gelatin capsules have been observed to be more sticky than tablets, delaying transit time or even adhering to the esophageal lining, and this is affected by the amount of fluids ingested with the pills.An erythematous rash in sun-exposed areas has been reported to occur in 7.3–21.2% of persons74in vitro study found that tetracycline may also enhance Candida albicans virulence factors, but the authors note that it is still unclear as to what degree this contributes to clinical vaginitis.C. albicans after taking tetracyclines has been shown.C. albicans increased from 7% to 24%.Antibiotics such as tetracyclines are believed to suppress vaginal bacterial flora, resulting in overgrowth of candida.Clostridium difficile-associated diarrhea (CDAD) has been reported in three patients taking doxycycline for malaria prophylaxis for between 2 and 6 weeks.94There are other AEs that have been reported in association with tetracyclines. These include case reports of benign intracranial hypertension with tetracycline, minocycline, and doxycycline, but this has been observed to resolve upon discontinuation of the medication and has not been described in someone taking doxycycyline for malaria prevention.There are varying reports of the comparative tolerability of doxycycline. Several non-randomized survey-based studies in military personnel have found a higher occurrence of photosensitivity, GI symptoms, headache, and light-headedness with doxycycline as compared with mefloquine, whereas others have found that doxycycline is better tolerated than mefloquine.15N = 623 evaluable persons).The strongest evidence for doxycycline's relatively good tolerability is found in three randomized, double-blinded trials. Two of the studies found little or no difference in the frequency of AEs between doxycycline  and mefloquine, and all AEs were mild and transient.Doxycycline is contraindicated in anyone with a history of hypersensitivity to any of the tetracyclines.There are very limited studies on the use of doxycycline for malaria prophylaxis for a prolonged period of time. The current FDA product insert approves use for up to 4 months.Although long-term safety and tolerability studies have not been performed with doxycycline as a malaria chemoprophylaxis, similar drugs in the same class, tetracycline and minocycline, have been used in equivalent doses  for a few months to several years in the treatment of various dermatological conditions. This suggests that doxycycline can be used for long-term malaria chemoprophylaxis. When used at this dosage, tetracyclines were found in some studies to cause no abnormalities in renal and hepatic function when taken for as long as 5 years and in one study, for as long as 13 years.Tetracycline antibiotics are relatively non-toxic. Exceeding the recommended dosage of doxycycline may result in increased incidence of ADRs. There has been one case report of an individual who took 1 g of doxycycline/day for 12 years.95in vitro or in vivo studies.in vitro studies with phase I metabolizing enzymes (cytochrome P450), phase II enzymes, and transporter mediated drug interactions has not been reported. On the basis of a single in vitro study showing that doxycycline inhibits the metabolism of quinine to 3-hydroxyquinine, the major metabolic pathway known to be enzymatically catalyzed by CYP 3A4, doxycycline is thought to be a modest inhibitor of CYP 3A4. Because CYP 3A4 is the most common phase 1 enzyme involved in drug metabolism, the spectrum of potential drug-drug interactions is wide and is not well defined. Furthermore, in vitro interactions do not always predict clinically significant effects. Information on drug-drug interactions with doxycycline is based on clinical observations and predictions are based on limited data accumulated over the last 40 years of clinical use, as described below.Doxycycline was introduced into clinical practice long before the recognition of drug-drug interactions and without currently required pre-licensure Tetracyclines can chelate divalent and trivalent cations in certain medications that may cause a decreased absorption of the tetracycline and the medication when given concurrently.108Although a possible interaction with oral contraceptives (OCs) has been cited in older literature,Tetracyclines may potentiate the effect of oral anticoagulants by impairing the use of prothrombin and decreasing vitamin K production by intestinal bacteria.3in vitro,As a bacteriostatic drug, tetracyclines may interfere with the bactericidal action of aminoglycosides and penicillinFatal nephrotoxicity has been reported in patients receiving methoxyflurane anesthesia and tetracycline.Concurrent use of digoxin and oral antibiotics such as tetracyclines may increase serum digoxin concentrations in some individuals. It is believed that the tetracycline alters gut flora, diminishing digoxin conversion to inactive metabolites, resulting in increased serum digoxin concentrations.Barbiturates, carbamazepine, and phenytoin are believed to decrease the half-life of doxycycline by inducing microsomal enzyme activity.120Salmonella Ty21a bacteria used in the vaccine. However, there is no experimental data in animals or humans to support this position. Nevertheless, most experts advise not giving doxycycline within 24 hours of the oral typhoid vaccine.Salmonella typhi,Theoretically, antibiotics such as doxycycline could decrease the effectiveness of the oral typhoid vaccine Ty21a by preventing replication of the live attenuated There are no known drug interactions between doxycycline and antiretrovirals.Doxcycycline is not recommended for children < 8 years of age because tetracyclines have been shown to cause permanent discoloration of the teeth.129Of note, doxycycline is considered the drug of choice for treatment of infections in which the risks of tooth discoloration is outweighed by the benefit of disease treatment, such as presumed or confirmed rickettsial infection, ehrlichiosis, murine typhus, or inhalational anthrax.The formation of the tetracycline and calcium orthophosphate complex in developing bone is thought to cause growth retardation, but the evidence is limited. Decreased long bone growth rate has been observed in premature infants given tetracycline but is reversible upon discontinuation of the drug.P. falciparum when no other treatment options are available. No congenital malformations have been associated with its use,Doxycycline is a category D drug; therefore, the possibility of pregnancy in a woman of childbearing age must be addressed before administration of this drug.Tetracyclines have also been associated with fatal liver necrosis in pregnancy.146For women planning on becoming pregnant, there is no need to wait a specific period of time after doxycycline use before attempting to conceive, based on the short half-life (15–25 hours) of the drug.147Data are extremely limited on the use of doxycycline in breastfeeding women. Doxycycline is excreted in breast milk at concentrations 30–40% of that found in maternal blood,In patients with impaired renal or hepatic function, the dose of doxycycline does not have to be adjusted because it is excreted as an inactive chelated product by a complex process of back diffusion in the small intestine.151In patients with seizure disorders in whom mefloquine is contraindicated, doxycycline can be given as an alternative.Doxycyline (Vibramycin) syrup contains metabisulfite, which can cause allergic reactions, including anaphylaxis and asthmatic episodes in susceptible individuals.3Persons being treated for skin conditions with minocycline and who are in need of malaria prophylaxis should stop minocycline and start doxycycline. There is more data on the antimalarial efficacy of doxycycline compared with minocycline, therefore minocycline is not recommended as a replacement for doxycycline. Additionally, there have been case reports of autoimmune hepatitis in persons taking 100 mg or more of minocycline daily, whereas doxycycline has been observed to have less hepatotoxicity than other tetracyclines and less adverse events than minocycline.The drug cost for doxycycline varies by doxycycline salt, enteric formulation, or brand name versus generic. One article examining the cost considerations of malaria chemoprophylaxis looked at the average wholesale price (prices from 2006) of various antimalarial medications and found that generic doxycycline hyclate was much cheaper  than generic doxycycline monohydrate  and that generic doxycycline monohydrate was much cheaper than the brand name .154P. falciparum malaria) with tetracycline (with atovaquone) has been associated with approximately a 40% reduction in plasma concentrations of atovaquone,”in vitro studies of atovaquone plus doxycycline and atovaquone plus tetracycline showed potentiation between the two drugs, which was later confirmed in clinical studies by improved cure rates when compared with atovaquone alone.Plasmodium life cycle. The Centers for Disease Control and Prevention (CDC) currently recommends that the atovaquone/proguanil is continued for up to 4 weeks after leaving the endemic area depending on when the change was made.There is very limited information on the best regimen for changing from doxycycline to other antimalarials, and recommendations for doing so are based on considering several issues such as drug-drug interactions, half-life differences, and differences in mechanism of action. If changing between atovaquone/proguanil and doxycycline, note that the current atovaquone-proguanil label lists a pharmacokinetic interaction of “concomitant treatment (of