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Background Naturally acquired immunity (NAI), which is characterized by protection against overt clinical disease and high parasitaemia, is acquired with age and transmission intensity. The role of NAI on the efficacy of anti-malarial drugs, including artemisinin-based combinations used as the first-line treatment for uncomplicated Plasmodium falciparum, has not been fully demonstrated. This study investigated the role of NAI in response to artemisinin-based combination therapy (ACT), in symptomatic patients living in western Kenya, a high malaria transmission area. Methods Sera samples from malaria immune participants (n = 105) in a therapeutic efficacy study were assessed for in vitro growth inhibitory activity against the 3D7 strain of P. falciparum using a fluorescent-based growth inhibition assay (GIA). Participants’ age and parasite clearance parameters were used in the analysis. Pooled sera from malaria naïve participants (n = 6) with no Plasmodium infection from malaria non-endemic regions of Kenya was used as negative control. Results The key observations of the study were as follows: (1) Sera with intact complement displayed higher GIA activity at lower (1%) serum dilutions (p p = 0.05) and slope half-life (p = 0.025); and (3) age was a confounding factor when comparing the GIA activity with parasite clearance kinetics. Conclusion This study demonstrates for the first time there is synergy of complement, pre-existing immunity, and drug treatment in younger patients with symptomatic malaria in a high-transmission area.

Reliably assessing exposure to mosquitoes carrying malaria parasites continues to be a challenge due to the lack of reliable, highly sensitive diagnostics with high-throughput potential. Here, we describe an approach that meets these requirements by simultaneously measuring immune responses to both disease vector and pathogen, using an electro-chemiluminescence-based multiplex assay platform. While using the same logistical steps as a classic ELISA, this platform allows for the multiplexing of up to ten antigens in a single well. This simple, reproducible, quantitative readout reports the magnitude, incidence, and prevalence of malaria infections in residents of malaria-endemic areas. By reporting exposure to both insect vectors and pathogen, the approach also provides insights into the efficacy of drugs and/or other countermeasures deployed against insect vectors aimed at reducing or eliminating arthropod-borne diseases. The high throughput of the assay enables the quick and efficient screening of sera from individuals for exposure to Plasmodium even if they are taking drug prophylaxis. We applied this assay to samples collected from controlled malaria infection studies, as well as those collected in field studies in malaria-endemic regions in Uganda and Kenya. The assay was sensitive to vector exposure, malaria infection, and endemicity, demonstrating its potential for use in malaria serosurveillance.

Human immunodeficiency virus (HIV) and malaria infection rates overlap across sub-Saharan Africa, but factors influencing their co-occurrence are unclear. In a case-control study, we investigated whether malaria exposure increases risk of type 1 (HIV-1) acquisition. Prior to seroconverting, HIV-positive cases had significantly higher malaria-associated antibodies compared to HIV-negative controls, linking malaria exposure to HIV-1 acquisition.

Background Malaria and schistosomiasis present considerable disease burden in tropical and sub-tropical areas and severity is worsened by co-infections in areas where both diseases are endemic. Although pathogenesis of these infections separately is well studied, there is limited information on the pathogenic disease mechanisms and clinical disease outcomes in co-infections. In this study, we investigated the prevalence of malaria and schistosomiasis co-infections, and the hematologic and blood chemistry abnormalities in asymptomatic adults in a rural fishing community in western Kenya. Methods This sub-study used samples and data collected at enrollment from a prospective observational cohort study (RV393) conducted in Kisumu County, Kenya. The presence of malaria parasites was determined using microscopy and real-time-PCR, and schistosomiasis infection by urine antigen analysis (CCA). Hematological analysis and blood chemistries were performed using standard methods. Statistical analyses were performed to compare demographic and infection data distribution, and hematologic and blood chemistry parameters based on different groups of infection categories. Clinically relevant hematologic conditions were analyzed using general linear and multivariable Poisson regression models. Results From February 2017 to May 2018, we enrolled 671 participants. The prevalence of asymptomatic Plasmodium falciparum was 28.2% (157/556) and schistosomiasis 41.2% (229/562), with 18.0% (100/556) of participants co-infected. When we analyzed hematological parameters using Wilcoxon rank sum test to evaluate median (IQR) distribution based on malarial parasites and/or schistosomiasis infection status, there were significant differences in platelet counts (p = 0.0002), percent neutrophils, monocytes, eosinophils, and basophils (p Conclusions Our study demonstrates the high burden of asymptomatic malaria parasitemia and schistosomiasis infection in this rural population in Western Kenya. Asymptomatic infection with malaria or schistosomiasis was associated with laboratory abnormalities including neutropenia, leukopenia and thrombocytopenia. These abnormalities could be erroneously attributed to other diseases processes during evaluation of diseases processes. Therefore, evaluating for co-infections is key when assessing individuals with laboratory abnormalities. Additionally, asymptomatic infection needs to be considered in control and elimination programs given high prevalence documented here.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.

Background Rapid diagnostic tests (RDT) are routinely used in screening for HIV infection. More complex diagnostic algorithms incorporating fourth-generation screening and confirmatory HIV-1/HIV-2 differentiation immunoassays (IA) may be used to confirm HIV infection. Co-infections and autoimmune diseases may lead to falsely reactive HIV diagnostic test results. Case presentation A Kenyan man with asymptomatic schistosomiasis and low risk factors for HIV infection demonstrated an inconsistent and discordant pattern of reactivity on HIV RDT, repeated reactivity on fourth-generation IA and positive at a single time-point for HIV-1 on the Geenius HIV1/HIV2 confirmatory assay during the course of a prospective cohort study with HIV repeat testing. The individual initiated antiretroviral therapy following HIV diagnosis. However, his bi-annual behavioral questionnaire suggested low-risk factors for infection. Supplementary confirmatory serologic and nucleic acid tests were performed and gave discordant results. The participant was determined to be HIV uninfected using cell-associated HIV-1 DNA/RNA testing and antiretroviral therapy was discontinued. Discussion and conclusions Sole reliance on diagnostic test results may result in misdiagnosis of HIV infection, social harm and potential antiretroviral induced drug toxicity. Interpretation of HIV test results should incorporate multiple parameters.

The role of humoral immunity on the efficacy of artemisinin combination therapy (ACT) has not been investigated, yet naturally acquired immunity is key determinant of antimalarial therapeutic response. We conducted a therapeutic efficacy study in high transmission settings of western Kenya, which showed artesunate-mefloquine (ASMQ) and dihydroartemisinin-piperaquine (DP) were more efficacious than artemether-lumefantrine (AL). To investigate the underlying prophylactic mechanism, we compared a broad range of humoral immune responses in cohort I study participants treated with ASMQ or AL, and applied machine-learning (ML) models using immunoprofile data to analyze individual participants’ treatment outcome. We showed ML models could predict treatment outcome for ASMQ but no AL with high (72-92%) accuracy. Simulated PK profiling provided evidence demonstrating specific humoral immunity confers protection in the presence of sub-therapeutic residual mefloquine concentration. We concluded patient humoral immunity and partner drug interact to provide long prophylactic effect of ASMQ.

Objective Good quality microscopy is critical for accurate detection and confirmation of malaria parasite infections. Microscopy relies on the skills of technicians to prepare and read slides, high quality reagents, and a good program of internal and external quality control (EQA), which are lacking in most malaria endemic settings. This study was undertaken between January 2016 and December 2018 to pilot an EQA of microscopy for improved diagnosis of malaria and patient care in Tanzanian Military health facilities. Results Of all blood smears crosschecked (n = 4000) at baseline, only 38.5% were incorrectly diagnosed by laboratory staff with false positive and negative rates of 46.7% and 16.4%, respectively. During the implementation of EQA, false positive and negative results decreased due to increased quality index of slide preparation and reading through supportive supervision, and retraining of laboratory personnel. There was a gradual increase of quarterly and annual total quality index for all laboratories, from 60% in 2016 to 78% in 2017 and 90% in 2018. The mean proficiency testing performance scores also increased from 75% in 2016 to 82% in 2017 and to 90% in 2018. Poor blood smear preparation and staining contributed to high false positive and negative rates while EQA helped in improvement of diagnostics.

BackgroundThe epidemiology and severity of non-falciparum malaria in endemic settings has garnered limited attention. We aimed to characterize the prevalence, interaction, clinical risk factors and temporal trends of non-falciparum malaria in endemic settings of Kenya.MethodsWe diagnosed and analyzed infecting malaria species via PCR in 2027 clinical samples collected between 2008 and 2016. Descriptive statistics were used to describe the prevalence and distribution of Plasmodium species. A statistical model was designed and used for estimating the frequency of Plasmodium species and assessing inter-species interactions. Mixed effect linear regression models with random intercepts for each location was used to test for change in prevalence over time.Findings72•5% of the samples were P. falciparum single species infections, 25·8% were mixed infections and only 1•7% occurred as single non-falciparum species infections. 23•1% were mixed infections containing P. ovale. A likelihood-based model calculation of the population frequency of each species estimated a significant within-host interference between P. falciparum and P. ovale curtisi. Mixed-effect logistic regression models identified a significant increase of P. ovale wallikeri and P. ovale curtisi species over time with reciprocal decrease in P. falciparum single species and P. malariae. The risk of P. falciparum infections presenting with fever was 0•43 times less likely if co-infected with P. malariae.InterpretationFindings show higher prevalence of non-falciparum malaria than expected. The proportion of infections that were positive for infection by P. ovale wallikeri and P. ovale curtisi was observed to significantly increase over the period of study which could be due to attenuated responsiveness to malaria drug treatment on these species. The increase in frequency of P. ovale spp in Kenya could threaten malaria control effort in Kenya and pose increased risk of malaria to travelers.FundingAFHSB and its GEIS Section

The development of artemisinin (ART)-resistant parasites in Southeast Asia (SEA) threatens malaria control globally. Mutations in the Kelch 13 (K13)-propeller domain have been useful in identifying ART resistance in SEA. ART combination therapy (ACT) remains highly efficacious in the treatment of uncomplicated malaria in Sub-Saharan Africa (SSA). However, it is crucial that the efficacy of ACT is closely monitored. Toward this effort, this study profiled the prevalence of K13 nonsynonymous mutations in different malaria ecological zones of Kenya and in different time periods, before (pre) and after (post) the introduction of ACT as the first-line treatment of malaria. Nineteen nonsynonymous mutations were present in the pre-ACT samples (N = 64) compared with 22 in the post-ACT samples (N = 251). Eight of these mutations were present in both pre- and post-ACT parasites. Interestingly, seven of the shared single-nucleotide polymorphisms were at higher frequencies in the pre-ACT than the post-ACT parasites. The A578S mutation reported in SSA and the V568G mutation reported in SEA were found in both pre- and post-ACT parasites, with their frequencies declining post-ACT. D584Y and R539K mutations were found only in post-ACT parasites; changes in these codons have also been reported in SEA with different amino acids. The N585K mutation described for the first time in this study was present only in post-ACT parasites, and it was the most prevalent mutation at a frequency of 5.2%. This study showed the type, prevalence, and frequency of K13 mutations that varied based on the malaria ecological zones and also between the pre- and post-ACT time periods.

Background Although microscopy is a standard diagnostic tool for malaria and the gold standard, it is infrequently used because of unavailability of laboratory facilities and the absence of skilled readers in poor resource settings. Malaria rapid diagnostic tests (RDT) are currently used instead of or as an adjunct to microscopy. However, at very low parasitaemia (usually

Edible insects are part of the diets of a significant proportion of rural populations in the tropics especially Africa and Asia, and their use as source of key nutrients for better nutrition is re-emerging. Indigenously, elemental methods are used to process the insects before they are consumed or sold in retail outlets. In recent years, better knowledge of processing, packaging and storage has become necessary because of commercialisation needs. A common processing approach involves drying after a brief heat-treatment step, and then milling into a powdered product which is sold to manufacturers or consumers as ingredient for processing final products. The hydration properties of dried powders of edible house cricket and black soldier fly larvae (BSFL) were studied with the aim of predicting shelf-life stability under typical packaging and storage temperatures experienced in the tropics. Moisture adsorption isotherms were determined gravimetrically at 25, 30 and 35 °C, over 0.11–0.97 water activity (aW) range, and the data fitted to various models. Sorption isotherms were of type II according to Brunauer classification indicating monolayer-multilayer sorption behaviour. Cricket powder exhibited higher hydration capacity, and aW of this product was less sensitive to temperature variation as compared to BSFL powder. In the two products, water exhibited transitions from bound- to free- state at ~5 g/100 g moisture content. Based on Heiss-Eichner model, a shelf-life of 7 months at 25 °C can be achieved if the cricket and BSFL powders are dried to ca. 5 g/100 g moisture content and packaged in 80 μm thick polyethylene films. At 35 °C the shelf-life of the cricket product is shortened three- to four-fold whereas the BSFL powder is unable to store.

Catabacter hongkongensis, an increasingly recognized bacteria in clinical samples, was identified by direct metagenomic sequencing of positive blood culture fluid from a 55-year-old patient with colonic perforation. The bacteremia was cleared by both antibiotic treatment and surgical intervention. This is the first case report of C. hongkongensis infection in the US.

Background Naturally acquired immunity (NAI), which is characterized by protection against overt clinical disease and high parasitaemia, is acquired with age and transmission intensity. The role of NAI on the efficacy of anti-malarial drugs, including artemisinin-based combinations used as the first-line treatment for uncomplicated Plasmodium falciparum, has not been fully demonstrated. This study investigated the role of NAI in response to artemisinin-based combination therapy (ACT), in symptomatic patients living in western Kenya, a high malaria transmission area. Methods Sera samples from malaria immune participants (n = 105) in a therapeutic efficacy study were assessed for in vitro growth inhibitory activity against the 3D7 strain of P. falciparum using a fluorescent-based growth inhibition assay (GIA). Participants’ age and parasite clearance parameters were used in the analysis. Pooled sera from malaria naïve participants (n = 6) with no Plasmodium infection from malaria non-endemic regions of Kenya was used as negative control.Results The key observations of the study were as follows: (1) Sera with intact complement displayed higher GIA activity at lower (1%) serum dilutions (p < 0.0001); (2) there was significant relationship between GIA activity, parasite clearance rate (p = 0.05) and slope half-life (p = 0.025); and (3) age was a confounding factor when comparing the GIA activity with parasite clearance kinetics. Conclusion This study demonstrates for the first time there is synergy of complement, pre-existing immunity, and drug treatment in younger patients with symptomatic malaria in a high-transmission area.

Ebb and flow of parasite populations The population genetics of the malaria parasite Plasmodium falciparum across Africa is poorly understood but important to know for grasping the risks and dynamics of the spread of drug resistance. Harnessing the power of genomics, Amambua-Ngwa et al. of the Plasmodium Diversity Network Africa found substantial population structure within Africa that is consistent with human and vector population divergence (see the Perspective by Sibley). Specific signatures of selection by antimalarial drugs were detected, along with indications of the effect of colonization and slavery. Furthermore, whole-genome sequencing showed that there is extensive gene flow among the different regions and that Ethiopia has a distinctive population of P. falciparum , which may be indicative of coexistence with another malaria parasite, P. vivax. Science , this issue p. 813 ; see also p. 752

Bacteria often exist in polymicrobial communities where they compete for limited resources. Intrinsic to this competition is the ability of some species to inhibit or kill their competitors. This phenomenon is pervasive throughout the human body where commensal bacteria block the colonization of incoming microorganisms. In this regard, molecular epidemiological and microbiota-based studies suggest that species-specific interactions play a critical role in the prevention of nasal colonization of the opportunistic pathogen Staphylococcus aureus. Despite this, S. aureus exists as part of the microbiota of ∼25% of the population, suggesting that the interplay between S. aureus and commensals can be complex. Microbiota studies indicate that several bacterial genera are negatively correlated with S. aureus colonization. While these studies paint a broad overview of bacterial presence, they often fail to identify individual species-specific interactions; a greater insight in this area could aid the development of novel antimicrobials. As a proof of concept study designed to identify individual bacterial species that possess anti-S. aureus activity, we screened a small collection of clinical isolates from the Walter Reed National Military Medical Center for the ability to inhibit multiple S. aureus strains. We found that the majority of the isolates (82%) inhibited at least one S. aureus strain; 23% inhibited all S. aureus strains tested. In total, seven isolates mediated inhibitory activity that was independent of physical contact with S. aureus, and seven isolates mediated bactericidal activity. 16S rRNA based-sequencing revealed that the inhibitory isolates belonged to the Acinetobacter, Agromyces, Corynebacterium, Microbacteria, Mycobacterium, and Staphylococcus genera. Unexpectedly, these included seven distinct Acinetobacter baumannii isolates, all of which showed heterogeneous degrees of anti-S. aureus activity. Defined mechanistic studies on specific isolates revealed that the inhibitory activity was retained in conditioned cell free medium (CCFM) derived from the isolates. Furthermore, CCFM obtained from S. saprophyticus significantly decreased mortality of S. aureus-infected Galleria mellonella caterpillars. While future studies will seek to define the molecular mechanisms of the inhibitory activities, our current findings support the study of polymicrobial interactions as a strategy to understand bacterial competition and to identify novel therapeutics against S. aureus and other pathogens.

Objective Cotrimoxazole prevents opportunistic infections including falciparum malaria in HIV-infected individuals but there are concerns of cross-resistance to other antifolate drugs such as sulphadoxine-pyrimethamine (SP). In this study, we investigated the prevalence of antifolate-resistance mutations in Plasmodium falciparum that are associated with SP resistance in HIV-infected individuals on antiretroviral treatment randomized to discontinue (STOP-CTX), or continue (CTX) cotrimoxazole in Western Kenya. Design Samples were obtained from an unblinded, non-inferiority randomized controlled trial where participants were recruited on a rolling basis for the first six months of the study, then followed-up for 12 months with samples collected at enrollment, quarterly, and during sick visits. Method Plasmodium DNA was extracted from blood specimens. Initial screening to determine the presence of Plasmodium spp. was performed by quantitative reverse transcriptase real-time PCR, followed by genotyping for the presence of SP-resistance associated mutations by Sanger sequencing. Results The prevalence of mutant haplotypes associated with SP-resistant parasites in pfdhfr (51I/59R/108N) and pfdhps (437G/540E) genes were significantly higher (P = 0.0006 and P = 0.027, respectively) in STOP-CTX compared to CTX arm. The prevalence of quintuple haplotype (51I/59R/108N/437G/540E) was 51.8% in STOP-CTX vs. 6.3% (P = 0.0007) in CTX arm. There was a steady increase in mutant haplotypes in both genes in STOP-CTX arm overtime through the study period, reaching statistical significance (P < 0.0001). Conclusion The frequencies of mutations in pfdhfr and pfdhps genes were higher in STOP-CTX arm compared to CTX arm, suggesting cotrimoxazole effectively controls and selects against SP-resistant parasites. Trial registration ClinicalTrials.gov NCT01425073, Author summary Cotrimoxazole, an antifolate, is a fixed-dose trimethoprim-sulfamethoxazole used to prevent opportunistic infections including malaria in HIV-infected individuals. There are concerns that widespread use of cotrimoxazole for prophylaxis may result in selection of P. falciparum parasites with cross-resistance to other antifolate drugs such as sulphadoxine-pyrimethamine (SP), which is used as intermittent preventive treatment of malaria in pregnancy (IPTp) and in infants (IPTi) in Africa. This sub-study used samples from a clinical trial in which HIV-infected individuals on antiretroviral treatment were randomized to discontinue (STOP-CTX) or continue (CTX) cotrimoxazole prophylaxis for 12 months. The sub-study was designed to assess whether taking cotrimoxazole increased the risk of selecting for parasites with SP-resistant mutations in HIV-infected individuals. Samples were genotyped by sequencing to assess the prevalence of mutations associated with SP-resistance. We found there was no risk of selecting for parasites with SP-resistance mutations while on cotrimoxazole. In fact, the opposite was true; cotrimoxazole controlled parasites carrying SP-resistance mutations as evident by the gradual increase in the prevalence of parasites with mutant alleles in the STOP-CTX arm and not in the CTX-arm. We concluded that cotrimoxazole remains effective in controlling malaria infection despite of the high prevalence of SP-resistant parasites, and its use does not select for SP mutations.

Human adenoviruses (HAdVs) are known to cause respiratory illness outbreaks at basic military training (BMT) sites. HAdV type-4 and -7 vaccines are routinely administered at enlisted BMT sites, but not at military academies. During August-September 2016, U.S. Naval Academy clinical staff noted an increase in students presenting with acute respiratory illness (ARI). An investigation was conducted to determine the extent and cause of the outbreak. During 22 August-11 September 2016, 652 clinic visits for ARI were identified using electronic health records. HAdV-4 was confirmed by realtime polymerase chain reaction assay in 18 out of 33 patient specimens collected and 1 additional HAdV case was detected from hospital records. Two HAdV-4 positive patients were treated for pneumonia including 1 hospitalized patient. Molecular analysis of 4 HAdV-4 isolates identified genome type 4a1, which is considered vaccine-preventable. Understanding the impact of HAdV in congregate settings other than enlisted BMT sites is necessary to inform discussions regarding future HAdV vaccine strategy.

447-52D (447) is a human monoclonal antibody that recognizes a conserved epitope in the crown region of the third variable loop (V3) of HIV-1 gp120, and like many anti-HIV-1 antibodies with broad neutralization capabilities, it has a long heavy-chain complementarity determining region (CDRH3). Here, we use a combination of computational mutagenesis and modeling in tandem with fluorescence polarization assays to interrogate the molecular basis of 447 CDRH3 length and the individual contribution of selected CDRH3 residues to affinity. We observe that 447 CDRH3 length provides a large binding surface area and the best enthalpic contributions derived from hydrophobic packing, main-chain hydrogen bonds, electrostatic and van der Waals interactions. We also found out that CDRH3 residue Try100I is critical to 447 binding affinity.

Background HIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell–cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined. Results To study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-∆V1V2. The Env-isfGFP-∆V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell–cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS. Conclusions Env and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS. Electronic supplementary material The online version of this article (10.1186/s12977-019-0464-3) contains supplementary material, which is available to authorized users.

Background Clinical laboratory reference intervals (RIs) are essential for diagnosing and managing patients in routine clinical care as well as establishing eligibility criteria and defining adverse events in clinical trials, but may vary by age, gender, genetics, nutrition and geographic location. It is, therefore, critical to establish region-specific reference values in order to inform clinical decision-making. Methods We analyzed data from a prospective observational HIV incidence cohort study in Kombewa, Kenya. Study participants were healthy males and females, aged 18–35 years, without HIV. Median and 95% reference values (2.5th percentile to 97.5th percentile) were calculated for laboratory parameters including hematology, chemistry studies, and CD4 T cell count. Standard Deviation Ratios (SDR) and Bias Ratios (BR) are presented as measures of effect magnitude. Findings were compared with those from the United States and other Kenyan studies. Results A total of 299 participants were analyzed with a median age of 24 years (interquartile range: 21–28). Ratio of males to females was 0.9:1. Hemoglobin range (2.5th—97.5th percentiles) was 12.0–17.9 g/dL and 9.5–15.3 g/dL in men and women respectively. In the cohort, MCV range was 59-95fL, WBC 3.7–9.2×103/μL, and platelet 154–401×103/μL. Chemistry values were higher in males; the creatinine RI was 59–103 μmol/L in males vs. 46–76 μmol/L in females (BRUL>.3); and the alanine transferase range was 8.8–45.3 U/L in males vs. 7.5–36.8 U/L in females (SDR>.3). The overall CD4 T cell count RI was 491–1381 cells/μL. Some parameters including hemoglobin, neutrophil, creatinine and ALT varied with that from prior studies in Kenya and the US. Conclusion This study not only provides clinical reference intervals for a population in Kisumu County but also highlights the variations in comparable settings, accentuating the requirement for region-specific reference values to improve patient care, scientific validity, and quality of clinical trials in Africa.

Transmission intensity, movement of human and vector hosts, biogeographical features, and malaria control measures are some of the important factors that determine Plasmodium falciparum parasite genetic variability and population structure. Kenya has different malaria ecologies which might require different disease intervention methods. Refined parasite population genetic studies are critical for informing malaria control and elimination strategies. This study describes the genetic diversity and population structure of P. falciparum parasites from the different malaria ecological zones in Kenya. Twelve multi-locus microsatellite (MS) loci previously described were genotyped in 225 P. falciparum isolates collected between 2012 and 2013 from five sites; three in lowland endemic regions (Kisumu, Kombewa, and Malindi) and two in highland, epidemic regions (Kisii and Kericho). Parasites from the lowland endemic and highland epidemic regions of western Kenya had high genetic diversity compared to coastal lowland endemic region of Kenya [Malindi]. The Kenyan parasites had a mean genetic differentiation index (FST) of 0.072 (p=0.011). The multi-locus genetic analysis of the 12 MS revealed all the parasites had unique haplotypes. Significant linkage disequilibrium (LD) was observed in all the five parasite populations. Kisumu had the most significant index of association values (0.16; p

A respiratory outbreak associated with human adenovirus type 7 (HAdV-7) occurred among unvaccinated officer candidates attending initial military training. Respiratory infections associated with HAdV-7 can be severe, resulting in significant morbidity. Genomic sequencing revealed HAdV-7d, a genome type recently remerging in the United States as a significant respiratory pathogen, following reports from Southeast Asia. Twenty-nine outbreak cases were identified; this likely represents an underestimate. Although the HAdV type 4 and 7 vaccine is currently given to US military enlisted recruit trainees, it is not routinely given to officer candidates. Administration of the HAdV type 4 and 7 vaccine may benefit this cohort.

IntroductionInternal and external quality control (QC) of rapid diagnostic tests (RDTs) is important to increase reliability of RDTs currently used to diagnose malaria. However, cross-checking of used RDTs as part of quality assurance can rarely be done by off-site personnel because there is no guarantee of retaining visible test lines after manufacturers' recommended reading time. Therefore, this study examined the potential of using Fionet™ technology for remote RDT quality monitoring at seven clinics, identifying reasons for making RDT processing and interpretation errors, and taking corrective actions for improvement of diagnosis and consequently improved management of febrile patients.MethodsThe study was conducted at seven military health facilities in Mainland Tanzania and utilized RDTs capable of detecting Plasmodium falciparum specific Histidine-rich protein 2 (Pf-HRP2) and the genus specific Plasmodium lactate dehydrogenase (pLDH) for other species of plasmodium (P. vivax, P. malariae or P. ovale; pan-pLDH). Patients' data and images of processed RDTs from seven clinics were uploaded on a Fionet web portal and reviewed regularly to monitor preparation procedures and visual interpretation of test results compared to automated analysis using the Deki reader of RDT. Problems detected were rapidly communicated to remote laboratory personnel at the clinic for corrective action and follow-up of patients who were falsely diagnosed as negative and missed treatment. Factors contributing to making errors in visual interpretation of RDT results were analyzed during visits to the health facilities.ResultsA total of 1,367 (1.6%) out of 83,294 RDT test images uploaded to the Fionet portal had discordant test results of which 822 (60.1%) and 545 (39.9%) were falsely reported as negative and positive, respectively. False negative and false positive test results were common for a single test line in 515 (62.7%) and 741 (54.2%) tests, respectively. Out of 1,367 RDT images assessed, 98 (7.2%) had quality problems related to preparation procedures of which 95(96.9%) errors were due to putting too much blood on the sample well or insufficient buffer in the respective wells. The reasons for discrepant results included, false reporting of none existent lines in 526 (38.5%) tests, missing a faint positive line in 493 (36.1%), missing a strong positive line in 248(18.1%) and errors caused by poorly processed RDTs in 96 (7.2%) tests. Among the false negative tests (n = 822), 669 (48.9%) patients were eligible for follow-up and only 339 (48.5%) were reached and 291 (85.8%) received appropriate anti-malaria therapy.ConclusionFionet technology enabled remote monitoring of RDT quality issues, identifying reasons contributing to laboratory personnel making errors and provided a rapid method to implement corrective actions at remote sites to improve malaria diagnosis and consequently improved health care management of febrile patients infected with malaria.

Background There are concerns that resistance to artemisinin-based combination therapy might emerge in Kenya and sub-Saharan Africa (SSA) in the same pattern as was with chloroquine and sulfadoxine–pyrimethamine. Single nucleotide polymorphisms (SNPs) in critical alleles of pfmdr1 gene have been associated with resistance to artemisinin and its partner drugs. Microsatellite analysis of loci flanking genes associated with anti-malarial drug resistance has been used in defining the geographic origins, dissemination of resistant parasites and identifying regions in the genome that have been under selection. Methods This study set out to investigate evidence of selective sweep and genetic lineages in pfmdr1 genotypes associated with the use of artemether–lumefantrine (AL), as the first-line treatment in Kenya. Parasites (n = 252) from different regions in Kenya were assayed for SNPs at codons 86, 184 and 1246 and typed for 7 neutral microsatellites and 13 microsatellites loci flanking (± 99 kb) pfmdr1 in Plasmodium falciparum infections. Results The data showed differential site and region specific prevalence of SNPs associated with drug resistance in the pfmdr1 gene. The prevalence of pfmdr1 N86, 184F, and D1246 in western Kenya (Kisumu, Kericho and Kisii) compared to the coast of Kenya (Malindi) was 92.9% vs. 66.7%, 53.5% vs. to 24.2% and 96% vs. to 87.9%, respectively. The NFD haplotype which is consistent with AL selection was at 51% in western Kenya compared to 25% in coastal Kenya. Conclusion Selection pressures were observed to be different in different regions of Kenya, especially the western region compared to the coastal region. The data showed independent genetic lineages for all the pfmdr1 alleles. The evidence of soft sweeps in pfmdr1 observed varied in direction from one region to another. This is challenging for malaria control programs in SSA which clearly indicate effective malaria control policies should be based on the region and not at a country wide level. Electronic supplementary material The online version of this article (10.1186/s12936-018-2534-8) contains supplementary material, which is available to authorized users.

Introduction. Microbacterium spp. are yellow-pigmented Gram-positive coryneform rods found in various environmental sources, such as soil and water samples. They rarely cause human infection, mostly infecting immunocompromised patients and catheter insertion sites, making them challenging to identify in clinical settings. Case presentation. We report a case of a 61-year-old female on long-term prednisone therapy for sarcoidosis with minimal exposure to environmental sources, who presented with an overtly infected Hickman catheter site and presyncope. The patient had a central venous catheter (CVC) that had been in place for the previous 6 years for treatment of refractory hypertension and congestive heart failure. Blood cultures obtained from the CVC on initial presentation were positive for a mixed infection, which was subcultured and grew Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter radioresistens and Leifsonia aquatica based on the Becton Dickinson Phoenix Automated Microbiology System. The L. aquatica, designated as isolate 4120, was further analysed, since infections associated with this organism are uncommon, and it was the only organism to grow from the patient’s catheter tip. Matrix-assisted laser desorption ionization–time of flight MS identified isolate 4120 as Microbacterium paraoxydans. To resolve the conflicting results, additional analyses of isolate 4120 were carried out and compared to several reference strains. Isolate 4120 was found to have intermediate susceptibility to ciprofloxacin and non-susceptibility to vancomycin. Morphology, susceptibility, biochemical characteristics and whole-genome sequencing confirmed the clinical isolate as Microbacterium paraoxydans. Conclusion. In this case, we identified an organism that is rarely seen in clinical settings and characterized it with a comprehensive laboratory analysis. The patient in our case responded to replacement of the CVC, and treatment with levofloxacin by mouth and intravenous vancomycin.

The development of artemisinin (ART)-resistant parasites in Southeast Asia (SEA) threatens malaria control globally. Mutations in the Kelch 13 (K13)-propeller domain have been useful in identifying ART resistance in SEA. ART combination therapy (ACT) remains highly efficacious in the treatment of uncomplicated malaria in Sub-Saharan Africa (SSA). However, it is crucial that the efficacy of ACT is closely monitored. Toward this effort, this study profiled the prevalence of K13 nonsynonymous mutations in different malaria ecological zones of Kenya and in different time periods, before (pre) and after (post) the introduction of ACT as the first-line treatment of malaria. Nineteen nonsynonymous mutations were present in the pre-ACT samples (N = 64) compared with 22 in the post-ACT samples (N = 251). Eight of these mutations were present in both pre- and post-ACT parasites. Interestingly, seven of the shared single-nucleotide polymorphisms were at higher frequencies in the pre-ACT than the post-ACT parasites. The A578S mutation reported in SSA and the V568G mutation reported in SEA were found in both pre- and post-ACT parasites, with their frequencies declining post-ACT. D584Y and R539K mutations were found only in post-ACT parasites; changes in these codons have also been reported in SEA with different amino acids. The N585K mutation described for the first time in this study was present only in post-ACT parasites, and it was the most prevalent mutation at a frequency of 5.2%. This study showed the type, prevalence, and frequency of K13 mutations that varied based on the malaria ecological zones and also between the pre- and post-ACT time periods.

International audience; Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.

Insects have been used as food, medicine and in rituals by a number of communities in the East African region comprising of Kenya, Uganda and Tanzania over centuries. Progressively, farmed edible insects mainly crickets and grasshoppers are gaining popularity within the region. However the utilization of the edible insects is hampered by lack of storage and preservation facilities in the rural areas leading to high postharvest losses. Sun drying and roasting have been the main processing methods applied for decades by communities consuming edible insects such as the Luo from Kenya. Recently there has been incorporation of insects as an ingredient in processing of baked products and complementary foods. Culture, taboos, customs and ethnic preferences have highly influenced the consumption of edible insects in East Africa. Edible insects such as grasshoppers, mayfly and termites that are consumed in this region have been shown to be source of both macro and micro nutrients and other components such as chitin which has been linked to improved health and better management of chronic diseases. Therefore edible insects promises to be a part of the solution to food and nutrition security within the East African region.

The prevalence of a genetic polymorphism(s) at codon 268 in the cytochrome b gene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227 Plasmodium falciparum parasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC 50 ) and parasite genetic polymorphism; mutant alleles had higher IC 50 s than the wild type.

We describe a case of attenuated-responsiveness to Artemether-Lumefantrine (AL) in western Kenya. A 4-year old patient with 14% parasitemia was treated with Coartem® but on day 7 still had 5% parasitemia. Parasite genetic profile had similar genetic polymorphism as that selected for when AL is administered. In vitro analysis revealed elevated IC 50 s for AL components compared to the reference P. falciparum clone 3D7 and samples collected at this site over the last 5 years. There is need for continued global surveillance to effectively manage emergence and spread of artemisinin drug resistance which has now been found in Southeast Asia. Keywords: Coartem; resistance; malaria; artemether-lumefantrine; treatment-failure. 1. INTRODUCTION malaria and are Artemether-Lumefantrine (AL) is the Artemisinin Combination Therapy (ACT) currently recommended first-line therapy for falciparum malaria since 2006 by Kenyan Ministry of Health (MoH). Artemether -lumefantrine is used in more than 80 countries worldwide and was the first World Health Organization (WHO) prequalified ACT anti-malarial drug, manufactured by Novartis under the brand name Coartem®. Artemether half-life is ~ 1.5 h and lumefantrine is 4 to 6 days, both for the hard and dispersible tablets. AL is administered twice daily for three days as tablets containing 20 mg of artemether plus 120 mg of lumefantrine as follows: 1 tablet (for patients weighing 5–14 kg), 2 tablets (for patients weighing 15–24 kg), 3 tablets (for patients weighing 25–34 kg) and 4 tablets (for patients weighing ≥35 kg). The efficacy of AL has remained > 95% with mild adverse events, most commonly GI (vomiting and diarrhea) and hematologic (anemia and eosinophilia). Although ACTs continue to have excellent cure rates in Africa, there are now confirmed reports of artemisinin-resistance malaria in Southeast Asia [1,2]. A recent report from the coast of Kenya showed that responsiveness to ACTs is declining [3]. This report was the first showing declining responsiveness to artemisinin outside Southeast Asia. The US Army Medical Research Unit – Kenya (USAMRU-K) has an approved active study protocol to study the epidemiology of malaria and drug sensitivity patterns in Kenya. The primary objective of the study is to describe the molecular and

Hoseah M Akala, Angela O Achieng, Fredrick L Eyase, Dennis W Juma, Luiser Ingasia, Agnes C Cheruiyot, Charles Okello, Duke Omariba, Eunice A Owiti, Catherine Muriuki, Redemptah Yeda, Ben Andagalu, Jacob D Johnson, Edwin Kamau Global Emerging Infections Surveillance Program, United States Army Medical Research Unit-Kenya, Kenya Medical Research Institute, Walter Reed Project, Kisumu and Nairobi, Kenya Background: The renewed malaria eradication efforts require an understanding of the seasonal patterns of frequency of polymorphic variants in order to focus limited funds productively. Although cross-sectional studies in holoendemic areas spanning a single year could be useful in describing parasite genotype status at a given point, such information is inadequate in describing temporal trends in genotype polymorphisms. For Plasmodium falciparum isolates from Kisumu District Hospital, Plasmodium falciparum chloroquine-resistance transporter gene (Pfcrt-K76T) and P. falciparum multidrug resistance gene 1 (PfMDR1-N86Y), were analyzed for polymorphisms and parasitemia changes in the 53 months from March 2008 to August 2012. Observations were compared with prevailing climatic factors, including humidity, rainfall, and temperature. Methods: Parasitemia (the percentage of infected red blood cells per total red blood cells) was established by microscopy for P. falciparum malaria-positive samples. P. falciparum DNA was extracted from whole blood using a Qiagen DNA Blood Mini Kit. Single nucleotide polymorphism identification at positions Pfcrt-K76T and PfMDR1-N86Y was performed using real-time polymerase chain reaction and/or sequencing. Data on climatic variables were obtained from http://www.tutiempo.net/en/. Results: A total of 895 field isolates from 2008 (n=169), 2009 (n=161), 2010 (n=216), 2011 (n=223), and 2012 (n=126) showed large variations in monthly frequency of PfMDR1-N86Y and Pfcrt-K76T as the mutant genotypes decreased from 68.4%±15% and 38.1%±13% to 29.8%±18% and 13.3%±9%, respectively. The mean percentage of parasitemia was 2.61%±1.01% (coefficient of variation 115.86%; n=895). There was no correlation between genotype or parasitemia and climatic factors. Conclusion: This study shows variability in the frequency of Pfcrt-K76T and PfMDR1-N86Y polymorphisms during the study period, bringing into focus the role of cross-sectional studies in describing temporal genotype trends. The lack of correlation between genotypes and climatic changes, especially precipitation, emphasizes the cost of investment in genotype change. Keywords: PfMDR1-N86Y, Pfcrt-K76T, polymorphism, parasitemia

In combination with antibiotics, quinine is recommended as the second-line treatment for uncomplicated malaria, an alternative first-line treatment for severe malaria, and for treatment of malaria in the first trimester of pregnancy. Quinine has been shown to have frequent clinical failures, and yet the mechanisms of action and resistance have not been fully elucidated. However, resistance is linked to polymorphisms in multiple genes, including multidrug resistance 1 (Pf mdr1 ), the chloroquine resistance transporter (Pf crt ), and the sodium/hydrogen exchanger gene (Pf nhe1 ). Here, we investigated the association between in vitro quinine susceptibility and genetic polymorphisms in Pf mdr1 codons 86 and 184, Pf crt codon 76, and Pf nhe1 ms4760 in 88 field isolates from western Kenya. In vitro activity was assessed based on the drug concentration that inhibited 50% of parasite growth (the IC 50 ), and parasite genetic polymorphisms were determined from DNA sequencing. Data revealed there were significant associations between polymorphism in Pf mdr1 -86Y, Pf mdr1 -184F, or Pf crt -76T and quinine susceptibility ( P < 0.0001 for all three associations). Eighty-two percent of parasites resistant to quinine carried mutant alleles at these codons (Pf mdr1 -86Y, Pf mdr1 -184F, and Pf crt -76T), whereas 74% of parasites susceptible to quinine carried the wild-type allele (Pf mdr1 -N86, Pf mdr1 -Y184, and Pf crt -K76, respectively). In addition, quinine IC 50 values for parasites with Pf nhe1 ms4760 3 DNNND repeats were significantly higher than for those with 1 or 2 repeats ( P = 0.033 and P = 0.0043, respectively). Clinical efficacy studies are now required to confirm the validity of these markers and the importance of parasite genetic background.

The CH 2 Cl 2 /MeOH (1:1) extract of the stem bark of Millettia oblata ssp. teitensis showed antiplasmodial activity (IC 50 = 10–12 μg/mL) against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum . Chromatographic separation of the extract led to the isolation of a new isoflavone, 4′-prenyloxyderrone ( 1 ), together with known isoflavones (8- O -methylretusin, durmillone, maximaisoflavone B, maximaisoflavone H and maximaisoflavone J), a rotenoid (tephrosin) and a triterpene (lupeol). Similar investigation of Millettia leucantha resulted in the identification of the isoflavones afrormosin and wistin, and the flavone chrysin. The identification of these compounds was based on their spectroscopic data. Five of the isoflavones isolated from these plants as well as 11 previously reported compounds from Millettia dura were tested and showed good to moderate antiplasmodial activities (IC 50 = 13–53 μM), with the new compound, 4′-prenyloxyderrone, being the most active (IC 50 = 13–15 μM).

Plasmodium ovale is one of several clinically relevant malaria species known to cause disease in humans. However, in contrast to Plasmodium falciparum and Plasmodium vivax, which are responsible for most cases of human malaria, P. ovale has a wide distribution but low prevalence in tropical regions. Here, we report the case of a soldier returning from Liberia with P. ovale wallikeri malaria. This case highlights the limitations of both microscopy and the malaria rapid diagnostic test for diagnosing infection with P. ovale and for distinguishing P. ovale wallikeri from P. ovale curtisi. To our knowledge, this is the first case report in which quantitative real-time polymerase chain reaction amplification using the Cytochrome B gene, coupled with genomic sequencing of the potra locus, was used for definitive diagnosis of P. ovale wallikeri malaria.

The malaria SYBR green assay, which is used to profile in vitro drug susceptibility of Plasmodium falciparum , is a reliable drug screening and surveillance tool. Malaria field surveillance efforts provide isolates with various low levels of parasitemia. To be advantageous, malaria drug sensitivity assays should perform reproducibly among various starting parasitemia levels rather than at one fixed initial value. We examined the SYBR green assay standardized procedure developed by the Worldwide Antimalarial Resistance Network (WWARN) for its sensitivity and ability to accurately determine the drug concentration that inhibits parasite growth by 50% (IC 50 ) in samples with a range of initial parasitemia levels. The initial sensitivity determination of the WWARN procedure yielded a detection limit of 0.019% parasitemia. P. falciparum laboratory strains and field isolates with various levels of initial parasitemia were then subjected to a range of doses of common antimalarials. The IC 50 s were comparable for laboratory strains with between 0.0375% and 0.6% parasitemia and for field isolates with between 0.075% and 0.6% parasitemia for all drugs tested. Furthermore, assay quality (Z′) analysis indicated that the WWARN procedure displays high robustness, allowing for drug testing of malaria field samples within the derived range of initial parasitemia. The use of the WWARN procedure should allow for the inclusion of more malaria field samples in malaria drug sensitivity screens that would have otherwise been excluded due to low initial parasitemia levels.

The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24-48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long-term cultures, which indicates parasite genetic information obtained even in short cultures is likely to be different from the natural infection parasites.

Toxoplasma gondii is a globally ubiquitous pathogen that can cause severe disease in immunocompromised humans and the developing fetus. Given the proven role of Toxoplasma -secreted kinases in the interaction of Toxoplasma with its host cell, identification of novel kinase inhibitors could precipitate the development of new anti- Toxoplasma drugs and define new pathways important for parasite survival. We selected a small ( n = 527) but diverse set of putative kinase inhibitors and screened them for effects on the growth of Toxoplasma in vitro . We identified and validated 14 noncytotoxic compounds, all of which had 50% effective concentrations in the nanomolar to micromolar range. We further characterized eight of these compounds, four inhibitors and four enhancers, by determining their effects on parasite motility, invasion, and the likely cellular target (parasite or host cell). Only two compounds had an effect on parasite motility and invasion. All the inhibitors appeared to target the parasite, and interestingly, two of the enhancers appeared to rather target the host cell, suggesting modulation of host cell pathways beneficial for parasite growth. For the four inhibitors, we also tested their efficacy in a mouse model, where one compound proved potent. Overall, these 14 compounds represent a new and diverse set of small molecules that are likely targeting distinct parasite and host cell pathways. Future work will aim to characterize their molecular targets in both the host and parasite.

Expression of ribosomal proteins is controlled by the Target of Rapamycin (TOR) kinase. The Saccharomyces cerevisiae Forkhead-like transcription factor Fhl1 is important for this regulation, and its localization to ribosomal protein gene promoters requires the high mobility group protein HMO1. We show here that HMO1 expression is similarly controlled by TOR signaling. Reporter constructs in which lacZ is under control of the HMO1 promoter show that HMO1 promoter activity is repressed on inactivation of TOR and that HMO1 is required for this repression. Chromatin immunoprecipitation shows that Fhl1 localizes to the HMO1 promoter in an HMO1-dependent fashion and that it centers on a predicted Fhl1 site, and removal of the Fhl1 site in the HMO1 promoter attenuates the response to rapamycin. Taken together, our data show that the HMO1 promoter is regulated by TOR signaling, and that TOR can signal through an HMO1- and Fhl1-dependent mechanism, as proposed for TOR-mediated regulation of ribosomal protein expression. The shared mechanism of regulation further reinforces the central role of HMO1 in TOR-mediated regulation of ribosomal protein gene expression.

A highly sensitive genus-specific quantitative reverse transcriptase real-time PCR (qRT-PCR) assay for detection of Plasmodium has been developed. The assay amplifies total nucleic acids (RNA and DNA) of the 18S rRNA genes with a limit of detection of 0.002 parasite/μl using cultured synchronized ring stage 3D7 parasites. Parasite densities as low as 0.000362 parasite/μl were detected when analyzing clinical samples. Analysis of clinical samples showed that detection of 18S rRNA genes from total nucleic acids increased the analytical sensitivity of the assay by more than 1 log unit compared to DNA only. When clinical samples with no parasites present by microscopy were analyzed by qRT-PCR, 90% (117 of 130) were positive for the presence of Plasmodium nucleic acids. Quantification of clinical samples by qRT-PCR using total nucleic acid versus DNA was compared to microscopy. There was a significantly greater correlation of parasite density to microscopy when DNA alone was used than with total nucleic acid. We conclude that analysis of total nucleic acids by qRT-PCR is a suitable assay for detection of low parasite levels in patients with early-stage malaria and/or submicroscopic infections and could greatly benefit malaria diagnosis, intervention trials, and malaria control and elimination efforts.

Background The clinical significance of Staphylococcus aureus bacteriuria (SABU) is unclear and often presumed to represent hematogenous spread of occult S. aureus bacteremia (SAB). The aim of this study was to evaluate the clinical significance of SABU by assessing factors associated with the development of invasive S. aureus disease and 12-month mortality. We also describe the proportion of patients with methicillin-resistant S. aureus (MRSA), symptomatic urinary tract infection (UTI), asymptomatic bacteriuria, concomitant SAB, and calculate Charlson comorbidity scores. Methods This is a retrospective cohort study of all SABU isolates in a tertiary care hospital from 1 January 2007 to 31 December 2016. Cases were identified by screening specimens processed in the microbiology laboratory with records in the Phoenix/BG data system. Demographic data, Charlson comorbidity score, antimicrobial susceptibility profiles, urinary catheter use, residence in a long-term care facility, and UTI symptoms were obtained through chart review. Results A total of 356 patients with SABU were identified and 237 met inclusion criteria. Seventeen patients had concurrent invasive S. aureus infections and 220 patients had SABU without invasive infection or SAB. Of the 220 patients with SABU and no invasive S. aureus infection, none developed invasive disease. The 12-month mortality rate was 6.8% in those without concurrent SAB and 52.9% in those with invasive infection. In those without concurrent invasive S. aureus infection, male sex (P = 0.033), MRSA (P = 0.008), collection of blood cultures (P = 0.005), and Charlson comorbidity score (P ≤ 0.001) were identified as risk factors for 12-month mortality. Race, inpatient status, specimen type, pyuria, UTI symptoms, and urologic instrumentation were not significantly associated with mortality. Conclusion Patients without evident invasive S. aureus infection at the time of urine culture were not observed to present with invasive disease in the following year. Mortality increases with Charlson score and invasive infection in patients with SABU. Clinicians may consider isolated UTI, contamination, and colonization before embarking on extensive searches for occult staphylococcal infection in patients who present without obvious invasive infection, particularly in those without comorbid disease. Disclosures All authors: No reported disclosures.

High mobility group box 1 (HMGB1) protein functions in regulation of transcription, cellular activation and pro-inflammatory responses. However, the potential role of HMGB1 during viral infection has not been investigated. This study attempted to elucidate whether the HMGB1-mediated inflammatory response contributes to the pathogenesis of dengue virus (DENV) infection. Our data showed that HMGB1 was released at low DENV infection levels (m.o.i. of 1) under non-necrotic conditions by human dendritic cells (DCs). When DENV-infected DCs were co-cultured with autologous T cells, there was increased production of HMGB1 by both cell types. HMGB1 regulated tumour necrosis factor alpha, interleukin (IL)-6, IL-8 and alpha interferon secretion in DENV-infected DCs. Additionally, increased HMGB1 production was associated with reduced DENV replication titres in DCs. These results suggest that HMGB1 production influences DENV infection in susceptible hosts.

Genetic analysis of molecular markers is critical in tracking the emergence and/or spread of artemisinin resistant parasites. Clinical isolates collected in western Kenya pre- and post- introduction of artemisinin combination therapies (ACTs) were genotyped at SNP positions in regions of strong selection signatures on chromosome 13 and 14, as described in Southeast Asia (SEA). Twenty five SNPs were genotyped using Sequenom MassArray and pfmdr1 gene copy number by real-time PCR. Parasite clearance half-life and in vitro drug sensitivity testing were performed using standard methods. One hundred twenty nine isolates were successfully analyzed. Fifteen SNPs were present in pre-ACTs isolates and six in post-ACTs. None of the SNPs showed association with parasite clearance half-life. Post-ACTs parasites had significantly higher pfmdr1 copy number compared to pre-ACTs. Seven of eight parasites with multiple pfmdr1 were post-ACTs. When in vitro IC50s were compared for parasites with single vs. multiple gene copies, only amodiaquine and piperaquine reached statistical significance. Data showed SNPs on chromosome 13 and 14 had different frequency and trend in western Kenya parasites compared SEA. Increase in pfmdr1 gene copy is consistent with recent studies in African parasites. Data suggests genetic signature of artemisinin resistance in Africa might be different from SEA.

Edible insects are widely consumed in different parts of the world and can serve as an alternative nutritional source to conventional foods. Nonetheless, little attention has been given to their quality and shelf life in different packages when exposed to different storage environments. In this study, the effect of storage temperature, duration and type of packaging on the storage stability of the adult house cricket meal was examined. The samples were boiled, solar dried, milled and packaged into polypropylene (PP), plastic (PL) and polyethylene (PL) packages. The samples were then stored for six months in refrigerated and ambient conditions where by changes in physical and biochemical attributes were monitored. Iodine values significantly decreased in all the packages while peroxide, p-anisidine and saponification values significantly increased. SFA, MUFA and PUFA contents reduced during storage although a higher tendency for MUFA and PUFA values was observed in the refrigerated samples. Total viable count (TVC) and yeast and molds counts significantly increased in storage. Three types of fungi; Aspergillus spp., Alternaria spp. and Penicillium spp. were isolated in all the packages. Overall color change steadily decreased with increase in storage time. Deterioration was higher in samples stored in ambient conditions than in refrigeration. The degree of deterioration in the two storage environments among the different packages was in the order; PP>PE>PL. Although the PL package outperformed the other packages it is recommended to carry out sensory analysis and avoid post-processing contamination that can adversely affect the product quality and safety during storage.

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa.

Ethnopharmacological relevance The plant Neoboutonia macrocalyx has been reported in traditional medicine to be used in the treatment of malaria. Aim of the study To study the in vitro antiplasmodial activity of compounds from the stem bark of Neoboutonia macrocalyx. Materials and methods Compounds were extracted and purified from stem bark of Neoboutonia macrocalyx and their structure identified and confirmed by spectroscopic methods. The crude ethyl acetate extract, aqueous extract and the isolated compounds were evaluated for antiplasmodial activity against the chloroquine sensitive Sierra Leone I (D6) and chloroquine-resistant Indochina I (W2) strains of Plasmodium falciparum. Results Chemical investigation of the ethyl acetate extract of Neoboutonia macrocalyx bark resulted in the identification of one new diterpenoid; neoboutomacroin (1) in addition to the four known compounds which included, a phenanthrene; 3,6-dihyroxy-1,7-dimethyl-9-methoxyphenanthrene (2), a sterol; 3-O-Acetyloleuritolic acid (3) and two diterpenoids; simplexin (4) and montanin (5). Compounds 1 and 5 displayed good antiplasmodial activity of IC50 values less than 10 μg/mL against both strains. However, all the compounds tested displayed high cytotoxic activity against MRC5 cell line with IC50 less than 10 μM. Conclusions Despite an indirect in vitro antiplasmodial activity of some compounds isolated from the stem bark of Neoboutonia macrocalyx, the identification of these bioactive compounds indicates that they may play a role in the pharmacological properties of this plant.

Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pf mdt ) and P. falciparum GTPase tetQ (Pf tetQ ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by Pf tetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC 50 s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC 50 s of >35,000 nM relative to those with IC 50 s of P value of mdt gene had a median IC 50 of 6,971 nM, whereas those with a Pf mdt copy number of >1 had a median IC 50 of 9,912 nM ( P = 0.0245). Isolates with 1 copy of Pf tetQ had a median IC 50 of 6,370 nM, whereas isolates with a Pf tetQ copy number of >1 had a median IC 50 of 3,422 nM ( P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC 50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC 50 of 3,352 nM ( P = 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing.