Assessment of the Worldwide Antimalarial Resistance Network Standardized Procedure for In Vitro Malaria Drug Sensitivity Testing Using SYBR Green Assay for Field Samples with Various Initial Parasitemia Levels.

The malaria SYBR green assay, which is used to profilein vitrodrug susceptibility ofPlasmodium 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% (IC50) 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. falciparumlaboratory strains and field isolates with various levels of initial parasitemia were then subjected to a range of doses of common antimalarials. The IC50s 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.

Plasmodium interspecies interactions during a period of increasing prevalence of Plasmodium ovale in symptomatic individuals seeking treatment: an observational study.

BACKGROUND: The epidemiology and severity of non-falciparum malaria in endemic settings has garnered little attention. We aimed to characterise the prevalence, interaction, clinical risk factors, and temporal trends of non-falciparum Plasmodium species among symptomatic individuals presenting at health-care facilities in endemic settings of Kenya. METHODS: We diagnosed and analysed infecting malaria species (Plasmodium falciparum, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, and Plasmodium malariae) via PCR in clinical samples collected between March 1, 2008, and Dec 31, 2016, from six hospitals located in different regions of Kenya. We recruited patients aged 6 months or older who presented at outpatient departments with symptoms of malaria or tested positive for uncomplicated malaria by malaria rapid diagnostic test. 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 interspecies interactions. Mixed-effect linear regression models with random slopes for each location were used to test for change in prevalence over time. FINDINGS: Samples from 2027 symptomatic participants presenting at care facilities were successfully analysed for all Plasmodium species. 1469 (72·5%) of the samples were P falciparum single-species infections, 523 (25·8%) were mixed infections, and only 35 (1·7%) were single non-falciparum species infections. 452 (22·3%) were mixed infections containing P ovale spp. 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 in P ovale wallikeri (2·1% per year; p=0·043) and P ovale curtisi (0·7% per year; p=0·0002) species over time, with a reciprocal decrease in P falciparum single-species infections (2·5% per year; p=0·0065). The frequency of P malariae infections did not significantly change over time. Risk of P falciparum infections presenting with fever was lower if co-infected with P malariae (adjusted odds ratio 0·43, 95% CI 0·25-0·74; p=0·0023). INTERPRETATION: Our results show a prevalence of non-falciparum species infections of 27·5% among symptomatic individuals presenting at care facilities, which is higher than expected from previous cross-sectional surveys. The proportion of infections with P ovale wallikeri and P ovale curtisi was observed to significantly increase over the period of study, which could be due to attenuated responsiveness of these species to malaria drug treatment. The increase in frequency of P ovale spp could threaten the malaria control efforts in Kenya and pose increased risk of malaria to travellers. FUNDING: Armed Forces Health Surveillance Branch and its Global Emerging Infections Surveillance Section.

Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein.

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds.  Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modelled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: These interactions provide lead compounds for new anti-malarial molecules. Further in vivo testing is recommended.

Evaluation of the Field Performance of ImmunoCard STAT!® Rapid Diagnostic Test for Rotavirus in Dadaab Refugee Camp and at the Kenya-Somalia Border.

AbstractRotavirus commonly causes diarrhea in children, leading to hospitalization and even death. Rapid diagnostic tests are feasible alternatives for determining rotavirus outbreaks in refugee camps that have inadequate laboratory capacity. We evaluated the field performance of ImmunoCard STAT!® Rotavirus (ICS-RV) in Dadaab Refugee Camp and at the Kenya-Somalia border. From May to December 2014, we prospectively enrolled children aged < 5 years hospitalized with acute diarrhea, defined as ≥ 3 episodes of loose stool in 24 hours for < 7 days. Stool samples were collected and tested by trained surveillance clerks using ICS-RV per manufacturer's instructions. The field performance characteristics of ICS-RV were evaluated against the gold standard test, Premier™ Rotaclone® enzyme immunoassay. The operational characteristics were evaluated using World Health Organization (WHO) ASSURED criteria to determine whether ICS-RV is appropriate as a point-of-care test by administering a standard questionnaire and observing surveillance clerks performing the test. We enrolled 213 patients with a median age of 10 months (range = 1-48); 58.2% were male. A total of 71 (33.3%) and 60 (28.2%) patients tested positive for rotavirus infection by immunoassay and ICS-RV, respectively. The sensitivity, specificity, and positive and negative predictive values of ICS-RV compared with the immunoassay were 83.1% (95% confidence interval [CI] = 72.3-91.0), 99.3% (95% CI = 96.1-100), 98.3% (95% CI = 91.1-100), and 92.1% (95% CI = 86.6-95.5), respectively. The ICS-RV fulfilled the WHO ASSURED criteria for point-of-care testing. ICS-RV is a field-ready point-of-care test with good field performance and operational characteristics. It can be useful in determining rotavirus outbreaks in resource-limited settings.

Five-year tracking of Plasmodium falciparum allele frequencies in a holoendemic area with indistinct seasonal transitions.

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.