Culture-adapted Plasmodium falciparum isolates from UK travellers: in vitro drug sensitivity, clonality and drug resistance markers.

BACKGROUND: The screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy. METHODS: Pre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC₅₀ estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines. RESULTS: Plasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC₅₀ estimates, and carried the CVIET haplotype at codons 72-76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC₅₀ estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86. CONCLUSIONS: This study describes the establishment in continuous culture, in vitro drug sensitivity testing and molecular characterization of a series of multiclonal P. falciparum isolates taken directly from UK malaria patients following recent travel to various malaria-endemic countries in Africa. These "HL" isolates are available as an open resource for studies of drug response, antigenic diversity and other aspects of parasite biology

Estimating the epidemiology of chronic Hepatitis B Virus (HBV) infection in the UK: what do we know and what are we missing?

Background: HBV is the leading global cause of cirrhosis and primary liver cancer. However, the UK HBV population has not been well characterised, and estimates of UK HBV prevalence and/or incidence vary widely between sources. We summarised datasets that are available to represent UK CHB epidemiology, considering differences between sources, and discussing deficiencies in current estimates. Methods: We searched for estimates of CHB case numbers in the UK (incorporating incidence and/or prevalence-like data) across a range of available sources, including UK-wide reports from government bodies, publications from independent bodies (including medical charities and non-governmental organisations) and articles in peer-reviewed scientific journals to collate estimated positivity rates. An alternative proxy for population prevalence was obtained via the UK antenatal screening programme which achieves over 95% coverage of pregnant women. Results: We identified six CHB case number estimates, of which three reported information concerning population subgroups, including number of infected individuals across age, sex and ethnicity categories. Estimates among sources reporting prevalence varied from 0.27% to 0.73%, congruent with an estimated antenatal CHB prevalence of <0.5%. Discussion: Estimates varied by sources of error, bias and missingness, data linkage, and substantial “blind spots” in consistent testing and registration of HBV diagnoses. The HBV burden in the UK is likely to be concentrated in vulnerable populations who may not be well represented in existing datasets including those experiencing socioeconomic deprivation, ethnic minorities, people experiencing homelessness and people born in high-prevalence countries. Together, these factors could lead to either under- or over-estimation of overall prevalence, and additional efforts are required to provide estimates that best reflect the whole population. Multi-parameter evidence synthesis and back-calculation model methods similar to those used to generate estimates of HCV ad HIV population-wide prevalence may be applicable to HBV

The Mu subunit of Plasmodium falciparum clathrin-associated adaptor protein 2 modulates in vitro parasite response to artemisinin and quinine.

The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance

Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe.

BACKGROUND: The replication-competent recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing a Zaire ebolavirus (ZEBOV) glycoprotein was selected for rapid safety and immunogenicity testing before its use in West Africa. METHODS: We performed three open-label, dose-escalation phase 1 trials and one randomized, double-blind, controlled phase 1 trial to assess the safety, side-effect profile, and immunogenicity of rVSV-ZEBOV at various doses in 158 healthy adults in Europe and Africa. All participants were injected with doses of vaccine ranging from 300,000 to 50 million plaque-forming units (PFU) or placebo. RESULTS: No serious vaccine-related adverse events were reported. Mild-to-moderate early-onset reactogenicity was frequent but transient (median, 1 day). Fever was observed in up to 30% of vaccinees. Vaccine viremia was detected within 3 days in 123 of the 130 participants (95%) receiving 3 million PFU or more; rVSV was not detected in saliva or urine. In the second week after injection, arthritis affecting one to four joints developed in 11 of 51 participants (22%) in Geneva, with pain lasting a median of 8 days (interquartile range, 4 to 87); 2 self-limited cases occurred in 60 participants (3%) in Hamburg, Germany, and Kilifi, Kenya. The virus was identified in one synovial-fluid aspirate and in skin vesicles of 2 other vaccinees, showing peripheral viral replication in the second week after immunization. ZEBOV-glycoprotein-specific antibody responses were detected in all the participants, with similar glycoprotein-binding antibody titers but significantly higher neutralizing antibody titers at higher doses. Glycoprotein-binding antibody titers were sustained through 180 days in all participants. CONCLUSIONS: In these studies, rVSV-ZEBOV was reactogenic but immunogenic after a single dose and warrants further evaluation for safety and efficacy. (Funded by the Wellcome Trust and others; ClinicalTrials.gov numbers, NCT02283099, NCT02287480, and NCT02296983; Pan African Clinical Trials Registry number, PACTR201411000919191.)

Haematological consequences of acute uncomplicated falciparum malaria: a WorldWide Antimalarial Resistance Network pooled analysis of individual patient data

Background: Plasmodium falciparum malaria is associated with anaemia-related morbidity, attributable to host, parasite and drug factors. We quantified the haematological response following treatment of uncomplicated P. falciparum malaria to identify the factors associated with malarial anaemia. Methods: Individual patient data from eligible antimalarial efficacy studies of uncomplicated P. falciparum malaria, available through the WorldWide Antimalarial Resistance Network data repository prior to August 2015, were pooled using standardised methodology. The haematological response over time was quantified using a multivariable linear mixed effects model with nonlinear terms for time, and the model was then used to estimate the mean haemoglobin at day of nadir and day 7. Multivariable logistic regression quantified risk factors for moderately severe anaemia (haemoglobin < 7 g/dL) at day 0, day 3 and day 7 as well as a fractional fall ≥ 25% at day 3 and day 7. Results: A total of 70,226 patients, recruited into 200 studies between 1991 and 2013, were included in the analysis: 50,859 (72.4%) enrolled in Africa, 18,451 (26.3%) in Asia and 916 (1.3%) in South America. The median haemoglobin concentration at presentation was 9.9 g/dL (range 5.0–19.7 g/dL) in Africa, 11.6 g/dL (range 5.0–20.0 g/dL) in Asia and 12.3 g/dL (range 6.9–17.9 g/dL) in South America. Moderately severe anaemia (Hb < 7g/dl) was present in 8.4% (4284/50,859) of patients from Africa, 3.3% (606/18,451) from Asia and 0.1% (1/916) from South America. The nadir haemoglobin occurred on day 2 post treatment with a mean fall from baseline of 0.57 g/dL in Africa and 1.13 g/dL in Asia. Independent risk factors for moderately severe anaemia on day 7, in both Africa and Asia, included moderately severe anaemia at baseline (adjusted odds ratio (AOR) = 16.10 and AOR = 23.00, respectively), young age (age < 1 compared to ≥ 12 years AOR = 12.81 and AOR = 6.79, respectively), high parasitaemia (AOR = 1.78 and AOR = 1.58, respectively) and delayed parasite clearance (AOR = 2.44 and AOR = 2.59, respectively). In Asia, patients treated with an artemisinin-based regimen were at significantly greater risk of moderately severe anaemia on day 7 compared to those treated with a non-artemisinin-based regimen (AOR = 2.06 [95%CI 1.39–3.05], p < 0.001). Conclusions: In patients with uncomplicated P. falciparum malaria, the nadir haemoglobin occurs 2 days after starting treatment. Although artemisinin-based treatments increase the rate of parasite clearance, in Asia they are associated with a greater risk of anaemia during recovery

Sensitivity and specificity of molecular methods for detecting markers of antimalarial drug resistance in clinical samples of Plasmodium falciparum: a systematic review

Background Each year infection with Plasmodium causes millions of clinical cases of malaria, and hundreds of thousands of deaths. Resistance to different antimalarial medications continues to develop and spread, threatening effective prophylaxis and treatment. Monitoring of resistance is required to inform health policy and preserve effective antimalarials; molecular methods can be used to determine likely parasite susceptibility. There is no consensus on the most accurate methods; large variation exists in practice. The goal of this systematic review was to identify the sensitivity and specificity of each molecular method for detecting antimalarial resistance markers. Methods All diagnostic accuracy studies that examine at least two molecular methods for detecting selected markers of antimalarial resistance in blood samples from patients diagnosed with, or suspected of having malaria were included. MEDLINE, EMBASE, BIOSIS, and Science Citation Index were searched. Methodological quality was evaluated using QUADAS-2. Sensitivity and specificity were calculated and results synthesised and compared narratively. Results 27,575 search results returned 18 eligible studies, examining 13 index tests, against five reference tests, for their accuracy in detecting 24 molecular markers. Some markers and tests were investigated multiple times, but rarely in the same combinations. Characteristics of studies were poorly reported. Generally, risk of bias and applicability concerns were unclear. Estimates of sensitivity and specificity were calculable for most evaluations, confidence intervals were generally wide. Discussion Estimates of the accuracy of molecular methods are generally imprecise, contributing to uncertainty. Many methods may be close to 100&amp;percnt; in sensitivity or specificity. Most methods require specialist equipment unlikely to be available in many low resource settings. High quality study design focusing on methods useable in low resource settings, prospective registration, and reporting according to STARD 2015 guidelines are essential for future studies. </p

Diagnostic accuracy of molecular methods for detecting markers of antimalarial drug resistance in clinical samples of Plasmodium falciparum: protocol for an update to a systematic review and meta-analysis

Abstract Background Each year, infection with Plasmodium causes millions of clinical cases of malaria and hundreds of thousands of deaths. Resistance to different antimalarial medications continues to develop and spread, threatening effective prophylaxis and treatment. Surveillance of resistance is required to inform health policy and preserve effective antimalarial drugs; molecular methods can be used to surveil likely parasite resistances. However, there is no consensus on the most accurate molecular methods, and large variation exists in practice. The objective of this update to this systematic review is to improve and update identification of the sensitivity and specificity of each molecular method for detecting selected antimalarial drug resistance markers. Methods We will include diagnostic accuracy studies that compare at least two of any molecular methods to examine blood samples from patients diagnosed with, or suspected of having malaria, to detect at least one selected marker of antimalarial drug resistance. We will search PubMed, EMBASE, BIOSIS, and Web of Science from 2000 to present. Two reviewers will independently screen all results, extract data, consider applicability, and evaluate the methodological quality of included studies using QUADAS-2. We will carry out a meta-analysis and use statistical methods to compare results from homogenous studies. We will use narrative to synthesise and compare results of heterogeneous studies. Discussion This review will help to identify sub-optimal molecular methods for antimalarial marker detection which may be discontinued and identify more sensitive and specific methods which may be adopted. More sensitive and specific detection of drug resistance can be used to improve the breadth and accuracy of surveillance. This would enable the identification of previously undiscovered areas of antimalarial resistances and susceptibilities, improve the precision of estimates of the prevalence of resistances, and improve our ability to detect smaller changes in these patterns. Higher-quality evidence generated by more accurate and detailed surveillance can be used to inform guidelines on the use of antimalarial drugs, leading to better outcomes for more patients. Systematic review registration This systematic review protocol was registered with PROSPERO on 22 November 2017 (registration number CRD42017082101)

Directional selection at the pfmdr1, pfcrt, pfubp1, and pfap2mu loci of Plasmodium falciparum in Kenyan children treated with ACT.

BACKGROUND: The efficacy of artemisinin-based combination therapy (ACT) for Plasmodium falciparum malaria may be threatened by parasites with reduced responsiveness to artemisinins. Among 298 ACT-treated children from Mbita, Kenya, submicroscopic persistence of P. falciparum on day 3 posttreatment was associated with subsequent microscopically detected parasitemia at days 28 or 42. METHODS: DNA sequences of resistance-associated parasite loci pfcrt, pfmdr1, pfubp1, and pfap2mu were determined in the Mbita cohort before treatment, on days 2 and 3 after initiation of treatment, and on the day of treatment failure. RESULTS: Parasites surviving ACT on day 2 or day 3 posttreatment were significantly more likely than the baseline population to carry the wild-type haplotypes of pfcrt (CVMNK at codons 72-76; P < .001) and pfmdr1 (NFD at codons 86, 184, 1246; P < .001). In contrast, variant alleles of the novel candidate resistance genes pfap2mu (S160N/T; P = .006) and pfubp-1 (E1528D; P < .001) were significantly more prevalent posttreatment. No genetic similarities were found to artemisinin-tolerant parasites recently described in Cambodia. CONCLUSIONS: Among treated children in western Kenya, certain P. falciparum genotypes defined at pfcrt, pfmdr1, pfap2mu, and pfubp1 more often survive ACT at the submicroscopic level, and contribute to onward transmission and subsequent patent recrudescence

Variability in white blood cell count during uncomplicated malaria and implications for parasite density estimation : a WorldWide Antimalarial Resistance Network individual patient data meta-analysis

Background: The World Health Organization (WHO) recommends that when peripheral malarial parasitaemia is quantified by thick film microscopy, an actual white blood cell (WBC) count from a concurrently collected blood sample is used in calculations. However, in resource-limited settings an assumed WBC count is often used instead. The aim of this study was to describe the variability in WBC count during acute uncomplicated malaria, and estimate the impact of using an assumed value of WBC on estimates of parasite density and clearance. Methods: Uncomplicated malaria drug efficacy studies that measured WBC count were selected from the WorldWide Antimalarial Resistance Network data repository for an individual patient data meta-analysis of WBC counts. Regression models with random intercepts for study-site were used to assess WBC count variability at presentation and during follow-up. Inflation factors for parasitaemia density, and clearance estimates were calculated for methods using assumed WBC counts (8000 cells/mu L and age-stratified values) using estimates derived from the measured WBC value as reference. Results: Eighty-four studies enrolling 27,656 patients with clinically uncomplicated malaria were included. Geometric mean WBC counts (x 1000 cells/mu L) in age groups &lt; 1, 1-4, 5-14 and &gt;= 15 years were 10.5, 8.3, 7.1, 5.7 and 7.5, 7.0, 6.5, 6.0 for individuals with falciparum (n = 24,978) and vivax (n = 2678) malaria, respectively. At presentation, higher WBC counts were seen among patients with higher parasitaemia, severe anaemia and, for individuals with vivax malaria, in regions with shorter regional relapse periodicity. Among falciparum malaria patients, using an assumed WBC count of 8000 cells/mu L resulted in parasite density underestimation by a median (IQR) of 26% (4-41%) in infants &lt; 1 year old but an overestimation by 50% (16-91%) in adults aged = 15 years. Use of age-stratified assumed WBC values removed systematic bias but did not improve precision of parasitaemia estimation. Imprecision of parasite clearance estimates was only affected by the within-patient WBC variability over time, and remained &lt; 10% for 79% of patients. Conclusions: Using an assumed WBC value for parasite density estimation from a thick smear may lead to underdiagnosis of hyperparasitaemia and could adversely affect clinical management; but does not result in clinically consequential inaccuracies in the estimation of the prevalence of prolonged parasite clearance and artemisinin resistance