Browsing by Author "Taylor, Steve M"

Background: Plasmodium falciparum has developed resistance against artemisinin and partner drugs that have been widely used globally as artemisinin-based combination therapies (ACT). Such resistance, poses the greatest challenge to the prospect of malaria elimination in the Greater Mekong Subregion (GMS). Genetic polymorphisms in P. falciparum that confer resistance to the drugs that are part of two most commonly used ACT in the GMS, artemisinin-lumefantrine (AL) and dihydroartemisisnin-piperaquine (DP). Single point mutations in Kelch propeller domain of falciparum chromosome 13 (K13) for artemisinin resistance, and copy number variations of plasmepsin 2/3 are associated with piperaquine resistance, and Pfmdr-1 for mefloquine and lumefantrine resistance. Although the efficacy is high for ACTs in Myanmar, molecular markers of resistance to either of the drugs in ACT can still be present and may indicate that the drugs will be at risk in the near future. Therefore, this study aimed to detect genetic polymorphisms in K13, Pfmdr-1, and plasmepsin 2 (Pfpm-2) that mediate ACT treatment outcomes in Myanmar. Methods: The current study uses a cross-sectional study design and retrospective analysis of laboratory samples collected in previous therapeutic efficacy studies (TES) conducted during 2014 to 2018 in nine sentinel malaria endemic remote townships in Myanmar. The ACTs used in these TES were artemether-lumefantrine, dihydroartemisinin-piperaquine, and pyronaridine-artesunate. 176 samples were randomly selected out of 651 samples from nine TES sites because of the time constraint. K13 genotyping was done by Sanger sequencing, and the copy numbers of Pfpm-2 and Pfmdr-1 were quantified by real-time polymerase chain reaction. Results: Among 176 randomly selected pre-treatment parasites, we observed non- synonymous mutation in the K13 gene in 25% (42/169; 95% CI: 18.3, 31.4). Overall, 23% (39/169; 95% CI: 16.7, 29.4) of infections harbored a K13 mutation that has been validated as associated with artemisinin resistance. Among these, 58.9% (23/39) encoded the F446I substitution. The prevalence of parasites harboring the C580Y mutation that is the most closely associated with artemisinin resistance was 6.5% (11/169; 95% CI: 2.8, 10.2), and was present in 4 out of 9 study sites. Only 1 sample 0.6% (1/172; 95% CI: 0, 1.7) harbored more than one copy of Pfpm-2; this parasite also contained the K13 C580Y mutation conferring artemisinin resistance. No parasites harbored more than one copy of Pfmdr-1. Conclusion: Consistent with the high efficacy of ACTs in Myanmar, there were little evidence of resistance to artemisinin or partner drugs by analysis of molecular markers. However, there was remarkable amount of K13 molecular markers (C580Y, F446I, R561H) seen in this study. These observed K13 markers have already been confirmed and validated by WHO. This finding may be a warning sign of developing artemisinin resistance which may, in turn, have an effect on the malaria elimination process in Myanmar. Emergence of drug resistant malaria in GMS threatens the malaria elimination effort in the region as well as globally. Continued monitoring of artemisinin and its partner drugs resistance is needed to prevent the spread of drug resistant malaria.

Dengue viruses (DENV) and other mosquito-borne flaviviruses are rapidly emerging human pathogens that threaten nearly half of the world’s population. There is currently no effective vaccine or antiviral therapeutics for the prophylaxis or treatment of DENV. While traditional drug development efforts have focused on inhibitors of viral enzymes, an alternative approach is to target host proteins that support virus replication. In an effort to identify novel human enzymes important for the DENV-2 life-cycle, we conducted a genome-wide RNAi screen and identified ERI3, a putative 3′-5′ exonuclease, as a novel DENV-2 host factor. Cell-free assays confirmed that purified ERI3 is capable of degrading single-stranded RNA in a 3′ to 5′ direction. We conducted a screen for compounds that inhibit ERI3 in vitro and identified small molecules that antagonized both exonuclease activity. In summary, we identified a host exonuclease that is important for DENV-2 replication and is a potential therapeutic target. Our approach illustrates the utility of identifying host enzymatic functions for development of anti-viral drugs.

Large-scale molecular epidemiologic studies of Plasmodium falciparum parasites have provided insights into parasite biology and transmission, can identify the spread of drug resistance, and are useful in assessing vaccine targets. The polyclonal nature infections in high transmission settings is problematic for traditional genotyping approaches. Next-generation sequencing approaches to parasite genotyping allow sensitive detection of minority variants, disaggregation of complex parasite mixtures, and scalable processing of large samples sets. Therefore, we designed, validated, and applied to field parasites a new approach that leverages sequencing of individually barcoded samples in a multiplex manner. We utilize variant barcodes, invariant linker sequences and modular template-specific primers to allow for the simultaneous generation of high-dimensional sequencing data of multiple gene targets. This modularity permits a cost-effective and reproducible way to query many genes at once. In mixtures of reference parasite genomes, we quantitatively detected unique haplotypes comprising as little as 2% of a polyclonal infection. We applied this genotyping approach to field-collected parasites collected in Western Kenya in order to simultaneously obtain parasites genotypes at three unlinked loci. In summary, we present a rapid, scalable, and flexible method for genotyping individual parasites that enables molecular epidemiologic studies of parasite evolution, population structure and transmission.

Background: Despite widespread access and use of insecticide-treated bed nets (ITN) in Bungoma County in Kenya, there has been little reduction in malaria infection rates. It has been hypothesized that this gap between theoretical and actual ITN efficacy is caused by improper use of ITNs, poor physical condition of ITNs, or insecticide resistance in local mosquitos. This study aims to examine potential factors that affect the efficacy of the ITNs in Western Kenya.

Methods: In order to assess the aforementioned aim, a longitudinal observational study was conducted. The study enrolled 9 households and performed weekly data and mosquito collections. Data and sample collection was conducted over an 8-week duration, from June 2016 to July 2016.

Results: The study found high ITN usage in the study households (99.3% coverage), a negative association between the number of mosquitoes collected and time, a high proportion of blood fed mosquitoes (0.409), and statistically significant associations with the proportion of blood fed mosquitos and twelve different predictor variables. Conclusion: This study shows that it is feasible to examine factors reducing ITN efficacy in the area and lays down a potential template to be scaled up to examine these factors more specifically.

Hydroxyurea, a mainstay of sickle cell management in the developed world, offers a range of potential benefits to children with sickle cell disease. There is strong evidence that hydroxyurea induces production of fetal hemoglobin (HbF) in red blood cells, which is generally associated with reduced morbidity and fewer incidents of clinical events in sickle cell patients. Based on literature from in vitro investigations, it has also been suggested that hydroxyurea may confer some protection against malaria parasitemia by inhibiting proliferation of the malaria-causing parasite - Plasmodium falciparum.

The purpose of this study was to examine the effects hydroxyurea use on P. falciparum infection, parasite density, HbF and morbidity among children with sickle cell disease living in a malaria endemic setting. We analyzed baseline data of 95 children (aged 1 – 10 years) enrolled in the EPiTOMISE clinical trial (Enhancing Preventive Therapy of Malaria in children with Sickle cell anemia in East Africa) between January 2018 and September 2018.

Our analyses showed no significant difference in the prevalence of P. falciparum infection between hydroxyurea users and non-hydroxyurea users, prevalence ratio [95% CI] = 1.14 [0.76, 1.71]. Among infected children, median (IQR) log parasites densities were also similar between hydroxyurea users, -0.96 (-1.67, 0.41) and hydroxyurea non-users, -0.12 (-1.32, 3.48), p-value=0.146.

We did observe substantial hematological benefits among hydroxyurea users including an approximate 1 unit increase in median hemoglobin concentration and a 2.7-fold increase in median percentage HbF. However, this observation did not translate to any meaningful difference in the prevalence of morbidity events.

In agreement with the few studies on hydroxyurea use in malaria endemic settings, these results suggest that there may be no added risk of P. falciparum infection to sickle cell patients who routinely use hydroxyurea. Furthermore, our results reflect that hydroxyurea use is associated with increased HbF and a better hematological profile in this population. There is need for more research on hydroxyurea use in sub-Saharan Africa to help resolve any existing concerns and conflicting data in the current body of literature.

Insecticide-treated bed nets (ITNs) are widely used in Kenya to prevent the mosquitoes, which can transmit malaria, from biting people at night. However, there is a concern that mosquitoes become resistant to the ITN environment by changing their feeding behavior. The research goal was to observe the current feeding behavior of female Anopheles in Webuye, Western Kenya. Prokopack aspirators, CDC light traps, and sticky barrier screens were used for mosquito collection during May – July 2018. 662 collected female Anopheles, most of which were Anopheles gambiae sensu lato, were included. Descriptive and statistical analyses were applied, and the results show that the feeding proportion was 79.3% in mornings and 13.7% at nights, which was not significantly affected by the frequency of bed net use. 35.9% and 4.3% of the female Anopheles were human and Plasmodium (P.) Falciparum gDNA positive, respectively. Most of the P.falciparum positives were also fed and human gDNA positive, meaning that most of the infected or infectious mosquitoes had human blood meals and may have already attended malaria transmission. The study failed to observe outdoor feeding behavior by sticky barrier screens, and behavioral adaptation may not be responsible for most of the persistence of transmission. The study implicates a full investigation of ITN condition, more research on chemical resistance, and further surveillance of mosquito biting time.

The sickle-cell trait hemoglobin genotype (HbAS) is known to protect against severe malaria caused by Plasmodium falciparum. However, the biological mechanisms behind this protection are not well understood. Cyclophilin 19B (PfCyP-19B) is a parasitic gene that produces the protein cyclophilin 19B, a member of the unfolded protein response that is important in parasitic protein folding and trafficking. We set out to measure the transcript expression level of PfCyP-19B to investigate its potential role in the mechanisms that confer protection for HbAS individuals. RNA was extracted from both in vivo samples collected from Malian children as well as in vitro samples harvested throughout a 48-hour incubation period. RNA extracts were reverse transcribed and transcript expression was measured via qPCR. Wilcoxon rank sum and bootstrapping methods were used to analyze transcript units between parasites grown in normal (HbAA) and HbAS red blood cells. The results from our cross-sectional in vivo data revealed under expression of PfCyP-19B among individuals with the HbAS genotype compared to those with the HbAA genotype (Wilcoxon rank sum p=0.006). In vitro time series results showed no significant difference in PfCyP-19B transcript expression levels between genotypes but did display a 24-hour pattern of peak expression for both HbAA and HbAS genotypes. The under expression of PfCyP-19B among HbAS individuals could be linked to impaired protein trafficking, interfering with the parasite’s ability to display surface proteins vital for cytoadherence and severe disease manifestation. The 24-hour peak transcript expression displayed in vitro roughly aligns with the P. falciparum parasite stage transition states, suggesting cyclophilin 19B may aid in parasitic transitions.