Melissa D. Conrad

Absence of Putative Artemisinin Resistance Mutations Among Plasmodium falciparum in Sub-Saharan Africa: A Molecular Epidemiologic Study

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasites K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance.

Multi-trait evolution in a cave fish, Astyanax mexicanus

SUMMARY When surface species colonize caves, a characteristic suite of traits eventually evolves over time, regardless of species. The genetic basis of the inevitable appearance of these very similar phenotypes was investigated through quantitative trait loci (QTL) mapping of 12 traits that differ significantly between the recently evolved (<1 Myr). Mexican cave tetra and its surface conspecific. The traits were a representative set, including eye size, pigment cell numbers, chemical sensitivity, body and skull morphology, standard length, and metabolism. We used both single‐ and multi‐trait models for QTL mapping. QTL effects of these traits were significantly clustered in the genome. We mapped 13 regions in the genome with QTL effects on from three to nine traits. These clusters could be multigenic or could represent single locus with pleiotropic alleles. Given the relatively short time available to construct clusters from unlinked genes through genomic rearrangement, and the counterintuitive polarities of some of the substitution effects, we argue that at least some of the clusters must have a pleiotropic basis.

Absence of putative Plasmodium falciparum artemisinin resistance mutations in sub-Saharan Africa: A molecular epidemiologic study

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasites K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance.

Synteny and candidate gene prediction using an anchored linkage map of Astyanax mexicanus

The blind Mexican cave tetra, Astyanax mexicanus, is a unique model system for the study of parallelism and the evolution of cave-adapted traits. Understanding the genetic basis for these traits has recently become feasible thanks to production of a genome-wide linkage map and quantitative trait association analyses. The selection of suitable candidate genes controlling quantitative traits remains challenging, however, in the absence of a physical genome. Here, we describe the integration of multiple linkage maps generated in four separate crosses between surface, cave, and hybrid forms of A. mexicanus. We performed exhaustive BLAST analyses of genomic markers populating this integrated map against sequenced genomes of numerous taxa, ranging from yeast to amniotes. We found the largest number of identified sequences (228), with the most expect (E) values <10−5 (95), in the zebrafish Danio rerio. The most significant hits were assembled into an “anchored” linkage map with Danio, revealing numerous regions of conserved synteny, many of which are shared across critical regions of identified quantitative trait loci (QTL). Using this anchored map, we predicted the positions of 21 test genes on the integrated linkage map and verified that 18 of these are found in locations homologous to their chromosomal positions in D. rerio. The anchored map allowed the identification of four candidate genes for QTL relating to rib number and eye size. The map we have generated will greatly accelerate the production of viable lists of additional candidate genes involved in the development and evolution of cave-specific traits in A. mexicanus.

Association between Trichomonas vaginalis and vaginal bacterial community composition among reproductive-age women

Objectives Some vaginal bacterial communities are thought to prevent infection by sexually transmitted organisms. Prior work demonstrated that the vaginal microbiota of reproductive-age women cluster into 5 types of bacterial communities; 4 dominated by Lactobacillus species (L. iners, L. crispatus, L. gasseri, L. jensenii) and 1 (termed community state type (CST) IV) lacking significant numbers of lactobacilli and characterized by higher proportions of Atopobium, Prevotella, Parvimonas, Sneathia, Gardnerella, Mobiluncus, and other taxa. We sought to evaluate the relationship between vaginal bacterial composition and Trichomonas vaginalis. Methods Self-collected vaginal swabs were obtained cross-sectionally from 394 women equally representing 4 ethnic/racial groups. T. vaginalis screening was performed using PCR targeting the 18S rRNA and &bgr;-tubulin genes. Vaginal bacterial composition was characterized by pyrosequencing of barcoded 16S rRNA genes. A panel of 11 microsatellite markers was used to genotype T. vaginalis. The association between vaginal microbiota and T. vaginalis was evaluated by exact logistic regression. Results T. vaginalis was detected in 2.8% of participants (11/394). Of the 11 T. vaginalis-positive cases, 8 (72%) were categorized as CST-IV, 2 (18%) as communities dominated by L. iners, and 1 (9%) as L. crispatus-dominated (P = 0.05). CST-IV microbiota were associated with an 8-fold increased odds of detecting T. vaginalis compared with women in the L. crispatus-dominated state (OR: 8.26, 95% CI: 1.07–372.65). Seven of the 11 T. vaginalis isolates were assigned to 2 genotypes. Conclusion T. vaginalis was associated with vaginal microbiota consisting of low proportions of lactobacilli and high proportions of Mycoplasma, Parvimonas, Sneathia, and other anaerobes.

Comparative Impacts Over 5 Years of Artemisinin-Based Combination Therapies on Plasmodium falciparum Polymorphisms That Modulate Drug Sensitivity in Ugandan Children

BACKGROUND Artemisinin-based combination therapies, including artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP), are recommended to treat uncomplicated falciparum malaria. Sensitivities to components of AL and DP are impacted by polymorphisms in pfmdr1 and pfcrt. We monitored changes in prevalences of polymorphisms in Tororo, Uganda, from 2008 to 2012. METHODS Polymorphic loci in pfmdr1 and pfcrt were characterized in samples from 312 children randomized to AL or DP for each episode of uncomplicated malaria (50 samples per arm for each 3-month interval) utilizing a fluorescent microsphere assay. Treatment outcomes and impacts of prior therapies were also characterized. RESULTS Prevalence increased significantly over time for pfmdr1 N86 (AL: odds ratio [OR], 2.08 [95% confidence interval {CI}, 1.83-2.38]; DP: 1.41 [95% CI, 1.25-1.57]), pfmdr1 D1246 (AL: 1.46 [95% CI, 1.29-1.64]; DP: 1.36 [95% CI, 1.23-1.50]), and pfcrt K76 (AL: 3.37 [95% CI, 1.85-6.16]; DP: 5.84 [95% CI, 1.94-17.53], and decreased for pfmdr1 Y184 (AL: 0.78 [95% CI, .70-.86]; DP: 0.84 [95% CI, .76-1.50]); changes were consistently greater in the AL arm. Recent AL treatment selected for pfmdr1 N86, D1246, and 184F in subsequent episodes; DP selected for the opposite alleles. CONCLUSIONS Genotypes with decreased sensitivity to AL components increased over time. This increase was greater in children receiving AL, suggesting that the choice of treatment regimen can profoundly influence parasite genetics and drug sensitivity. CLINICAL TRIALS REGISTRATION NCT00527800.

Extensive Genetic Diversity, Unique Population Structure and Evidence of Genetic Exchange in the Sexually Transmitted Parasite Trichomonas vaginalis

Background Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasites genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes. Methodology/Principal Findings Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages. Conclusions/Significance Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.

Microsatellite polymorphism in the sexually transmitted human pathogen Trichomonas vaginalis indicates a genetically diverse parasite

Given the growing appreciation of serious health sequelae from widespread Trichomonas vaginalis infection, new tools are needed to study the parasites genetic diversity. To this end we have identified and characterized a panel of 21 microsatellites and six single-copy genes from the T. vaginalis genome, using seven laboratory strains of diverse origin. We have (1) adapted our microsatellite typing method to incorporate affordable fluorescent labeling, (2) determined that the microsatellite loci remain stable in parasites continuously cultured for up to 17 months, and (3) evaluated microsatellite marker coverage of the six chromosomes that comprise the T. vaginalis genome, using fluorescent in situ hybridization (FISH). We have used the markers to show that T. vaginalis is a genetically diverse parasite in a population of commonly used laboratory strains. In addition, we have used phylogenetic methods to infer evolutionary relationships from our markers in order to validate their utility in future population analyses. Our panel is the first series of robust polymorphic genetic markers for T. vaginalis that can be used to classify and monitor lab strains, as well as provide a means to measure the genetic diversity and population structure of extant and future T. vaginalis isolates.

Temporal Changes in Prevalence of Molecular Markers Mediating Antimalarial Drug Resistance in a High Malaria Transmission Setting in Uganda.

Standard therapy for malaria in Uganda changed from chloroquine to chloroquine + sulfadoxine-pyrimethamine in 2000, and artemether-lumefantrine in 2004, although implementation of each change was slow. Plasmodium falciparum genetic polymorphisms are associated with alterations in drug sensitivity. We followed the prevalence of drug resistance-mediating P. falciparum polymorphisms in 982 samples from Tororo, a region of high transmission intensity, collected from three successive treatment trials conducted during 2003-2012, excluding samples with known recent prior treatment. Considering transporter mutations, prevalence of the mutant pfcrt 76T, pfmdr1 86Y, and pfmdr1 1246Y alleles decreased over time. Considering antifolate mutations, the prevalence of pfdhfr 51I, 59R, and 108N, and pfdhps 437G and 540E were consistently high; pfdhfr 164L and pfdhps 581G were uncommon, but most prevalent during 2008-2010. Our data suggest sequential selective pressures as different treatments were implemented, and they highlight the importance of genetic surveillance as treatment policies change over time.

Longitudinal Outcomes in a Cohort of Ugandan Children Randomized to Artemether-Lumefantrine Versus Dihydroartemisinin-Piperaquine for the Treatment of Malaria

BACKGROUND Artemisinin-based combination therapy (ACT) has become the standard of care for the treatment of uncomplicated Plasmodium falciparum malaria. Although several ACT regimens are approved, data guiding optimal choices of ACTs are limited. We compared short- and long-term outcomes in a cohort of young Ugandan children randomized to 2 leading ACTs. METHODS Overall, 312 children were randomized to artemether-lumefantrine or dihydroartemisinin-piperaquine (DP) at the time of the first episode of uncomplicated malaria (median age, 10.5 months). The same treatment was given for all subsequent episodes of uncomplicated malaria and children were followed until they reached 5 years of age. The cohort included a subgroup that was human immunodeficiency virus (HIV) infected (n = 44) or HIV exposed (n = 175) and prescribed trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis. Outcomes included time to recurrent malaria following individual treatments and the overall incidences of treatments for malaria, complicated malaria, and hospitalizations. RESULTS Among children not prescribed TMP-SMX prophylaxis, 4443 treatments for malaria were given over 790 person-years following randomization. Treatment with DP was associated with a lower hazard of recurrent malaria over the 84 days after treatment (hazard ratio, 0.66; 95% confidence interval [CI], .61-.70; P < .001). Children randomized to DP had a lower incidence of all treatments for malaria (incidence rate ratio [IRR], 0.85; 95% CI, .75-.96; P = .01), complicated malaria (IRR, 0.12; 95% CI, .04-.39; P < .001), and hospitalizations (IRR, 0.31; 95% CI, .13-.77; P = .01). Among children prescribed TMP-SMX prophylaxis, there were no significant differences in longitudinal outcomes. CONCLUSIONS Compared to artemether-lumefantrine, the use of DP to treat uncomplicated malaria delayed the time to recurrent malaria and reduced the incidences of treatments for malaria, complicated malaria, and hospitalizations. CLINICAL TRIALS REGISTRATION NCT00527800.