Tonya Mixson-Hayden

Origin and evolution of sulfadoxine resistant Plasmodium falciparum.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

Suppression of interferon‐α signaling by hepatitis E virus

The interferon (IFN) system is integral to the host response against viruses, and many viruses have developed strategies to overcome its antiviral effects. The effects of hepatitis E virus (HEV), the causative agent of hepatitis E, on IFN signaling have not been investigated primarily because of the nonavailability of an efficient in vitro culture system or small animal models of infection. We report here the generation of A549 cell lines persistently infected with genotype 3 HEV, designated as HEV‐A549 cells and the effects HEV has on IFN‐α–mediated Janus kinase–signal transducer and activator of transcription (JAK–STAT) signaling. Treatment of HEV‐A549 cells with 250, 500, and 1000 U/mL of IFN‐α for 72 hours showed a dose‐dependent reduction in HEV RNA levels by 10%, 20%, and 50%, respectively. IFN‐α–stimulated genes coding for the antiviral proteins dsRNA‐activated protein kinase (PKR) and 2′,5′‐oligoadenylate synthetase (2′,5′‐OAS) were down‐regulated in IFN‐α–treated HEV‐A549 cells. HEV infection also prevented IFN‐α–induced phosphorylation of STAT1. Regulation of STAT1 by HEV was specific, as phosphorylation of STAT2, tyrosine kinase (Tyk) 2, and Jak1 by IFN‐α was unaltered. Additionally, STAT1 levels were markedly increased in HEV‐A549 cells compared with naive A549 cells. Furthermore, binding of HEV open reading frame (ORF)3 protein to STAT1 in HEV‐A549 cells was observed. HEV ORF3 protein alone inhibited IFN‐α–induced phosphorylation of STAT1 and down‐regulated the IFN‐α–stimulated genes encoding PKR, 2′,5′‐OAS, and myxovirus resistance A. Conclusion: HEV inhibits IFN‐α signaling through the regulation of STAT1 phosphorylation in A549 cells. These findings have implications for the development of new strategies against hepatitis E. (HEPATOLOGY 2012 )

pfmdr1 Amplification and Fixation of pfcrt Chloroquine Resistance Alleles in Plasmodium falciparum in Venezuela

ABSTRACT Molecular tools are valuable for determining evolutionary history and the prevalence of drug-resistant malaria parasites. These tools have helped to predict decreased sensitivity to antimalarials and fixation of multidrug resistance genotypes in some regions. In order to assess how historical drug policies impacted Plasmodium falciparum in Venezuela, we examined molecular changes in genes associated with drug resistance. We examined pfmdr1 and pfcrt in samples from Sifontes, Venezuela, and integrated our findings with earlier work describing dhfr and dhps in these samples. We characterized pfmdr1 genotypes and copy number variation, pfcrt genotypes, and proximal microsatellites in 93 samples originating from surveillance from 2003 to 2004. Multicopy pfmdr1 was found in 12% of the samples. Two pfmdr1 alleles, Y184F/N1042D/D1246Y (37%) and Y184F/S1034C/N1042D/D1246Y (63%), were found. These alleles share ancestry, and no evidence of strong selective pressure on mutations was found. pfcrt chloroquine resistance alleles are fixed with two alleles: StctVMNT (91%) and SagtVMNT (9%). These alleles are associated with strong selection. There was also an association between pfcrt, pfmdr1, dhfr, and dhps genotypes/haplotypes. Duplication of pfmdr1 suggests a potential shift in mefloquine sensitivity in this region, which warrants further study. A bottleneck occurred in P. falciparum in Sifontes, Venezuela, and multidrug resistance genotypes are present. This population could be targeted for malaria elimination programs to prevent the possible spread of multidrug-resistant parasites.

Laboratory-based Surveillance for Hepatitis E Virus Infection, United States, 2005–2012

Clinicians should consider this virus in the differential diagnosis of hepatitis, regardless of patient travel history.

South American Plasmodium falciparum after the Malaria Eradication Era: Clonal Population Expansion and Survival of the Fittest Hybrids

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradications successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999–2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006–2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.

Evidence of Selective Sweeps in Genes Conferring Resistance to Chloroquine and Pyrimethamine in Plasmodium falciparum Isolates in India

ABSTRACT Treatment of Plasmodium falciparum is complicated by the emergence and spread of parasite resistance to many of the first-line drugs used to treat malaria. Antimalarial drug resistance has been associated with specific point mutations in several genes, suggesting that these single nucleotide polymorphisms can be useful in tracking the emergence of drug resistance. In India, P. falciparum infection can manifest itself as asymptomatic, mild, or severe malaria, with or without cerebral involvement. We tested whether chloroquine- and antifolate drug-resistant genotypes would be more commonly associated with cases of cerebral malaria than with cases of mild malaria in the province of Jabalpur, India, by genotyping the dhps, dhfr, pfmdr-1, and pfcrt genes using pyrosequencing, direct sequencing, and real-time PCR. Further, we used microsatellites surrounding the genes to determine the origins and spread of the drug-resistant genotypes in this area. Resistance to chloroquine was essentially fixed, with 95% of the isolates harboring the pfcrt K76T mutation. Resistant genotypes of dhfr, dhps, and pfmdr-1 were found in 94%, 17%, and 77% of the isolates, respectively. Drug-resistant genotypes were equally likely to be associated with cerebral malaria as with mild malaria. We found evidence of a selective sweep in pfcrt and, to a lesser degree, in dhfr, indicating high levels of resistance to chloroquine and evolving resistance to pyrimethamine. Microsatellites surrounding pfcrt indicate that the resistant genotypes (SVMNT) were most similar to those found in Papua New Guinea.

Characterization of the Bacterial Communities of Life Stages of Free Living Lone Star Ticks ( Amblyomma americanum )

The lone star tick (Amblyomma americanum) is an abundant and aggressive biter of humans, domestic animals, and wildlife in the southeastern-central USA and an important vector of several known and suspected zoonotic bacterial pathogens. However, the biological drivers of bacterial community variation in this tick are still poorly defined. Knowing the community context in which tick-borne bacterial pathogens exist and evolve is required to fully understand the ecology and immunobiology of the ticks and to design effective public health and veterinary interventions. We performed a metagenomic survey of the bacterial communities of questing A. americanum and tested 131 individuals (66 nymphs, 24 males, and 41 females) from five sites in three states. Pyrosequencing was performed with barcoded eubacterial primers targeting variable 16S rRNA gene regions 5–3. The bacterial communities were dominated by Rickettsia (likely R. amblyommii) and an obligate Coxiella symbiont, together accounting for 6.7–100% of sequences per tick. DNAs from Midichloria, Borrelia, Wolbachia, Ehrlichia, Pseudomonas, or unidentified Bacillales, Enterobacteriaceae, or Rhizobiales groups were also detected frequently. Wolbachia and Midichloria significantly co-occurred in Georgia (p<0.00001), but not in other states. The significance of the Midichloria-Wolbachia co-occurrence is unknown. Among ticks collected in Georgia, nymphs differed from adults in both the composition (p = 0.002) and structure (p = 0.002) of their bacterial communities. Adults differed only in their community structure (p = 0.002) with males containing more Rickettsia and females containing more Coxiella. Comparisons among adult ticks collected in New York and North Carolina supported the findings from the Georgia collection despite differences in geography, collection date, and sample handling, implying that the differences detected are consistent attributes. The data also suggest that some members of the bacterial community change during the tick life cycle and that some sex-specific attributes may be detectable in nymphs.

One-step real-time PCR assay for detection and quantitation of hepatitis D virus RNA.

Hepatitis D virus (HDV) is a defective virus which requires hepatitis B virus (HBV) surface antigen (HBsAg) for its assembly. Hepatitis B infected individuals co-infected or superinfected with HDV often present with more severe hepatitis, progress faster to liver disease, and have a higher mortality rate than individuals infected with HBV alone. Currently, there are no commercially available clinical tests for the detection and quantitation of HDV RNA in the United States. A one-step TaqMan quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for detection of HDV RNA, designing primers located in the region just downstream from the HDV antigen gene. The assay has the potential to detect all eight HDV genotypes. A quantifiable synthetic RNA control was also developed for use in the determination of HDV RNA titers in clinical samples. The limit of detection of this assay is 7.5×10(2) HDV RNA copies/ml with a dynamic range of six logs. Most clinical specimens tested (40/41) fell within the linear range of the assay. The median HDV RNA titer of the tested specimens was 6.24×10(6) copies/ml, with a range of 8.52×10(3)-1.79×10(9) copies/ml. Out of 132 anti-HDV-positive specimens 41 (31.1%) were positive for HDV RNA.

Hepatitis B Virus and Hepatitis C Virus Infections in United States-Bound Refugees from Asia and Africa

The aim of this study was to determine the prevalence of active hepatitis B and C virus infections among refugees from various countries in Africa and Asia. Pre-admission serum samples collected during 2002-2007 from refugees originating from Bhutan (N = 755), Myanmar (N = 1076), Iraq (N = 1137), Laos (N = 593), Thailand (N = 622), and Somalia (N = 707) were tested for hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA. The HBV DNA (genotypes A, B, C, and G) was detected in 12.1% of samples negative for anti-HBs. Highest HBV prevalence was found among Hmong; lowest among Bhutanese. The HCV RNA (genotypes 1a, 1b, 1c, 3b, 6n, and 6m) was detected in 1.3% of the samples. Highest HCV prevalence was found among Hmong from Thailand; lowest among Iraqis. Screening specific refugee groups at high risk for viral hepatitis infections will identify infected individuals who could benefit from referral to care and treatment and prevent further transmissions.

Historical Shifts in Brazilian P. falciparum Population Structure and Drug Resistance Alleles

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.