Genetic profiling of drug resistance in Plasmodium falciparum

Thesis (Ph. D.)--University of Washington, 2007.Malaria is an infectious febrile illness caused by four species of Plasmodium parasite, with Plasmodium falciparum responsible for the greatest number of fatalities. Unfortunately, drug resistant strains of P. falciparum are increasingly prevalent. Resistance to sulfadoxine-pyrimethamine (SP) is due to point mutations in the gene that encodes dihydrofolate reductase (dhfr). Patients infected with a parasite carrying three mutations in dhfr (N51I/C59R/S108N) are at elevated risk of failing SP treatment. Prior studies of the extended haplotype encompassing dhfr suggest that a single triple-mutant allele of dhfr emerged in Asia and spread to Africa. However, it is unclear whether this "Asian" strain replaced triple-mutants that had previously evolved in Africa, or simply invaded a population devoid of triple-mutants. In addition, prior studies were hampered by an inability to study infections containing DNA from multiple parasite strains. This dissertation presents a novel method for using yeast to separate haplotypes in mixed malaria infections. Second, it investigates the history of the Asian strain in Africa by analyzing samples collected in Kilifi, Kenya, from 1987-88 and 1993-95, periods immediately before and after the widespread use of SP. We genotyped each sample at dhfr and flanking microsatellite loci. All of the triple-mutants had the same haplotype, and it matched the haplotype of the Asian strain from previous studies. The wild-type parasites had a variety of haplotypes, none related to the triple-mutant haplotype. Each double-mutant (N51I/S108N or C59R/S108N) had a single haplotype. Both of the double-mutant haplotypes shared some alleles with wild-type samples and with each other, but neither shared any alleles with the triple-mutant haplotype. In addition, the Asian triple-mutant was present in 1988, well before the widespread use of SP in Kenya. These results indicate that the Asian triple-mutant arrived in Kenya before any SP use and rose to high frequency as soon as SP was introduced

Using Engineered Bacteria to Characterize Infection Dynamics and Antibiotic Effects In Vivo

Synthetic biology has focused on engineering microbes to synthesize useful products or to serve as living diagnostics and therapeutics. Here we utilize a host-derived Escherichia coli strain engineered with a genetic toggle switch as a research tool to examine in vivo replicative states in a mouse model of chronic infection, and to compare in vivo and in vitro bacterial behavior. In contrast to the effect of antibiotics in vitro, we find that the fraction of actively dividing bacteria remains relatively high throughout the course of a chronic infection in vivo and increases in response to antibiotics. Moreover, the presence of non-dividing bacteria in vivo does not necessarily lead to an antibiotic-tolerant infection, in contrast to expectations from in vitro experiments. These results demonstrate the utility of engineered bacteria for querying pathogen behavior in vivo, and the importance of validating in vitro studies of antibiotic effects with in vivo models. Using Escherichia coli engineered with a genetic toggle switch, Certain et al. demonstrate ongoing bacterial replication in a mouse model of chronic infection, even with antibiotic treatment. This finding runs contrary to the expectation that antibiotics preferentially eliminate actively dividing bacteria and highlights the in vivo utility of synthetic biology.Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-15-1-0051

General Assembly, Treatment, Multidisciplinary Issues: Proceedings of International Consensus on Orthopedic Infections

Periprosthetic joint infection (PJI) significantly increases the utilization of hospital and physician resources compared to primary cases or aseptic revisions. There is evidence to support that primary total joint replacements performed in a specialized center may have lower complications and lower reoperations than those performed in nonspecialized centers