Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae, the cause of leprosy. In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii. However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii. Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a-alpha sexual cycle, the majority outbreak clone appears to have descended from two alpha mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms.
Heteroduplex tracking assays (HTAs) of Plasmodium falciparum merozoite surface protein 1 block-2 were used to assess complexity of infection and treatment efficacy in a trial of three antimalarial treatments in 141 Malawian pregnant women. An elevated complexity of infection (COI) was associated with anemia, parasite burden, and human immunodeficiency virus infection but was not associated with age or gravidity. Comparisons of HTA patterns before and after treatment allowed the classification of 20 of 30 (66%) recurrent episodes as either definite treatment failures or reinfections. An elevated COI was strongly associated with treatment failure (P ؍ 0.003). An algorithm was developed to assign a probability of failure for the 10 indeterminate participants, some of whose infections shared a single variant of high prevalence (>10%). By summing these probabilities, treatment efficacy was estimated.More than 500 million people suffer from Plasmodium falciparum malaria each year. The majority of all malaria infections occur in sub-Saharan Africa, where there is often intense year-round exposure to infected mosquitoes (20). Probably as a result of frequent infectious bites, malaria infections in subSaharan Africa are usually polyclonal (2, 3, 13). Individual patients have been infected with as many as 14 genetically distinct parasite subpopulations or "variants" (3, 6, 11). The complexity of infection (COI), which is the number of variants in an individual host, has been measured by a variety of molecular methods. These methods include nested PCR, restriction fragment length polymorphism, microsatellite analysis, pyrosequencing, and PCR with a fluorescence primer followed by capillary electrophoresis (7,14,19,21).Using these molecular tools, a higher COI has been associated with symptomatic malaria manifestation in pregnant women (3). In a recent study, the risk of treatment failure in children was positively correlated with the COI (13).The current World Health Organization protocol recommends that all clinical efficacy studies with more than 14 days of follow-up use PCR-based methods to determine whether recurrent infection is due to drug failure (recrudescence) or a newly acquired infection (reinfection) (25). The inherent limitations of conventional nested PCR genotyping, such as sensitivity only to size differences, and different interpretations of the PCR comparisons can both have a dramatic impact on the treatment failure rate (18). Therefore, more definitive methods are needed.We have previously used the heteroduplex tracking assay (HTA) in an area of low malaria transmission to characterize the COI in Southeast Asia (15, 26). In the HTA, radiolabeled P. falciparum merozoite surface protein 1 (PfMSP1) probe is annealed to amplicons from a patient whose PfMSP1 block-2 region has undergone a single round of PCR amplification. Double-stranded complexes are formed between the probe and host amplicon(s); these complexes, termed heteroduplexes, migrate on a nondenaturing gel at various speeds based on the compleme...
Biological diversity has been estimated for various phyla of life, such as insects and mammals, but in the microbe world is has been difficult to determine species richness and abundance. Here we describe a study of species diversity of fungi with a yeast-like colony morphology from the San Juan Islands, a group of islands that lies southeast of Vancouver Island, Canada. Our sampling revealed that the San Juan archipelago biosphere contains a diverse range of such fungi predominantly belonging to the Basidiomycota, particularly of the order Tremellales. One member of this group, Cryptococcus gattii, is the etiological agent of a current and ongoing outbreak of cryptococcosis on nearby Vancouver Island. Our sampling did not, however, reveal this species. While the lack of recovery of C. gattii does not preclude its presence on the San Juan Islands, our results suggest that the Strait of Juan de Fuca may be serving as a geographical barrier to restrict the dispersal of this primary human fungal pathogen into the United States.
The polychaete annelid Paralvinella pandoraeDesbruyères and Laubier 1986 is endemic to hydrothermal vents in the northeast Pacific, and is found at almost all vents sites along the 500‐km long Juan de Fuca ridge (JdF) system. The sperm morphology of P. pandorae indicates that fertilization occurs internally or in the worm's tube, and the maximum observed oocyte size of 215 μm suggests that a dispersive larval phase is short or non‐existent. Size frequency analyses of populations of P. pandorae suggest continuous or semi‐continuous recruitment of juveniles. Given our limited knowledge of the species’ life history, we predicted that populations of P. pandorae would exhibit a decline in genetic similarity with increasing distance among populations along the JdF. While our attempts to use amplified fragment length polymorphisms to test this prediction were not successful, our analysis of cytochrome oxidase I gene sequences provided insights into the phylogeography of the species. For 31 individuals from five sites along the JdF there is little sequence variation among individuals and no phylogeographic pattern among haplotypes from populations separated by distances of up to 210 km. These results indicate that gene flow occurs among all sites in the analyses, i.e. despite the very limited dispersal potential inferred from life history characteristics of this worm, there is no evidence for isolation‐by‐distance across the geographical scale of the study. Demersal larvae dispersed by near‐bottom currents might explain the gene flow among sites, as well as the establishment of populations of P. pandorae at new vents within a year.
Public health emergencies, such as hurricanes and the constant threat of an influenza pandemic, present public health responders with many ethical issues and little time to think them through. We interviewed 13 responders in the Epidemiology Section of the North Carolina Division of Public Health to learn how they have identified and addressed ethical issues in public health emergencies affecting the state and to identify potential means of improving those processes for North Carolina and other states. The Epidemiology Section staff demonstrated an awareness of several ethical issues in public health emergencies and an ability to identify and address issues through group interactions. However, few study participants in the section had received any training in public health ethics. Perhaps for this reason, the range of ethical issues they identified excluded several mentioned in the Public Health Code of Ethics. Moreover, their ethical decision making could be enhanced by a more detailed understanding of the ethical issues they named. We recommend seven practical steps that the Epidemiology Section can take to improve their ability to identify and address ethical issues in a public health emergency. The recommendations are likely relevant to many state, city, and county public health departments throughout the United States.
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