Jessica A. Hill

Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata.

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential.

Genetic and Genomic Architecture of the Evolution of Resistance to Antifungal Drug Combinations

The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast Saccharomyces cerevisiae and the leading human fungal pathogen Candida albicans with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, PDR1, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, MOT3, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in LCB1, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the C. albicans lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance.

Using combination therapy to thwart drug resistance

Drug combination therapy is a promising strategy to extend the lifespan of our antimicrobials. Drug combinations used in treatment must be carefully selected to minimize the evolution of resistance, either by carefully determining drug pairs that hinder the acquisition of resistance mechanisms, or by screening for combinations that inhibit growth and show reduced vulnerability to resistance. Modeling of interactions between drugs has provided intriguing insights into strategies for combination therapy deployment. Ultimately, more rigorous clinical trials need to be performed to evaluate the laboratory and modeling results and advance treatment options.

Cancer genetics education in a low- to middle-income country: Evaluation of an interactive workshop for clinicians in Kenya

Background Clinical genetic testing is becoming an integral part of medical care for inherited disorders. While genetic testing and counseling are readily available in high-income countries, in low- and middle-income countries like Kenya genetic testing is limited and genetic counseling is virtually non-existent. Genetic testing is likely to become widespread in Kenya within the next decade, yet there has not been a concomitant increase in genetic counseling resources. To address this gap, we designed an interactive workshop for clinicians in Kenya focused on the genetics of the childhood eye cancer retinoblastoma. The objectives were to increase retinoblastoma genetics knowledge, build genetic counseling skills and increase confidence in those skills. Methods The workshop was conducted at the 2013 Kenyan National Retinoblastoma Strategy meeting. It included a retinoblastoma genetics presentation, small group discussion of case studies and genetic counseling role-play. Knowledge was assessed by standardized test, and genetic counseling skills and confidence by questionnaire. Results Knowledge increased significantly post-workshop, driven by increased knowledge of retinoblastoma causative genetics. One-year post-workshop, participant knowledge had returned to baseline, indicating that knowledge retention requires more frequent reinforcement. Participants reported feeling more confident discussing genetics with patients, and had integrated more genetic counseling into patient interactions. Conclusion A comprehensive retinoblastoma genetics workshop can increase the knowledge and skills necessary for effective retinoblastoma genetic counseling.

Knowledge, experiences and attitudes concerning genetics among retinoblastoma survivors and parents

Clinical genetic services are increasingly providing a more nuanced understanding of genetic disease diagnostics and future risk for patients. Effectively conveying genetic information is essential for patients to make informed decisions. This is especially important for survivors of heritable cancers such as retinoblastoma (childhood eye cancer), where survivors who carry a germline mutation in the RB1 gene are at increased risk of second cancers in adulthood, and of passing on the disease risk to future offspring. We conducted focus groups with adult survivors of retinoblastoma and parents of children with retinoblastoma, to uncover their knowledge of, experiences with and attitudes about retinoblastoma genetics and related impacts of the cancer. Results revealed that participants understood that retinoblastoma was a genetic disease, but often misunderstood the implications of genetics on cancer phenotype and risk. Experiences with genetic testing and counseling were generally positive, however, participants reported challenges in accessing genetic information and psychosocial support. Participants suggested more educational resources, peer-to-peer counseling, and psychosocial support would enhance uptake of important genetic information. The results of the study will inform patient-oriented approaches to deliver comprehensive genetic healthcare.

In Their Own Words: A Qualitative Study of Kenyan Breast Cancer Survivors’ Knowledge, Experiences, and Attitudes Regarding Breast Cancer Genetics

Introduction Breast cancer ranks among the most common adult cancers in Kenya. Individuals with a family history of the disease are at increased risk. Mutations most commonly associated with breast cancer affect BRCA1 and BRCA2; mutations in several other genes may also confer breast cancer risk. Genetic testing and counseling can help patients understand their risk and assist clinicians in choosing therapies. We aimed to uncover what patients know, experience, and think with regard to breast cancer genetics in Kenya. Methods Participants included breast cancer survivors age > 18 years. Participants completed a demographic questionnaire before participating in focus group discussions to uncover knowledge of, experiences with, and attitudes toward the genetics of breast cancer. Data were analyzed by inductive thematic analysis. Results Four focus groups were conducted. Participants had rudimentary knowledge about genetics and cancer development, and although they understood breast cancer could be familial, many suspected environmental factors causing spontaneous disease. They reported limited experience with counseling about genetic risk, perceiving that their physicians were too busy to provide comprehensive information. Many indicated they promoted cancer screening among family to promote early diagnosis. Participants expressed a need for more comprehensive counseling and access to genetic testing, recognizing the added clarity it would bring to their families’ risk of cancer. Conclusion Improved communication from health care teams could clarify the risk of cancer for affected families. The introduction of affordable genetic testing and counseling for breast cancer in Kenya is welcomed by survivors.

“Where Does it Come from?” Experiences Among Survivors and Parents of Children with Retinoblastoma in Kenya

Genetic testing and counseling have become integral to the timely control of heritable cancers, like the childhood eye cancer retinoblastoma. This study aimed to determine attitudes, knowledge and experiences related to retinoblastoma genetics, among survivors and parents of children with retinoblastoma in Kenya. This qualitative study used focus groups as the primary data collection method, coupled with a brief demographic questionnaire. Study settings were Kenyatta National Hospital and Presbyterian Church of East Africa Kikuyu Hospital. Thematic analysis was used to identify key themes. Thirty-one individuals participated in five focus groups. Two main concepts emerged: (1) the origins of retinoblastoma are unclear, and (2) retinoblastoma is associated with significant challenges. The lack of clarity surrounding the origins of retinoblastoma was linked to limited knowledge of retinoblastoma genetics, and limited genetic counseling delivery and uptake. The challenges associated with retinoblastoma were discussed in terms of the impact of the diagnosis on individuals and families, and unmet healthcare needs related to the diagnosis. Next steps will incorporate these findings to develop evidence-informed and accessible cancer genetic services in Kenya.

Ethical, social, and cultural issues related to clinical genetic testing and counseling in low- and middle-income countries: a systematic review

PurposeWe performed a systematic review of the ethical, social, and cultural issues associated with delivery of genetic services in low- and middle-income countries (LMICs).MethodsWe searched 11 databases for studies addressing ethical, social, and/or cultural issues associated with clinical genetic testing and/or counselling performed in LMICs. Narrative synthesis was employed to analyze findings, and resultant themes were mapped onto the social ecological model (PROSPERO #CRD42016042894).ResultsAfter reviewing 13,308 articles, 192 met inclusion criteria. Nine themes emerged: (1) genetic counseling has a tendency of being directive, (2) genetic services have psychosocial consequences that require improved support, (3) medical genetics training is inadequate, (4) genetic services are difficult to access, (5) social determinants affect uptake and understanding of genetic services, (6) social stigma is often associated with genetic disease, (7) family values are at risk of disruption by genetic services, (8) religious principles pose barriers to acceptability and utilization of genetic services, and (9) cultural beliefs and practices influence uptake of information and understanding of genetic disease.ConclusionWe identified a number of complex and interrelated ethical, cultural, and social issues with implications implications for further development of genetic services in LMICs.