Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/µl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

Attitudes toward Web application supporting pharmacist-clinician comanagement of postexposure prophylaxis patients.

OBJECTIVES: To qualitatively explore clinician and pharmacist attitudes toward using a Web application virtual pharmacist-clinician partnership (VPCP) to assist with comanaged care of illicit drug-using patients prescribed postexposure prophylaxis (PEP). DESIGN: Qualitative, descriptive, nonexperimental study. SETTING: New York City (NYC) from February 2011 to March 2012. PARTICIPANTS: Four pharmacists and nine clinicians. INTERVENTION: In-depth interviews. MAIN OUTCOME MEASURES: Potential impact of the VPCP on pharmacist-clinician communication and potential barriers to use of the VPCP when comanaging PEP patients among pharmacists and clinicians. RESULTS: Pharmacists and clinicians were supportive of an interactive Web application that would expand the role of pharmacists to include assistance with PEP access and patient management. Participants noted that the VPCP would facilitate communication between pharmacists and clinicians and have potential to support adherence among patients. Pharmacists and clinicians were concerned about not having time to use the VPCP and security of patient information on the site. Pharmacist and clinician concerns informed final development of the VPCP, including creation of a user-friendly interface, linkage to users' e-mail accounts for timeline notification, and attention to security. CONCLUSION: Use of Web-based technology to support communication between pharmacists and clinicians was seen as being a potentially feasible method for improving patient care, particularly in the delivery of PEP to drug users and other high-risk groups. These findings highlight the need for further study of a technology-supported partnership, particularly for comanagement of patients who face challenges with adherence.