Sensitive test helps improve vaccine safety

June 27, 2012

Salmonella Typhi (S. Typhi) is the causative agent of typhoid fever, a serious health threat resulting in some 22 million new cases yearly and approximately 217,000 fatalities. A number of novel vaccine candidates using live attenuated strains of Salmonella are being developed, but care must be taken to ensure the bacteria are not excreted into the environment following vaccination.

Karen Brenneman and her colleagues at Arizona State University’s Biodesign Institute have been examining ways to detect the presence of S. Typhi in stool following inoculation with various vaccine strains. Their results recently appeared in the Journal of Microbiological Methods. Download Full Image

The group, overseen by Roy Curtiss III, who directs Biodesign’s Center for Infectious Diseases and Vaccinology, applied a test known as RapidChek SELECTTM Salmonella to detect the shedding of S. Typhi in stool, demonstrating a 10-fold to 1000-fold improvement in detection of attenuated strains compared with conventional methods. 

“This technology is of critical value to help us assess the degree and duration of shedding that occurs after immunization with any live vaccine. We have an ethical responsibility to make sure we understand the effects of vaccination not only on the individual that receives the vaccine, but also on those around him/her in the community,” Brenneman says.

Shedding of Salmonella species in stool following infection typically lasts 3-5 weeks, although typhoid fever infection often results in shedding of bacteria for up to 3 months and typhoid carriers can shed the pathogen indefinitely.  

New vaccines using attenuated versions of S. Typhi are able to confer protection against typhoid fever, triggering a robust, system-wide immune response. Further, the current research (combined with data from clinical trials) demonstrates that attenuated forms of the bacteria are shed for a much shorter time period than wild-type S. Typhi. A number of vaccine strains show early shedding for 1-3 days following vaccination. With some vaccine strains however, shedding persists for 7-11 days, potentially leading to vaccination of those not electing to be vaccinated or who, because of age or other conditions should not be vaccinated.

The fact that attenuated strains are significantly less capable of surviving outside their host is encouraging, though it presents a problem. Detection of attenuated strains in stool is difficult using conventional tests. While the attenuated strains of S. Typhi used in vaccines are considered safe for healthy adults, shedding of bacteria poses some risk for infants, elderly individuals or those with weakened immune systems. For this reason, patients involved in clinical vaccine trials must be carefully monitored to ensure shedding has completely abated.  Conventional detection tests for S. Typhi rely on culturing the bacteria in conjunction with additives designed to selectively inhibit the growth of competing flora. To do this, bile, heavy metals or dyes are added to the growth medium during the enrichment of S. Typhi. While the process works well for enriching and detecting wild-type S. Typhi, these additives have a negative effect on attenuated forms of the bacteria, often rendering them undetectable. 

In the current study, the group used an alternate method – the RapidChek SELECTTM Salmonella detection system, which has been applied successfully to detect the presence of Salmonella on poultry carcasses, eggs and other food products. The method enriches the bacterium in the presence of a bacteriophage cocktail, which reduces competitor microbes. This new, highly sensitive technique can be applied to subjects engaged in vaccine clinical trials, to ensure they are not excreting live vaccine microbes, which could then be passed between humans via the fecal-to-oral route.

Another advantage of RapidChek SELECTTM Salmonella is that it permits a rapid test for the presence of Salmonella just 6 hours after vaccine inoculation, using a Salmonella-specific antibody strip, sensitive to the bacterium’s lipopolysaccharide. RapidChek SELECTTM Salmonella is capable of detecting many different types of Salmonella. Here, the technique was used to assess its ability to detect a number of attenuated strains previously or currently being evaluated in phase I clinical trials with healthy young adults 18 to 40 years of age. 

Results showed that RapidChek SELECTTM Salmonella is highly sensitive to the presence S. Typhi vaccine in stool for the majority of clinically relevant attenuated vaccine strains. In every example, the technique yielded at least a 10-fold improvement in detection over traditional methods and in some cases yielded increases in sensitivity over 1000-fold. 

All vaccine strains examined showed some potential for shedding. Each strain exhibited a characteristic shedding profile, generally much shorter than for wild-type S. Typhi, though all those examined persisted for at least 4 days. Some experimental strains, like χ8444 on the other hand, resembled wild-type S. Typhi in their shedding behavior, in some cases persisting in stools for up to 2 weeks post-immunization. 

A suite of three new vaccine candidates belonging to a group known as regulated delayed attenuation vaccine strains, developed by the Curtiss lab, were among those tested. Their persistence resembled wild-type S. Typhi for the first 48 hours before rapidly declining to undetectable levels by day 5. These strains could not be cultured and enriched with conventional methods however, due to slower than normal growth rates and increased susceptibility to stress.  

To get an approximation of shedding potential, the regulated delayed attenuation vaccine strains were exposed to fecal flora common in the large intestine. These strains showed less ability to survive stress and lower competitive fitness, compared with other S. Typhi strains. The authors suggest that these survival rates in vitro provide a reasonable baseline to approximate actual shedding potential in stool – currently being evaluated in clinical trials.

While conventional methods of detection still have an edge over RapidChek SELECTTM Salmonella for detecting wild-type S. Typhi, the method showed a 10-fold to 100-fold improvement in detecting attenuated strains, (with the exception of a single strain – Ty21a) and 1000-fold improvement in the case of the 3 regulated delayed attenuation vaccine strains tested in the study. 

The research further solidified evidence that attenuation of vaccine strains exerts a profound negative impact on bacterial fitness, thereby improving their safety for vaccine use. Future work is aimed at further reducing the potential for person-to-person transmission of a live attenuated vaccine strain, following immunization.

Richard Harth

Science writer, Biodesign Institute at ASU


FlashFood team looks at going global with venture to help alleviate hunger

June 28, 2012

A team of three recent Arizona State University graduates and one undergraduate are on their way to Sydney, Australia to represent the United States in the premiere international student technology competition July 6-10.

The team named FlashFood earned the trip to the Microsoft Imagine Cup World Finals by winning the 2012 U.S. Imagine Cup finals in April at the Microsoft company headquarters in Redmond, Wash. FlashFood Imagine Cup Download Full Image

It’s the second year in a row a team of ASU students won the U.S. Imagine Cup finals and will go to the world finals.

The prizes for FlashFood’s win in competition with more than 20 student teams from throughout the country included $8,000 to help fund the team’s entrepreneurial venture, as well as a $10,000 donation to ASU. Now FlashFood will compete for additional support against teams from about 75 other countries in the world finals.

FlashFood’s winning project involves a mobile phone application to help establish a network to connect restaurants, hotels, catering and banquet services with teams of people who would collect leftover and excess food and transport to community centers, churches and other neighborhood locations where it can be distributed to people in need.

The idea for the endeavor emerged from the Engineering Projects in Community Service program (EPICS) in ASU’s Ira A. Fulton Schools of Engineering, and from team member Jake Irvin’s involvement with Students In Free Enterprise, a global entrepreneurial non-profit organization with affiliate groups at more than 1,500 universities.

Irvin, a recent marketing and sustainability graduate, will go to the Imagine Cup World Finals with teammates Eric Lenhardt, the team leader and a recent biomedical engineering graduate, Katelyn Keberle, a junior studying materials science and engineering, and computer science graduate Steven Hernandez.

Richard Filley, director of the EPICS program and the team’s faculty adviser, will accompany the students to Australia.

Microsoft requires teams to send only four representatives to the Imagine Cup competitions. Other FlashFood members are Loni Amundson, a recent sustainability graduate, and computer science graduate Ramya Baratam.

With the focus of the competition expanding to a global scale, the members of FlashFood have been working to adapt their venture to an operations model that could be applied to various types of communities throughout the world.

They are working with international groups and organizations to research the issue of food insecurity on a global scale.

“We've been holding focus groups, and generating dialogue with friends and colleagues from countries around the world,  so that FlashFood can fight hunger and food waste internationally,” Keberle says.

Competing in the Imagine Cup Worldwide Finals will give the team “an opportunity to expand our product and form it into something that can be applied in any country,” she says.

“Around the world, there are certain similarities that could make FlashFood effective in different countries,” Keberle says. “Regardless of location, individuals in poverty do have access to cell phones, and there are several international hotel chains that face similar food-waste challenges.”

Through the Imagine Cup, the multinational Microsoft computer-technologies and services corporation encourages students to develop creative technology-based solutions that solve real-world problems.

 The program began in 2003 with only 1,000 students on participating teams. It has since grown into one of the largest student technology competitions in the world. By last year about 350,000 students from 183 countries were members of teams that entered the first rounds of Imagine Cup competitions.
FlashFood is going into the Imagine Cup World Finals with an impressive track record. The team has won awards and support from several competitions and programs, including the ASU Innovation Challenge, the Entrepreneurs @ ASU Elevator Pitch, the ASU 10,000 Solutions Leap Day Challenge and the international Dell Social Innovation Challenge People’s Choice Awards.

The team has also won a $12,000 grant from ASU’s Edson Student Entrepreneur Initiative, which brought them support in the way of office space and mentoring from experienced entrepreneurs

In addition, FlashFood was recently selected as a semifinalist in the 2012 Global Sustainability Challenge, a national competition Nov 7-9 in Louisville, Ky., hosted by YUM!

YUM! is the world’s largest multinational fast-food restaurant company. Its Global Sustainability Challenge supports students in efforts to develop innovative ways to implement sustainability practices in the areas of waste and recycling, product packaging, water and energy consumption and green building design and construction.

By Joe Kullman and Natalie Pierce

Joe Kullman

Science writer, Ira A. Fulton Schools of Engineering