Micro revolution: a radical new way to diagnose disease

Speed matters when lives are on the line and helping them requires knowing what's wrong. That's why Cornell's Animal Health Diagnostic Center (AHDC) is in the business of getting answers quickly, getting them right, and pioneering ways to do both better. The AHDC's Bacteriology Section has radically transformed the way and speed with which it identifies bacteria, becoming one of the first labs in the country to use a machine invented for other purposes to diagnose disease.

Because there are hundreds of proteins in each bacterium, thousands of bacteria on each testing plate, and often many different kinds of bacteria in a sample, the protein map obtained from bacteriology samples is so complex that using it to diagnose was very difficult. Thanks to Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass-spectrometer (MALDI-TOF for short), the process is much more doable.

Mass spectrometry is a powerful analytical technique. By mapping out the block-by-block structure of a sample, a mass spectrometer can identify unknown molecules. This new process identifies bacteria by recognizing patterns in the complex maps mass spectrometry makes of microorganisms using a robust database of common patterns that can be used like fingerprints for specific species. Now when a MALDI-TOF analyzes a bacterial sample, a computer can match the resulting map to that of a known species with accuracy equal to that of microbiology’s old system.

In traditional clinical microbiology, researchers streak a sample onto an agar plate and try to identify microorganisms with several different biochemical tests. Best case scenario, this takes 24 hours, but it takes longer if the sample is small or contains colonies of more than one kind of organism. Then each colony must be separated, purified, and re-grown large enough to accurately run the test.

The MALDI-TOF works with much smaller samples, making purification unnecessary. Even if a plate contains multiple different colonies, a diagnostician can focus on one tiny colony without having to isolate and regrow. After one overnight growth, answers come in minutes.

At the AHDC, speed is the real advantage for us. In some cases the MALDI-TOF will enable the Center to identify bacteria straight from a sample without having to grow them on the plate.

Mystery-Solving MALDI

Even before the Cornell University Animal Health Diagnostic Center's Bacteriology Laboratory had the verification data accepted and approved for the Bruker MALDI- Biotyper, this amazing technology made a significant impact for one of our clients by guiding us in the identification of a pathogenic organism we would have otherwise missed.

Two animals belonging to the collection of one of our zoological clients died suddenly, within two days of each other. When the necropsies were performed, they found evidence of bacterial sepsis in both animals. Samples were submitted to our laboratory for bacterial culture, consisting of a lymph sac swab from an exotic toad, and swabs of peritoneal fluid and brain from a lesser panda.

The bacterial cultures from these samples were evaluated by different technicians, with a similar enteric organism being isolated from both cases. Initially, these organisms were unidentifiable by our conventional methods.  The Sensititre® automated identification system gave a result of "No Identification Possible." Additionally, screening tests set with conventional biochemicals lead to reactions that did not afford many clues to the identification.

We decided to run the isolates on our MALDI Biotyper, as we had already completed operational training and were actively gathering verification data from the instrument. The identifications were received within minutes for the isolates, and were both identified as Salmonella species! The identification given by the MALDI Biotyper was questioned because the biochemical reactions were so uncharacteristic for Salmonella species. We still needed to pursue additional avenues to confirm the actual identification of the organism.

We collaborated with a research laboratory in the Cornell College of Veterinary Medicine who has an established BAX System PCR Assay specifically for Salmonella species. The test was positive for isolates from both cases.  Isolates were then referred to the National Veterinary Services Laboratory in Ames, Iowa and the identification was confirmed as an uncharacterized novel strain of Salmonella species.

Communication was maintained with the client continuously throughout the identification process. The new technology of the MALDI Biotyper allowed us to give the client extremely valuable information, the ability for them to mitigate potential spread of this organism within the facility, and identified a significant pathogen that would have been missed by conventional identification methods.

Q & A with Melissa Aprea

How are bacteria normally identified?

Many 'tools' to help identify bacteria have emerged over the years, and to ensure the accuracy of results, the AHDC Bacteriology laboratory has developed protocols that involve nearly all available techniques. A sample is placed on a series of specialized nutritive and differential agars in order to provide optimal growth conditions for any bacterial organisms that may be present. Agar plates are then incubated and if bacteria are present in the sample, they will grow on the agar in the form of bacterial colonies. Much can be inferred about an organism from this simple step alone, but identification to the species level requires numerous additional tests, ranging from quick simple biochemical reactions to lengthy automated or manual phenotypic assays. If identification of an organism cannot be determined by these methods, the isolate can usually be identified by DNA sequencing methods, results of which are not available immediately.

Why is using the MALDI-TOF to identify bacteria a better approach?

The use of MALDI-TOF mass spectrometry provides yet another analytical tool to help aid in identification...this time using a microorganism's unique protein profile. A single bacterial colony is all that is required. Identification by MALDI-TOF is extremely fast (minutes compared to hours!) and will improve efficiency when used early in the identification protocol. Additional conventional techniques can then be reserved for isolates not identified by MALDI-TOF with high confidence. In most cases, culture results can potentially be reported to the client one day earlier. This in turn could help stop the spread of disease not only to other animals in a herd situation, but also to the people associated with them in certain situations!

How has having the MALDI-TOF changed the way you do things or what you do during the day? Does it allow you to do more? To investigate other novel approaches? To redefine your role as a microbiologist?

The AHDC employs a team of highly qualified technologists in the Bacteriology Laboratory who are not only trained to simply 'find the pathogen', but to also investigate potentially new and emerging pathogens. Each culture is unique and we never know what we will find when we open those agar plates! The use of MALDI-TOF mass spectrometry will certainly aid in the rapid identification of anything unusual. Our role as microbiologists will remain the same due to the inherent curiosity of investigation in our field. This will never be lost and I believe we will become even more important in the rapid identification and diagnoses of emerging bacterial diseases.

What kind of samples do you need?

Bacterial culturing can be performed on nearly any sample type imaginable, literally from head to toe and beyond, including environmental samples. The AHDC maintains a high standard to provide quality results to our clients and we strongly believe in 'Quality in, Quality out'. When sampling, all surfaces should be assumed to contaminated with bacteria, most of which could be normal commensal or environmental organisms. It is especially important when sampling from animal patients to prepare the sample site properly. Ideally, samples submitted overnight on swabs in an agar transport media will provide the best chance for recovery of any potential pathogens.

Where can I find information on how to submit the sample?

Information on sample submission for bacterial culture can be found on our website. Information regarding purchasing of supplies (such as bacterial transport media and sterile containers) and discounted shipping labels can also be found there.

What information do you need with the submitted samples?

It is important to provide all patient demographic information such as species and age of the patient, as well as type of sample and date obtained. We also strongly suggest providing a complete history on the patient, including any previous bacterial culture results, antibiotic treatments and whether the patient has had any chronic conditions. All this information is necessary to help determine the significance of any bacteria isolated.

It is important to note that antibiotic susceptibility testing is an additional cost to bacterial culture. If you know you would like antibiotic susceptibility testing on significant isolates, please indicate this at the time of submission to ensure you receive your results as quickly as possible.

How much does it cost?

Bacterial identification by MALDI-TOF is included in the cost of the numerous types of bacterial cultures we offer, including aerobic and anaerobic bacterial culture, urine culture and Salmonella culture to name few. All of our available Bacteriology cultures and prices can be found on our website.

How will the results be returned?

Results are available by the method most convenient for you, including fax, mail and email. Additionally, we provide online access to accounts and test results through the 'AHDC Online Portal'. Simply register for this service by submitting the electronic request form here.