Monday, June 16, 2014

New Mass Spec Method Quickly And Accurately Identifies Pathogens

A mass-spectrometry-based method can rapidly identify the species and strain of bacteria and fungi responsible for infections (Anal. Chem. 2014, DOI: 10.1021/ac501075f). Doctors could use such a technique to find the microbial culprits behind life-threatening infections in patients, allowing them to save precious time and pick the most effective antibiotics for the specific species or strain, the researchers say.

The standard way to identify a microorganism in the clinic is to grow cultures in the laboratory and look for characteristic physical properties, a process that can take days. In recent years, culture-free methods have emerged that identify microbes based on their characteristic 16S ribosomal RNA sequences. However, these approaches often require extensive sample preparation and still sometimes fail to make an unequivocal designation, limiting their usefulness.

Mass spectrometry could be a quick alternative to gene sequencing, says Zoltan Takats of the Imperial College London. He and his colleagues envisioned using the technique to obtain profiles of the lipids in different microbes and then using those molecular signatures to identify species. Such profiles may also provide additional information, such as the capacity of a microorganism to cause disease, he says.

To test their idea, Takats’s team used a method called rapid evaporative ionization mass spectrometry (REIMS) to analyze 28 species of bacteria grown under a variety of conditions. They picked up colonies they wanted to analyze with a device that looks like a pair of forceps. The tip of the forceps is wired: When the researchers flip a switch, current pulses through the device, generating a cloud of mostly lipids from the selected colony. The airborne lipids are then drawn into a fluid line embedded in the body of the forceps, directing the molecules to the spectrometer for detection. Takats says the forceps could be adapted to gather samples taken from a patient’s mucosal membranes.

A computer program analyzed the spectra and compared the colonies’ lipid profiles, allowing Takats’s team to successfully distinguish all 28 species of bacteria and five species of Candida fungi. They could make the identifications regardless of growth conditions and often at greater than 95% accuracy. The identification process, from picking the colony to running the program, took as little as three to five seconds. To see if they could identify different strains of the same types of bacteria, the researchers analyzed seven strains of Escherichia coli and found they could distinguish them with around 90% accuracy.

The technique’s ability to distinguish microorganisms based on their lipid profiles is surprising and exciting, says Charles Y. Chiu of the University of California, San Francisco. However, he points out that the study was done with pure colonies, which have a high concentration of organisms. “In clinical samples, it’s more of a needle and a haystack,” Chiu says.

Chiu also wonders if the method will be able to distinguish between dangerous and harmless versions of the same microorganism. Takats says that is his goal, and he is currently working to discriminate between pathogenic and innocuous strains of Clostridium difficile.

The reference is provided below, and a full PDF is available for download.

Rapid Characterization and Identification of Clinically Relevant Microorganisms Using Rapid Evaporative Ionization Mass Spectrometry

Nicole Strittmatter , Monica Rebec , Emrys A Jones , Ottmar M.H. Golf , Alireza Abdolrasouli , Julia Balog , Volker D Behrends , Kirill A. Veselkov , and Zoltan Takats.

Anal. Chem., Just Accepted Manuscript
DOI: 10.1021/ac501075f
Publication Date (Web): June 4, 2014


Rapid evaporative ionization mass spectrometry was investigated for its suitability as a general identification system for bacteria and fungi. Strains of 28 clinically relevant bacterial species were analyzed in negative ion mode and corresponding data was subjected to unsupervised and supervised multivariate statistical analyses. The created supervised model yielded correct cross-validation results of 95.9%, 97.8% and 100% on species-, genus- and Gram-stain-level, respectively. These results were not affected by the resolution of the mass spectral data. Blind identification tests were performed for strains cultured on different culture media and analyzed using different instrumental platforms which led to 97.8-100% correct identification. Seven different Escherichia coli strains were subjected to different culture conditions and were distinguishable with 88% accuracy. In addition, the technique proved suitable to distinguish five pathogenic Candida species with 98.8% accuracy without any further modification to the experimental workflow. These results prove that REIMS is sufficiently specific to serve as a culture condition-independent tool for the identification and characterization of microorganisms.

Monday, June 2, 2014

Suspect Strep Throat? Re-check Negative Rapid Test Results With Lab Culture

Clinical guidelines conflict on testing teens and adults whose symptoms point to a possible strep throat. A chief contention is whether negative tests results from a rapid analysis of a throat swab, done in a doctor's office, should be confirmed through a follow-up laboratory culture.

The rapid test detects certain antigens, one of the body's efforts to fight off strep bacteria. Attempting to grow bacteria from a throat specimen double checks for the presence or absence of Group A Streptococcus bacteria, as well as a few other bacterial infections.

A study published May 27 in Clinical Infectious Diseases indicates that performing a laboratory culture could help doctors and patients avoid both under-treating and over-treating sore throats.

Several guidelines on diagnosing and treating Group A Streptococcus sore throats in adults have been published by medical and scientific professional societies, including the College of American Pathologists and the Infectious Diseases Society of America.

The study findings call into serious question clinical guidelines that rely only on the rapid test, according to Dr. Ferric Fang, professor of microbiology and laboratory medicine at the University of Washington, and senior author of the paper.

Fang says, "Each year nearly seven million Americans seek medical attention for a sore throat, making it one of the most common reasons to see a doctor."

About one in 10 of these patients has a strep throat. The rest are due to viruses or other causes. Although most cases of strep throat heal on their own, antibiotic treatment reduce symptoms decrease transmission to others, and can prevent rare but serious complications such as damage to heart valves.

Accurately diagnosing that the sore throat is not bacterial is also important, Fang says. Antibiotics for a strep throat don't work against a viral sore throat, and can produce harmful side effects not outweighed by benefits. Unnecessary use of antibiotics also contributes to antibiotic-resistance.

Fang explained that rapid tests, while convenient, miss up to 1 out of 4 cases of strep throat. Some physicians will recommend a throat culture even when the rapid test is negative, if the patient's symptoms seem to warrant it.

Doctors check a patient with a suspected strep throat for tender glands in the neck below the ears, lack of a cough, difficulty opening the jaw, painful swallowing; oozing, swollen, tonsils; fever and a high white blood cell count.

Despite the constellation of suspicious symptoms in the Centor score for strep throat likelihood, it is still difficult to distinguish a viral sore throat from a strep throat. However, if doctors depend simply on symptom presentation to diagnose, without any testing, the tendency is to over-prescribed antibiotics. Doctors who rely just on the rapid test may end up undertreating strep throat.

The controversy on diagnosing strep throat prompted Fang and his UW colleagues, Dr. Tanis Dingle, senior fellow in laboratory medicine, and Dr. April Abbot, assistant professor of laboratory medicine and director of clinical microbiology at UW Medicine, to review the anonymous records of more than 25,000 teens and adults seen for sore throats at Harborview Medical Center and University of Washington Medical Center in Seattle. The records went back 11 years. The researchers found that more than 1,000 patients whose rapid throat cultures were negative actually turned out to have strep in their laboratory throat cultures.

"These cases would have been undetected if their doctors hadn't performed cultures," Fang says.

Most of these patients had moderate to severe symptoms; a very few had serious complications such as a tonsil abscess or rheumatic fever. Most of the patients were given antibiotics. The throat cultures, Fang said, helped guide treatment decisions in half of the cases.

From their results, the researchers extrapolated that, each year in the United States more than 250,000 patients with strep throat would not receive beneficial treatment if doctors followed those clinical guidelines that advise relying on the rapid test alone. The findings support those guidelines that mandate back-up lab cultures.

The researchers concluded, "Appropriate use of rapid and culture-based diagnostic tests can reduce inappropriate use of antibiotics for sore throats, while avoiding under-treatment of patients who can benefit from antibiotics."

Source: UW Medicine

Sunday, June 1, 2014

Pharmig Hosts Rapid Methods Conference in Ireland

On May 14, 2014, Pharmig hosted their Annual Irish Microbiology Conference and Rapid Microbiological Methods (RMM) Symposium and Exhibition.  I had the honor of presenting a number of talks covering the myths of implementing RMMs in a regulated industry, the new PDA Technical Report No. 33, and a case study on using a real-time environmental monitoring technology.

Amy McDaniel, Director of Technical Operations, Pfizer, provided an overview and case study of implementing an automated, growth-based RMM for environmental monitoring, including surface sampling and viable air monitoring. She discussed the installation of the technology in a controlled manufacturing environment and the benefits Pfizer realized. Amy also discussed the role of bioprocess technicians when the technology was incorporated into their routine EM program.

Matthias Fischer, Manager Microbiology/Biochemistry at Eurofins, discussed rapid methods in the microbiological quality control of specialized nutrition products. The validation of an impedance technology to detect Salmonella, Enterobacteriaceae, Cronobacter, Enterococci and total viable count was discussed. Next, Matthias provided an overview of his work with PCR amplification and bioluminescence technologies.

The afternoon session allowed attendees to visit more than eight different vendor booths where hands-on demonstrations of their technologies and applications were provided.

The meeting provided participants with many opportunities to ask questions, share their own experiences and network with fellow microbiologists who have implemented RMMs i their facilities.  Watch for more raid method conferences from Pharmig in the future.

New Rapid Micro Method Papers

When new rapid microbiological method case studies and technology overviews are published, we will add them to our Reference Page.  Four new papers have recently been added, and the topics span real-time environmental monitoring using light-scattering technologies, the implementation of PCR in developing countries and the utilization of resazurin for the detection of viable microorganisms.

Full citations and abstracts are provided below.

2014. Ragheb, S.M.; Jimenez, L. Polymerase Chain Reaction/Rapid Methods Are Gaining a Foothold in Developing Countries. PDA Journal of Pharmaceutical Science and Technology. 68(3): 239-255.

Detection of microbial contamination in pharmaceutical raw materials and finished products is a critical factor to guarantee their safety, stability, and potency. Rapid microbiological methods—such as polymerase chain reaction— have been widely applied to clinical and food quality control analysis. However, polymerase chain reaction applications to pharmaceutical quality control have been rather slow and sporadic. Successful implementation of these methods in pharmaceutical companies in developing countries requires important considerations to provide sensitive and robust assays that will comply with good manufacturing practice.

2014. Eaton, T.; Wardle, C.; Whyte, W. Use of a Real-Time Microbial Air Sampler for Operational Cleanroom Monitoring. PDA Journal of Pharmaceutical Science and Technology. 68(2): 172-184.

A sampler that detects and counts viable particles in the air of cleanrooms in real-time was studied. It was found that when the sampler was used to monitor airborne particles dispersed from a number of materials used in cleanrooms, including garments, gloves, and skin, the number of viable particles dispersed from these materials was greater than anticipated. It was concluded that a substantial proportion of these viables were of a non-microbiological origin. When the sampler was used to monitor a non-unidirectional airflow cleanroom occupied by personnel wearing cleanroom garments, it was found that the airborne viable concentrations were unrealistically high and variable in comparison to microbe-carrying particles simultaneously measured with efficient microbial air samplers. These results confirmed previously reported ones obtained from a different real-time sampler. When the real-time sampler was used in a workstation within the same cleanroom, the recorded viables gave results that suggest that the sampler may provide an effective airborne monitoring method, but more investigations are required.

2014. Gurramkonda, C.; Mupparapu, K.; Abouzeid, R.; Kostov, Y.; Rao, G. Fluorescence-Based Method and a Device for Rapid Detection of Microbial Contamination. PDA Journal of Pharmaceutical Science and Technology. 68(2): 164-171.

A method for rapid detection of microbial detection is presented. It uses the reduction of resazurin to resorufin as an indication of the presence of viable cells. The method is highly sensitive (limit of detection 1 CFU/mL) and rapid (detection time 180 s). A portable device that could allow the detection to be performed in the field is also described.

2014. Sandle, T.; Leavy, C.; Jindal, H.; Rhodes, R. Application of Rapid Microbiological Methods for the Risk Assessment of Controlled Biopharmaceutical Environments. Journal of Applied Microbiology. 116(6): 1495-1505.

To assess the different operational states within a biopharmaceutical grade clean room, using a rapid microbiological method. The method was a novel system, based on spectrometry, designed for sampling, discriminating, and enumerating airborne particles. Central to the study was the aim to determine the microbiological levels as a clean room went from standard use through maintenance and shutdown, disinfection, and then back to standard use. The objective was to evaluate whether a rapid method could replace conventional environmental monitoring using growth‐based media.