Ways to move quickly on bloodstream infection

Other advantages of MALDI-TOF MS are that it is not necessary to predetermine whether an organism is gram-positive or gram-negative and it has the potential for future susceptibility testing.

MALDI-TOF MS can have some difficulty differentiating very similar organisms such as Shigella spp from E. coli. Some systems have difficulty with differentiating Streptococcus pneu­moniae and S. mitis/oralis. However, “The Vitek MS can very accurately differentiate these organisms,” Dr. Ginocchio says.

Several studies evaluating the use of MALDI-TOF MS for the identification of bacteria and yeast from positive blood culture bottles have been published. Using one of the two major instruments, the Bruker BioTyper, a Dutch group analyzed 101 positive BacT/Alert bottles. Correct identification at the species level was obtained in 78 percent of specimens—64 percent of gram-positive cocci and 96 percent of gram-negative rods (Loonen AJ, et al. Eur J Clin Microbiol Infect Dis. 2012;31:1575–1583). In a similar study using the BioMérieux Vitek MS system, a group at Emory University was able to obtain a correct identification to species level in 73 percent of 259 bottles (Fothergill A, et al. J Clin Microbiol. 2013;51: 805–809).

Another Dutch group showed that incorporating MALDI-TOF MS into the identification system increased appropriate antibiotic prescribing. Using an alternate-month protocol, they found that, in the months when MALDI-TOF MS was part of the system, time to species identification was reduced by almost 30 hours. In addition, there was an 11.3 percent increase in the proportion of patients receiving appropriate antibiotic treatment 24 hours after blood culture positivity (64 percent in the control period versus 75.3 percent in the intervention period) (Vlek AL, et al. PLoS One. 2012;7:e32589).

A Swiss group found that MALDI-TOF MS had a positive impact on more cases than did Gram stain. Using impact on empirical antibiotic choice as the primary outcome, they found that, among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 20.8 percent of cases, while MALDI-TOF MS identification led to a modification of empirical therapy in 35.1 percent. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum, the investigators reported (Clerc O, et al. Clin Infect Dis. 2013; 56:1101–1107).

A five-center group evaluated the accuracy of Vitek MS for bacteria and yeast identification from colonies, using 16S ribosomal RNA gene sequencing and biochemical testing as the comparators.

Results for species-level identification were as follows:

• Gram-positive cocci: 92.8 percent (Rychert J, et al. J Clin Microbiol. 2013;51:2225–2231).
• Yeast: 96.1 percent (Westblade LF, et al. J Clin Microbiol. 2013;51: 2267–2272).
• Enterobacteriaceae: 83.8 percent (Richter SS, et al. Eur J Clin Microbiol Infect Dis. July 2, 2013, Epub ahead of print).
• non-Enterobacteriaceae gram-negative bacilli: 77.8 percent (Manji R, et al. Eur J Clin Microbiol Infect Dis. Sept. 10, 2013, Epub ahead of print).

Based in large part on these data, in August the FDA gave the Vitek MS 510(k) de novo clearance, the first such platform approved for clinical use in the United States.

Dr. Ginocchio was part of the five-center group that conducted the clinical trials for the Vitek MS. Her laboratory was validating it for clinical use and monitoring its performance while the application was under consideration at the FDA. “It performed quite excellently. You do not have to do extraction or up-front preparation prior to applying the organisms to the target slide. For bacteria, matrix is then added to the dried spot; for yeast, a drop of formic acid is added to the target slide prior to the addition of matrix,” she says.

With the FDA now having cleared the instrument, her laboratory will soon use it clinically. “We plan to switch to Vitek MS as the primary method for microbial identification.” Not all organisms will be identified, and supplemental tests may be required, she says. “However, for the 90-plus percent of the most frequently isolated aerobic and anaerobic bacteria and yeast, MALDI-TOF MS should provide a rapid and accurate identification.” Culture that would routinely take 48 to 72 hours can now be reported in 24 to 36 hours when combined with a rapid automated susceptibility test, she says.

Identification alone can provide initial critical information for guiding antimicrobial therapy for such pathogens as Pseudomonas, Acinetobacter, Stenotrophomonas, and fluconazole-resistant yeast, including Candida krusei.

“The direct identification of pathogens from positive blood cultures will allow a rapid assessment of the potential efficacy of empiric therapy and guide change in a time frame that should have a significant impact on patient morbidity and mortality,” Dr. Ginocchio says.

One issue she identified is the need to validate MALDI-TOF MS on positive blood cultures as an off-label use, since it is FDA cleared for identification of bacteria or yeast colonies from culture plates. “We have just completed our in-house validation on 1,000 positive blood cultures,” she says.

MALDI-TOF MS will miss mixed infections or not identify the predominant organism, she says.

Specific Technologies’ colorimetric sensor array technology was the last method for use on positive blood cultures that Dr. Ginocchio described. By sampling volatile organic compounds in the headspace of the culture, this technique produces an organism-specific difference map that can serve as a profile for identification. In one experiment Dr. Ginocchio illustrated, the Specific Technologies sensor identified organisms to the species level greater than 20 percent faster than the BioMérieux BacT/Alert 3D system gave a CO2 positive growth signal only.

Identifying pathogens directly from patient blood samples without having to culture would be the fastest way to diagnose bloodstream infection. While no method for this use is approved in the United States, Dr. Ginocchio spoke about the possibilities.

Although the Roche LightCycler SeptiFast Test is available for clinical use in Europe, Dr. Ginocchio told CAP TODAY that it was not going to be brought to the FDA because its sensitivity is not sufficiently high to meet FDA specifications. “It works well in children and newborns for sepsis because they have a high bacterial burden,” she says.

A product with promising sensitivity is T2 Biosystems’ T2MR assay. In this assay, microbial cells are lysed, followed by PCR and hybridization of the amplified pathogen DNA to capture probe-decorated nanoparticles. Hybridization with the superparamagnetic nanoparticles yields nanoparticle microclusters that cause large changes in the sample’s T2MR signal. Spiked human blood samples were used to show that the method detects five Candida species with a limit of detection as low as 1 CFU/mL—though for four of the species sensitivity was acceptable only at 2 to 3 CFU/mL—and a time to result of less than three hours (Neely LA, et al. Sci Transl Med. 2013; Apr 24;5[182] 182ra54). Agreement with blood culture was 97.8 percent for positive samples and 100 percent for negative samples.

“This method has the sensitivity you would need to work directly from whole blood patient samples. You can do it with any organism; Candida is just the first one they tried,” she says, adding that the company plans to develop versions for other organisms.

In September, T2 Biosystems announced the initiation of the direcT2 clinical trial of T2Candida, its flagship product for identifying five species of Candida.

Distinguishing rapidly between similar organisms, one of which can have a problematic antibiotic resistance, has important infection control implications. Dr. Ginocchio highlighted these pairs:

• Klebsiella pneumoniae (KPC) versus E. coli.
• Acinetobacter spp (MDRO) versus E. coli.
• S. aureus (MRSA) versus coagulase-negative staphylococci.
• E. faecalis (VRE) versus E. faecium.
• S. pneumoniae and S. bovis versus S. viridans.
• C. krusei versus C. albicans.

In closing, she reminded her ASM audience that rapid detection means nothing without rapid transmission of results, and she pointed to the results of a study in which one medical center instituted PNA FISH for coagulase-negative staphylococci but didn’t put in place an active notification program. The authors wrote, “Without active notification or antimicrobial stewardship intervention, a pre- and postimpact analysis showed no benefit of this assay with respect to the length of hospital stay or vancomycin use” (Holtzman C, et al. J Clin Microbiol. 2011;49:1581–1582).

“It’s not necessarily the laboratory’s issue,” Dr. Ginocchio says. “We can send the information to the doctor’s or the nurse’s phone, but if no one is there to receive and act on the information in a timely fashion, the result goes for naught.” There must be a highly collaborative interaction between the laboratory, pharmacy, and medical staff. To benefit from the use of rapid assays, all pieces must be in place.

William Check is a writer in Ft. Lauderdale, Fla.