Inside the microbiology lab: conventional and novel diagnostics coexisting

In this video Dr Paño, Infectious Diseases Physician introduces Bill’s case to Dr García, microbiologist.

Dr Paño has provided the microbiology lab with two specimens: a urine sample and blood specimen inoculated in blood culture bottles. He is interested to learn from Dr García how these samples are worked-up in the lab and what the turnaround time is for the identification and susceptibility of both samples:

Dr García (Microbiologist) : Urine, first. Sample is seeded on agar plates. Plates are read usually 24 hours later. If no bacterial or fungal growth is observed, the urine is said to be sterile. If significant growth. At this point, with either straightforward testing or with the help of some rapid tests we can know whether it’s a Grampositive, Enterobacteriaceae, non-fermenter Gramnegative bacilli or a yeast.

We then run definitive identification and phenotypical antimicrobial susceptibility testing. These take no less than 12 hours, but depending on each laboratory working hours it can take up to 72 hours (Bank holidays, weekends…).

Dr Paño (Infectious Diseases Physician) Thanks, Julio. What about blood cultures?

Dr García : Blood culture bottles are incubated. Once the incubator detects indirect signs of microbial growth, a Gram stain of the positive bottle is performed and at the same time we plate several Petri dishes. This takes more than 12 hours, but again this depends on laboratory working hours. Once growth in the Petri dish occurs the process is the same as with the urine culture. As you can see, with blood cultures there is an additional step which is the time until the blood culture bottle becomes positive which can take from hours to five days.

Dr Paño: Up to four days to final susceptibility results might be too much for Bill, given his current condition. How could this interval be improved?

Dr García: I agree, José. Four days is too much. First of all I have to say that this is not a Microbiology Lab-only mission: all of us can contribute to speed microbiological diagnosis. Obtaining good-quality clinical samples and making them reach the laboratory in best conditions as soon as possible is essential. As it is to communicate with the microbiologist, especially when caring for severely-ill patients, immunecompromised hosts or whenever there is a non-usual, specific etiological suspicion.

Dr Paño: Touché, Julio. Speeding the microbiological diagnosis starts out of the Microbiology laboratory, and us, clinicians, nurses and other healthcare professionals are key. But, assuming that all this pre-laboratory process has been optimised, what else can be done to improve the timing of microbiological diagnosis?

Dr García: New technology and more specifically how it is integrated in the Microbiology lab workflow, unquestionably, can help. For instance, we have integrated MALDI-TOF MS into our identification workflow leading to a decrease of 12-48 hours in the identification of bacterial and fungal isolates. MALDI-TOF MS stands for Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry. MALDI-TOF MS can identify bacteria and yeast within minutes directly from colonies grown on culture plates and even from positive blood culture bottles. Applying MALDI-TOF to positive blood culture bottles provides an accurate identification within 30-60 minutes from detected positivity instead of 12-36 hours of the conventional approach. Do you like it?

Dr Paño: Yes, I like it, Julio. But you this technology seems to be expensive.

Dr García: Unquestionably MALDI-TOF MS has significant acquisition costs. There are several companies working on this technology and they offer different marketing approaches for Microbiology Labs to adopt MALDI-TOF MS (purchase, lease…). From my perspective, the benefits of integrating MALDI-TOF to the identification workflow in the Microbiology Lab clearly outweigh its costs. Nevertheless I understand that there is a huge diversity of healthcare settings and the decisions have to be individualised.

Dr Paño: You have not talked about PCR (polymerase-chain reaction) yet. What do you think about it?

Dr García: PCR, and more specifically Multiplex-PCR can significantly speed up microbiological identification. Within a couple hours we can identify microorganisms and even some mechanisms of resistance on direct samples, even before any growth has been observed. Keys to successful implementation of PCR-based testing are the availability of real-time easy-to-operate kits and affordable cost per test to warrant 24/7 availability, as well as integration with antimicrobial stewardship programs. There are other genetic-based approaches apart from PCR (i.e. Peptide Nucleic Acid Fluorescent In Situ Hybridisation).

Dr Paño: To summarise, Julio. When are we expected to have definitive identification and antimicrobial susceptibility results to target Bill´s antimicrobial therapy?

Dr García: Regarding the urine, in 12 hours we will know if there is significant growth and in that case we would identify the microorganisms with MALDI-TOF in 30 additional minutes, approximately. If everything works fine we would need 12-24 hours to get final antimicrobial susceptibility results. For the blood culture, applying MALDI-TOF on the positive blood culture bottle, we might have identification within 30-60 minutes of bottle positivity and setting up direct susceptibility testing on an standardised aliquot of the positive bottle we are expected to have final susceptibility results 12 hours later.

Dr Paño: Thank you very much, Julio. I will come back often to the Microbiology Lab.

Dr García: You are welcome.

In the “see also” section below are a number of videos which explain the tests which Dr Paño and Dr García have been discussing. You may wish to watch these to expand your understanding of what these diagnostic tests can offer.