High-quality clinical microbiology testing requires optimizing activities at all phases of diagnostic testing, which begin at specimen ordering and end with result reporting. Figure 5-1 summarizes this process. Broadly, preanalytic activities are those that involve test ordering and specimen collection; analytic activities involve specimen processing and testing; and postanalytic activities encompass all aspects of test result reporting. We will discuss each category in turn.

One of the most important roles of the clinical microbiology laboratory is to clearly communicate correct specimen collection techniques and transportation requirements for laboratory submission. Incorrect specimen collection and transport can result in inaccurate test results, which may in turn lead to unnecessary labor in the laboratory, suboptimal clinical decisions by providers, and unnecessary patient charges. Best practices for specimen collection can be obtained from the literature and should be summarized in a specimen collection manual that is available hospitalwide.^2,3 Further, to reduce inappropriate and unnecessary testing, microbiology leadership should partner with colleagues in IP, AS, infectious diseases, and other stakeholder groups to develop practice guidance for appropriate indications for ordering microbiology tests.

Once a test has been ordered and a specimen is collected and received by the laboratory, laboratory staff will assess specimen quality to ensure that it meets testing specifications. Clinical microbiology laboratories may cancel test requests for a variety of reasons. Common reasons include the following: aerobic swabs are received for anaerobic cultures; toxigenic Clostridioides difficile testing is requested on formed stool; and stool cultures are requested after 72 hours of hospitalization (because hospital-acquired gastroenteritis due to other bacterial pathogens is exceptionally rare). Further, sputum samples usually should not proceed to culture if a direct Gram stain shows large numbers of epithelial cells (eg, >25 per lowpower field under a light microscope). Finally, most laboratories also cancel test requests on unlabeled or incorrectly labeled specimens and specimens that have been received after excessively prolonged transport.³

If a specimen has been deemed to be appropriate for testing, the analytic activities begin. Cultivation of organisms from patient specimens through bacterial, fungal, and mycobacterial cultures is the bedrock of clinical microbiology services. Most organisms relevant to healthcare-associated infections (HAI; eg, Staphylococcus spp., Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, and Candida spp.) grow easily and quickly on standard bacteriological media with colonies visible after 12-24 hours of incubation. Until recently, most laboratories assigned genus/species identifications to organisms using biochemical reactions that required at least 8-12 hours to complete. Automated biochemical identification instruments including BD Phoenix (BD, Sparks, MD), VITEK 2 (BioMérieux, Durham, NC), and MicroScan WalkAway (Beckman Coulter, Brea, CA) continue to be widely used throughout the world. These systems require preparation of an organism suspension from pure colonies for inoculation of a biochemical card or panel, which is then incubated in the instrument. Biochemical reactions are then read by the instrument and interpreted using proprietary software. In the last decade, however, automated biochemical identification has been increasingly supplanted by matrix-assisted laser desorption/ionization time-of-light mass spectrometry (MALDI-TOF MS), which has emerged as a faster, more accurate, and cost-efficient method of organism identification.⁴
“MALDI” refers to the matrix that assists in the desorption (lifting off of a surface) and ionization of microbial molecules through pulses of energy from a laser. The ionized proteins are directed through a positively charged electrostatic field into a time-of-flight (“TOF”) mass analyzer toward an ion detector with small molecules traveling fastest, followed by progressively larger ones. A mass spectrum is generated, representing the number of ions of a given mass impacting the ion detector over time. The spectrum is compared against those in a database containing spectra of known organisms.⁴ There are two U.S. Food and Drug Administration (FDA)-cleared MALDI-TOF MS systems that are commercially available in the United States: the Bruker Biotyper CA system (Bruker, Billerica, MA), and VITEK MS (BioMérieux). Both systems identify Gram-positive and Gram-negative organisms, anaerobes, and various yeast species within minutes. VITEK MS was also recently FDA-cleared for identification of various Nocardia spp., Mycobacterium spp., and molds.

After organism identification, antimicrobial susceptibility testing (AST) using methods such as disk diffusion, gradient diffusion (eg, E-test), broth dilution, and/or automated methods follows and typically requires an additional 18-24 hours to complete. BD Phoenix, VITEK 2, and MicroScan instruments also perform automated AST in addition to identification, but their performance may vary.^5,6 Laboratory directors must therefore keep current with the literature pertaining to the performance of automated AST systems and be prepared to implement alternative AST methods when there are performance problems reported.

While most HAI-associated organisms are easily cultivated, Mycobacterium spp. and some fungi can require days to weeks to grow in culture. Viral cultures are also labor intensive, insensitive, and slow. For detection of these pathogens, many laboratories have implemented molecular methods such as polymerase chain reaction (PCR) that detect microbial nucleic acids, expedite testing turnaround time, and in some cases improve sensitivity. For example, molecular detection of respiratory viruses underwent early and rapid development. Today, rapid, multiplexed, fully automated respiratory pathogen panels are commercially available and are widely used. They require minimal specimen processing, can be performed on-demand 24/7, and are particularly helpful in the detection of nosocomial viral transmission. Mycobacterium tuberculosis complex has also been considered a logical candidate for rapid molecular diagnostics due to its slow growth in culture. In 2015, the FDA cleared Xpert MTB/RIF (Cepheid, Sunnyvale, CA) for testing of sputum specimens. Rapid diagnosis of MTB can expedite decisions related to application of airborne precautions in patients admitted with possible pulmonary tuberculosis and assist with bed management.

Finally, postanalytic activities focus on ensuring that test reports are accurate and clinically actionable. Reports should provide clear and understandable information about test results and indicate the testing method used (eg, “positive for influenza A by real-time PCR”). Reports may also communicate specimen quality problems (eg, on a urine culture report, “mixed growth suggesting contamination”) and/or clinical recommendations (eg, on a report of a multidrug-resistant organism (MDRO), “this patient should be placed in contact precautions”).

Laboratory personnel should call the IP program directly to report some results to ensure that appropriate control measures are implemented. Examples of organisms requiring immediate notification include Neisseria meningitidis, Bordetella pertussis, Legionella, acid-fast bacilli, and emerging threats such as carbapenem-resistant Enterobacteriaceae (CRE) and Candida auris (see below). In addition, new or unusual pathogens or potential agents of bioterrorism (eg, Bacillus anthracis, Yersinia pestis, orthopoxviruses) should be reported immediately to the IP program. The list of organisms for immediate notification may vary from one institution to another and should be developed in consultation with IP and bioemergency preparedness personnel.

Monitoring of clinical laboratory reports is essential to case finding for HAI surveillance, including those that are subject to public reporting. The Centers for Medicare and Medicaid Services (CMS) mandates the reporting of certain HAIs through the National Healthcare Safety Network (NHSN).⁷ States and counties may also mandate active surveillance for MDROs and/or reporting of certain organisms through legislation or municipal ordinances.

To ensure compliance with these reporting requirements, it is crucial for microbiology laboratories to promptly and accurately report positive test results and ensure that they are transmitted to the IP team. This communication may occur via phone calls, faxes, emails, or electronic surveillance systems that interface directly with the laboratory information system (LIS). Such systems allow IP teams to configure alerts and efficiently monitor laboratory results in real time. The following summarizes laboratory developments that impact HAI detection and surveillance.