Sentinel Recognition and Referral of Select Agents

Handle primary patient specimens with biosafety level 2 (BSL-2) practices, because the laboratory usually has no warning. As soon as one of these organisms is suspected, all further work moves into a certified Class II biological safety cabinet under biosafety level 3 (BSL-3) practices: restricted room access, a solid front gown, gloves, eye and face protection, previously fit-tested respiratory protection, no open-vessel work on the bench, and taped plates. Where a BSL-3 laboratory is available, the work belongs there. Where no BSL-3 laboratory is available, BSL-3 practices inside a BSL-2 facility are the accepted minimum until the isolate is referred.^{1,21 Association of Public Health Laboratories. Recognize. 2018.2 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2024.}

The hazard is aerosol, and several routine bench steps generate one. Opening a plate, smelling a plate, heat fixing a slide, dispensing pipette tips, vortexing, centrifuging, subculturing a positive blood culture bottle, inoculating media, and even the catalase test all impart energy to a microbial suspension.^{11 Association of Public Health Laboratories. Recognize. 2018.} Replace the routine version of each step once one of these organisms is in the differential.

Do not use wet mount or hanging drop motility preparations. Semisolid medium without triphenyltetrazolium chloride gives a less subjective reading and greatly reduces the aerosol risk, so keep motility medium in stock for this purpose even when routine work no longer uses it.^{1,21 Association of Public Health Laboratories. Recognize. 2018.2 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2024.}

Automated and manual identification systems, including MALDI-TOF mass spectrometry, must not be used on a suspected isolate. Preparing the concentrated suspension these systems need is itself an aerosol-generating step, and the databases misidentify these organisms: Bacillus anthracis read as B. cereus, Yersinia pestis read as Y. pseudotuberculosis, Brucella read as Moraxella or a slow-growing Staphylococcus, Francisella tularensis read as Haemophilus influenzae.^{1,21 Association of Public Health Laboratories. Recognize. 2018.2 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2024.} When a system does report one of these organisms, or reports a result such as "Bacillus cereus group" that contains one of them, the result does not rule the organism out. Secure the isolate and begin the conventional rule-out and referral process.^{2,32 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2024.3 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

Fresh 10% household bleach, made daily and supplying roughly 5,000 ppm free available chlorine, decontaminates laboratory surfaces. Vegetative cells die within seconds. Spores need about 5 minutes of wet contact time. Organic material such as blood inactivates bleach, so remove visible soil first. Phenolics are not sporicidal at their usual working dilutions and cannot substitute where spores are a concern.^{22 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2024.}

Bacillus anthracis is a large, aerobic, spore-forming, nonmotile gram-positive rod, 1 to 1.5 by 3 to 5 µm, with squared ends. Its glutamyl polypeptide capsule blocks phagocytosis, and a tripartite toxin of protective antigen, edema factor, and lethal factor produces the clinical picture. Cell wall peptidoglycan drives a severe intravascular inflammatory response that can progress to shock.

Gram stain appearance depends on the source. Direct smears of clinical material show short chains of 2 to 4 encapsulated cells. Smears from colonies show long chains without capsule, and older cultures decolorize readily enough to look gram-negative. Spores are oval, central to subterminal, and do not swell the cell. Spores are rare in direct smears of clinical specimens. Finding spores in a direct smear suggests the specimen was delayed in transit.^{22 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2024.}

Current sentinel guidance calls the target of this rule-out the brucellosis-causing Brucella species, or BBS: B. melitensis (goats, sheep, camels), B. abortus (cattle), B. suis (swine), and B. canis (dogs). The narrower term exists because the Ochrobactrum genus was reclassified into Brucella in 2020, so the genus now contains both these pathogens and roughly 18 environmental species that are opportunists at most. An automated system reporting "Brucella" may therefore mean either one, so the rule-out must be completed before any identification or susceptibility result is released.^{33 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

HHS and USDA removed B. abortus, B. melitensis, and B. suis from the select agent list in a final rule published December 17, 2024 and effective January 16, 2025.^{55 Possession, et al. 89 Fed Reg 101941 (December 17, 2024) (effective January 16… 2024.} Delisting changes the biosecurity obligations, meaning the registration documentation, federal forms, and destruction deadlines. Biosafety handling does not change. BBS remains highly infectious and nationally notifiable, and it requires rule-out in the sentinel laboratory and referral to the LRN reference laboratory for definitive identification.^{33 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

These are small, nonmotile, aerobic, non-spore-forming gram-negative coccobacilli, classically 0.4 by 0.8 µm and faintly staining, seen singly and not in clusters. They may also stain gram-variable or frankly gram-positive when the cells retain crystal violet. A tiny coccobacillus on a smear from a sterile site is reason to begin containment. Because heat fixing aerosolizes, fix smears inside the cabinet and only then carry them out for staining and reading. As intracellular pathogens of macrophages and neutrophils, brucellae evade phagosomal killing and spread through the reticuloendothelial system, forming granulomas in liver, spleen, bone marrow, genitourinary tract, and lung. Acquisition is by inhalation, direct contact with infected animal material, or unpasteurized dairy products, with imported unpasteurized goat cheese an ongoing route in the United States. The incubation period averages 2 to 10 weeks and ranges from a few days to 6 months, and the illness is a nonspecific febrile syndrome, acute or insidious in onset, classically undulant. Brucellosis is among the most frequently reported laboratory-acquired infections, so the handling rules are strict.^{33 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

Recovery from blood is delayed relative to other bloodstream pathogens, peaking at 3 to 4 days compared with 6 to 36 hours for most organisms. Automated blood culture bottles are incubated for 10 days, and a blind or terminal subculture at 7 or 10 days increases yield when brucellosis is suspected. Keep plates inoculated directly with patient specimen for 7 days. MacConkey plates need only 3 days. Growth is dust-like overnight, pinpoint at 24 hours, and 0.5 to 1 mm by 48 hours. On blood agar the colonies are raised and convex, white, non-pigmented, non-hemolytic, non-mucoid, with an entire edge and shiny surface, and they are odorless. Do not smell plates. Growth occurs on blood and chocolate agar. Growth on MacConkey is absent, apart from occasional pinpoint colonies after incubation as long as 7 days, and nothing grows under anaerobic conditions.^{33 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

Rule-out uses three steps after the Gram stain. The organism grows on blood agar without needing to satellite around a Staphylococcus aureus ATCC 25923 streak, which separates it from Haemophilus. It is oxidase-positive and catalase-positive, though B. canis can be oxidase-variable. A tiny gram-negative coccobacillus that is oxidase-negative and catalase-negative is consistent with Francisella and is worked through the tularemia rule-out. It is urease-positive, sometimes within 5 minutes and sometimes only after 72 hours on Christensen's agar. An isolate meeting all of these cannot be ruled out and is referred. Any one of three findings rules BBS out: colony growth on MacConkey larger than 0.5 mm at 24 hours, mucoid colony morphology, or positive motility by a tube-based method.^{33 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

Species-level characterization by CO₂ requirement, oxidative metabolic profile, urease kinetics, dye sensitivity, and phage lysis is a reference laboratory function and must not be attempted on the bench, nor with MALDI-TOF or an automated system.^{33 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

Serology supplements culture, particularly in chronic or complicated disease. A titer of at least 1:160 by standard tube agglutination or Brucella microagglutination supports a presumptive diagnosis, and a fourfold rise between acute and convalescent sera collected at least 2 weeks apart is confirmatory. Brucella IgG detected by ELISA in a specimen drawn at least 2 weeks after onset is supporting evidence only, and enzyme immunoassay results must be confirmed by microagglutination or tube agglutination before the titer is interpreted. False positives arise from antibodies cross-reacting with other gram-negative bacilli, most often when IgM is measured or a non-reference method is used.^{33 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

F. tularensis is a strictly aerobic, fastidious, cysteine-requiring gram-negative coccobacillus and an HHS Tier 1 select agent. Its infectious dose is under 10 organisms and it transmits by aerosol, so it is both a bioterrorism concern and an occupational hazard in clinical microbiology.^{4,64 Federal Select Agent Program. Select agents and toxins list. 2025.6 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

Small rodents, hares, and voles are the primary reservoirs, with ticks, mosquitoes, and biting flies as vectors. Humans acquire infection most often through arthropod bite or direct contact with infected animals, and less commonly from contaminated soil, water, infected carcasses, or aerosolized particles. Incubation averages 3 to 5 days with a range of 1 to 21 days. About 80% of cases are ulceroglandular. The remaining cases present as glandular disease without a lesion, oculoglandular, oropharyngeal, typhoidal without lymphadenopathy, or pneumonic, the most severe form and the one an aerosol release would produce.^{66 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

On the Gram stain the cells are tiny, 0.2 to 0.5 by 0.7 to 1.0 µm, pleomorphic, mostly single, and counterstain so poorly with safranin that interpretation is difficult. Substituting basic fuchsin as the counterstain improves resolution, and acridine orange also helps. The organism is smaller than H. influenzae. It does not show bipolar staining.^{66 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

The cysteine requirement accounts for the culture picture and is the most useful rule-out clue. F. tularensis may grow on sheep blood agar on primary culture and fail to grow on subculture. Growth on blood agar is scant to absent, and growth on cysteine-supplemented media proceeds. Chocolate, Thayer-Martin, buffered charcoal yeast extract, and cysteine heart agar all support it. Colonies are usually invisible at 24 hours and reach 1 to 2 mm by 48 hours or later, appearing gray-white to bluish-gray, opaque, flat, smooth and shiny with an entire edge. There is no growth on MacConkey or eosin methylene blue agar.^{66 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

Rule-out combines that growth pattern with four reactions performed in the cabinet: satellite test negative, oxidase negative, catalase negative or only weakly positive, and beta-lactamase positive. Those tests address the two most common misidentifications. H. influenzae satellites and is X and V factor dependent. Aggregatibacter species are beta-lactamase negative. An isolate that is a faintly staining tiny gram-negative coccobacillus with scant to no growth on blood agar, gray-white growth on chocolate at 48 hours, no growth on MacConkey, no satellite growth, oxidase negative, catalase weak or negative, and beta-lactamase positive cannot be ruled out and is referred.^{66 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

Interpret serology against the clinical picture. A single titer does not confirm tularemia. Seroconversion between paired sera, ideally drawn in the first week of illness and again 2 to 3 weeks later, is confirmatory. A single positive serologic test is supporting evidence. Antibodies are often undetectable until 2 to 3 weeks after onset, so a negative early result does not exclude tularemia, and both IgM and IgG can persist for years after recovery. F. tularensis serology cross-reacts with Brucella antibodies.^{77 Centers for Disease Control and Prevention. Clinical testing and diagnosis for tularemia. 2024.}

Every Francisella isolate examined to date has been beta-lactamase positive, so penicillins and cephalosporins are ineffective and are not used to treat tularemia. Acquired resistance is rare. A CLSI reference broth microdilution method with susceptible-only breakpoints exists, but the testing is performed by a reference laboratory equipped for higher containment.^{66 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

Yersinia pestis is a nonmotile, slow-growing, facultatively anaerobic gram-negative organism in the order Enterobacterales, the agent of plague, and an HHS Tier 1 select agent. Direct smears show plump rods, 0.5 by 1 to 2 µm, mostly single or in pairs, with short chains in liquid media. Transmission is by infected flea bite, direct contact with contaminated tissue, or inhalation, with an incubation period of 2 to 10 days, and the disease presents in bubonic, septicemic, and pneumonic forms. Primary pneumonic plague is the form an aerosol release would produce, and it is the one form that transmits readily between people.^{4,84 Federal Select Agent Program. Select agents and toxins list. 2025.8 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

The bipolar, "safety pin" appearance is a Wright or Giemsa finding on peripheral blood or tissue, and the Gram stain frequently does not show it. Hematology or histopathology performs those stains. A bipolar staining rod noticed on a peripheral smear in a septic patient is a reason for hematology to call microbiology, so the specimen is handled under appropriate containment. Do not prepare additional smears from suspect material.^{88 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

On sheep blood agar at 35 °C, growth is pinpoint or absent at 24 hours. By 48 hours the colonies are 1 to 2 mm, gray-white to slightly yellow, translucent to opaque, with little or no hemolysis. At about 96 hours and under magnification, older cultures take on a fried-egg or hammered-copper appearance.^{11 Association of Public Health Laboratories. Recognize. 2018.} Growth on MacConkey or eosin methylene blue agar is slow and non-lactose-fermenting and may be invisible at 24 hours. Growth is optimal at 25 to 28 °C and slower at 35 to 37 °C. Keep plates 5 days, or 7 if the patient received antimicrobials before collection.^{88 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

An isolate from blood, sputum, or lymph node aspirate showing that morphology and colony pattern, and reacting catalase-positive with oxidase, indole, and urease negative, cannot be ruled out as Y. pestis. Urease read at 35 °C is the reaction that separates it from Y. pseudotuberculosis and Y. enterocolitica, both urease-positive. Y. pseudotuberculosis and Y. enterocolitica give stronger urease reactions at 25 to 28 °C, though that temperature is not required to demonstrate the enzyme. If the rule-out criteria are not met, identification proceeds by established methods for the other Yersinia species.^{88 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

When an isolate cannot be ruled out, the laboratory reports to the physician that the organism cannot be excluded and that the isolate has been referred, using wording such as "Possible Brucella species submitted to LRN Reference Level Laboratory for confirmatory testing." The isolate is not identified further and is not tested for susceptibility.^{1,31 Association of Public Health Laboratories. Recognize. 2018.3 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2025.}

Notification goes to several places at once. Telephone the designated LRN reference laboratory immediately. It directs what to retain and what to send. Public health authorities are notified according to state and local communicable disease reporting requirements, and the public health laboratory or health department is the body that notifies state and federal law enforcement when that is warranted. Inside the hospital, infection prevention and the infectious disease service are notified so the patient is treated appropriately, precautions are applied, and the exposure history is investigated.^{6,86 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.8 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

Preserve original specimens in case a criminal investigation follows. Document the blood culture bottles, tubes, plates, and original clinical material, and either ship them or keep them until the reference laboratory confirms the identification. Secure all remaining material against unauthorized access in the meantime.^{1,81 Association of Public Health Laboratories. Recognize. 2018.8 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

Ship nothing without prior arrangement with the receiving laboratory. Isolates and specimens of these agents travel as Category A infectious substances under a triple packaging system, and the packaging and shipping must comply with International Air Transport Association and Department of Transportation regulations. A sentinel laboratory meets this requirement by keeping enough staff currently trained and certified for Category A and Category B shipping. Shipping by an untrained staff member is a regulatory violation and a containment failure.^{1,91 Association of Public Health Laboratories. Recognize. 2018.9 American Society for Microbiology, et al. Sentinel level clinical laboratory guidelines for suspected… 2021.}

Federal select agent obligations apply in addition to the clinical reporting. B. anthracis, B. cereus biovar anthracis, F. tularensis, and Y. pestis are Tier 1 select agents. The brucellosis-causing Brucella species are not select agents.^{4,54 Federal Select Agent Program. Select agents and toxins list. 2025.5 Possession, et al. 89 Fed Reg 101941 (December 17, 2024) (effective January 16… 2024.} A sentinel laboratory does not have to register with the Federal Select Agent Program to perform diagnostic testing, including on Tier 1 agents, provided it follows the reporting and disposition rules when it cannot rule an agent out. Identification of a select agent is reported on APHIS/CDC Form 4. Transfer of the agent uses Form 2. Theft, loss, or release uses Form 3. Residual specimens and cultures of a confirmed select agent must be destroyed or transferred to a registered entity within 7 days of the confirmed identification, and the reference laboratory advises on which route to take. Destruction is by autoclaving or on-site incineration, or by submission to the reference laboratory when the laboratory cannot destroy the material adequately.^{6,86 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.8 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}

Exposure response is a separate obligation. The exposure happens before anyone suspects the organism, so it is easy to overlook. Evaluate every potential exposure immediately, and document who worked with the material and who else was present. Risk classification of exposed staff is determined with the state public health laboratory and epidemiologist. Collect and submit serologic specimens when that assessment indicates it. Decisions about post-exposure prophylaxis and occupational vaccination belong to occupational health and the infectious disease service, informed by the reference laboratory and the health department. These decisions are not made at the laboratory bench and are not made by the reference laboratory alone. When you cannot exclude one of these organisms, stop testing, contain the isolate, and refer it to the reference laboratory.^{1,61 Association of Public Health Laboratories. Recognize. 2018.6 American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected… 2016.}