. 2013 Aug 15;57(4):e22–e121. doi: 10.1093/cid/cit278

Table IX-1.

Laboratory Diagnosis of Osteomyelitis

Etiologic Agents	Diagnostic Procedures	Optimum Specimens	Transport Issues; Optimal Transport Time
Hematogenous Seeding of Bone
Staphylococus aureus	Gram stain	Bone biopsy	Sterile container, RT, immediately
Salmonella spp^a	Aerobic bacterial culture
Streptococcus pneumoniae^b
Brucella spp^c
Pseudomonas spp^d
Mycobacterium tuberculosis^e	Acid fast smear	Bone biopsy	Sterile container, RT, 2 h
	AFB culture
	M. tuberculosis NAAT^e
Blastomyces dermatitidis	Calcofluor-KOH stain	Bone biopsy	Sterile container, RT, 2 h
Coccidioides immitis	Fungus culture	Bone biopsy	Sterile container, RT, 2 h
Extension from a Contiguous Skin or Soft Tissue Site of Infection
Staphylococcus aureus	Gram stain	Bone biopsy	Sterile container, RT, immediately
Other bacteria^f	Aerobic bacterial culture	Bone biopsy	Sterile container, RT, immediately
Mixed aerobic and anaerobic bacterial flora of the oral cavity including Actinomyces spp^g	Gram stain	Bone biopsy	Sterile anaerobic transport container
	Aerobic and anaerobic bacterial culture	Bone biopsy	RT, immediately
Mixed bacterial flora in diabetic patients with skin and soft tissue extremity infections^h	Gram stain	Bone biopsy	Sterile anaerobic transport container, RT, immediately
	Aerobic and anaerobic bacterial culture	Bone biopsy	Sterile anaerobic transport container, RT, immediately
Nocardia spp, other aerobic actinomycetes and soil filamentous fungi in patients with mycetomaⁱ	Gram stain	Bone biopsy or sinus tract specimen (curetting or tissue biopsy)	Sterile container, RT, immediately
	Aerobic bacterial culture		Sterile container, RT, immediately
	Silver stain	Bone biopsy or sinus tract specimen	Sterile container, RT, 2 h
	Calcofluor-KOH stain
	Buffered charcoal yeast extract (BCYE) agar for Nocardia
	Fungus culture
Traumatic Inoculation
Staphylococcus aureus	Gram stain	Bone biopsy	Sterile anaerobic transport container, RT, immediately
Enterobacteriaceae	Aerobic and anaerobic bacterial culture
Pseudomonas aeruginosa^j
Bacterial flora of the skin
Bacteria found in the environment^k
Nontuberculous mycobacteria	Acid fast smear	Bone biopsy	Sterile container, RT, 2 h
Nontuberculous mycobacteria	AFB culture	Bone biopsy	Sterile container, RT, 2 h
Environmental moulds	Calcofluor-KOH stain	Bone biopsy or sinus tract specimen (curetting or tissue biopsy)	Sterile container, RT, 2 h
Environmental moulds	Fungus culture		Sterile container, RT, 2 h

Abbreviations: AFB, acid-fast bacillus; KOH, potassium hydroxide; NAAT, nucleic acid amplification test; RT, room temperature.

^a Salmonella osteomyelitis occurs most often in patients with sickle cell trait or disease [137].

^b Streptococcus pneumoniae as a cause of osteomyelitis occurs most often in pediatric patients, not infrequently in the setting of spontaneous pneumococcal bacteremia [138].

^c Brucella spp will be recovered in standard aerobic bacterial cultures, however, it is a slow growing bacterium and as a result, the laboratory should be notified when Brucella is considered to be a potential cause of osteomyelitis so that cultures can be held for examination over at least a one-week period and examined only in a biological safety cabinet. Concomitant blood cultures and serology testing are recommended (not necessary to hold blood cultures beyond standard incubation).

^d Hematogenous osteomyelitis caused by Pseudomonas aeruginosa and other Pseudomonas spp occurs most often in injection drug users [139].

^e The most common site of osteomyelitis due to M. tuberculosis is the vertebral bodies. This organism can also seed the clavicles and in this setting represents one of the most common causes of clavicular osteomyelitis. Commercial NAATs are not FDA-cleared for nonrespiratory sites, so a laboratory-validated test method must be used if NAATs are requested.

^f Infections of skin and soft tissues, especially with extension of infection into deeper tissue spaces, pose a risk for the development of osteomyelitis of adjacent bone. While S. aureus is the most commonly incriminated organism, essentially any bacterium capable of causing deep soft tissue infection can also cause osteomyelitis.

^g Chronic endodontic infections such as apical abscesses may extend into surrounding bone resulting in osteomyelitis of the maxilla or mandible. These infections are caused by the aerobic and anaerobic bacterial flora of the oral cavity and may be either monomicrobic or polymicrobic. Actinomyces spp is a recognized pathogen in this setting. When Actinomyces is suspected, specimens should be transported to the laboratory and then processed within 15 minutes or there is little chance of recovering Actinomyces in culture.

^h Diabetic extremity infections with underlying osteomyelitis can be caused by a diverse group of bacteria including S. aureus, Group B β-hemolytic streptococci, Enterococcus spp, the Enterobacteriaceae, Pseudomonas spp, Stenotrophomonas maltophilia and a variety of anaerobes. This represents one of the few settings in which osteomyelitis can be polymicrobial. Superficial debridement followed by deep sampling at the advancing margin of the lesion is essential to avoid being misled by surface colonizing contaminants.

ⁱ Mycetoma is a chronic soft tissue infection of the extremities which can also extend into contiguous bone and connective tissue. It occurs most often in tropical and subtropical climates and may be characterized by the development of draining sinuses. The etiologic agents (see table) are derived from the soil. Sinus tract drainage material, when present, may be representative of the etiology of underlying osteomyelitis. In addition to the stains and cultures noted in the table, sinus drainage should also be examined grossly and microscopically for the presence of “sulfur granules” characteristic of this disease. Further, the laboratory should be notified of the possibility of Nocardia as a pathogen so that appropriate media, eg Neisseria selective media and Legionella selective agar, can be inoculated which facilitate recovery of this organism.

^j Pseudomonas aeruginosa is the most common bacterial cause of calcaneal osteomyelitis in individuals who develop this infection after stepping on nails while wearing sneakers.

^k Direct trauma to bone such as may occur in open fractures with contamination of the site by soil, animal feces, water, etc, may lead to the development of osteomyelitis due to essentially any microorganism present in the contamination source. This includes the Enterobacteriaceae, Pseudomonas aeruginosa, unusual gram-negative bacilli, Bacillus spp, anaerobes such as Clostridium spp, Nocardia and other aerobic actinomycetes. This represents another form of osteomyelitis that can be polymicrobial.