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Staphylococcus aureus is also a major pathogen in this infectious disease. Alternatively, organisms may reach

joints during hematogenous spread from distant, infected sites.

 Diagnosis of joint infections requires an aspiration of joint fluid for culture and microscopic examination.

Inoculating the fluid directly into blood culture bottles may prevent the fluid from clotting. Some of the fluid

may be Gram stained and inoculated onto blood as well as chocolate and anaerobic media. The use of AFB

(acid fast bacteria) and fungal media must also be considered.

BONE:

Bone Marrow Aspiration or Biopsy:

Diagnosis of diseases, including brucellosis, histoplasmosis, blastomycosis, tuberculosis, and leishmaniasis, can

sometimes be made by detection of the organisms in the bone marrow. Brucella spp. can be isolated on culture,

as can fungi, but parasitic agents must be visualized in smears or sections made from bone marrow material.

Many of the etiologic agents associated with disseminated infections in patients with human immunodeficiency

virus (HIV) may be visualized or isolated from the bone marrow. Some of these organisms include

cytomegalovirus, Cryptococcus neoformans, and Mycobacterium avium complex.

Bone Biopsy:

A small piece of infected bone is occasionally sent to the microbiology laboratory to identify the etiologic agent

of osteomyelitis (infection of bone). Patients develop osteomyelitis from hematogenous spread of an infectious

agent, invasion of bone tissue from an adjacent site (e.g., joint infection, dental infection), breakdown of tissue

caused by trauma or surgery, or lack of adequate circulation followed by colonization of a skin ulceration with

microorganisms. Once established, infections in bone may progress toward chronicity, particularly if blood

supply is insufficient in the affected area.

Staphylococcus aureus, seeded during bacteremia, is the most common etiologic agent of osteomyelitis among

patients of all age groups. The toxins and enzymes produced by this bacterium, as well as its ability to adhere to

smooth surfaces and produce a protective glycocalyx coating, seem to contribute to the organism’s

pathogenicity. Osteomyelitis in younger patients is often associated with a single agent. Such infections are

usually of hematogenous origin. Other organisms recovered from hematogenously acquired osteomyelitis

include Salmonella spp., Haemophilus spp., Enterobacteriaceae, Pseudomonasspp., Fusobacterium

necrophorum, and yeasts. S. aureus or P. aeruginosa is often recovered from cases in patients with drug

addictions. Parasites or viruses are rarely, if ever, etiologic agents of osteomyelitis. Bone biopsies from

infections that have spread to a bone from a contiguous source or that are associated with poor circulation,

especially in patients with diabetes, are likely to yield multiple isolates. Gram-negative bacilli are increasingly

common among hospitalized patients; a break in the skin (surgery or intravenous line) may precede

establishment of gram-negative osteomyelitis.

Breaks in skin from other causes, such as a bite wound or trauma, also may be the initial event leading to

underlying bone infection. For example, a human bite may lead to infection with Eikenella corrodens, whereas

an animal bite may result in Pasteurella multocida osteomyelitis.

Poor oral hygiene may lead to osteomyelitis of the jaw with Actinomyces spp., Capnocytophaga spp., and other

oral flora, particularly anaerobes. Pigmented Prevotella and Porphyromonas, Fusobacterium, and

Peptostreptococcus spp. are often involved. Pelvic infection in the female may result in a mixed aerobic and

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anaerobic osteomyelitis of the pubic bone. Patients with neuropathy (pathologic changes in the peripheral

nervous system) in the extremities, notably patients with diabetes, who may have poor circulation, may

experience an unrecognized or notable trauma.

They develop ulcers on the feet that do not heal, become infected, and may eventually progress to involve

underlying bone. These infections are usually polymicrobial, involving anaerobic and aerobic bacteria.

Prevotella or Porphyromonas, other gram-negative anaerobes, including the Bacteroides fragilis group,

Peptostreptococcus spp., Staphylococcus aureus, and group A and other streptococci are frequently

encountered.

 Molecular testing, such as polymerase chain reaction, may be useful in determining the infectious organism

associated with the patient’s condition when the laboratory is unable to recover the organism by traditional

culture.

Solid tissues:

Pieces of tissue are removed from patients during surgical or needle biopsy procedures or may be collected at

autopsy. Any agent of infection may cause disease in tissue, and laboratory practices should be adequate to

recover bacteria, fungi, and viruses and detect the presence of parasites. Fastidious organisms (e.g., Brucella

spp.) and agents of chronic disease (e.g., systemic fungi and mycobacteria) may require special media and long

incubation periods for isolation. Some agents requiring special supportive or selective media are listed in Table

(5).

Table( 5 )Infectious Agents in Tissue Requiring Special Media:

Actinomyces spp.

Brucella spp.

Legionella spp.

Bartonella (Rochalimaea) henselae (cat-scratch disease bacilli)

Systemic fungi

Mycoplasma spp.

Mycobacterium spp.

Viruses

Laboratory diagnostic procedures:

Specimen collection and transport:

Requirements for the collection and transport of specimens from sterile body sites vary because of the

numerous types of specimens that can be collected and submitted to the laboratory for testing.

Fluids and Aspirates

Most specimens (pleural, peritoneal, pericardial, and synovial fluids) are collected by aspiration with a needle

and syringe. Collecting pericardial fluid is not without risk to the patient because the sample is collected from

the cavity immediately adjacent to the heart.

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Collection is performed by needle aspiration with electrocardiographic monitoring or as a surgical procedure.

Laboratory personnel should be alerted in advance of the procedure, ensuring that the appropriate media, tissue

culture media, and stain procedures are available immediately. Body fluids from sterile sites should be

transported to the laboratory in a sterile tube or airtight vial. From 1 to 5 mL of specimen is adequate for

isolation of most bacteria, but the larger the specimen, the better, particularly for isolation of M. tuberculosis

and fungi; at least 5 mL should be submitted for recovery of these organisms. Ten milliliters of fluid is

recommended for the diagnosis of peritonitis. Anaerobic transport vials are available from several sources.

These vials are prepared in an oxygenfree atmosphere and are sealed with a rubber septum or short stopper

through which the fluid is injected. Transportation of fluid in a syringe capped with a sterile rubber stopper is

not recommended. Most clinically significant anaerobic bacteria survive adequately in aerobic transport

containers (e.g., sterile, screw-capped tubes) for short periods if the specimen is purulent and of adequate

volume. However, collection in anaerobic transport media is recommended, and procedures vary in different

laboratories. Specimens received in anaerobic transport vials should be inoculated to routine aerobic (an

enriched broth, blood, chocolate, and sometimes MacConkey agar plates) and anaerobic media as quickly as

possible.

Specimens for recovery of fungi or mycobacteria may be transported in sterile, screw-capped tubes. At least 5

to 10 mL of fluid are required for adequate recovery of small numbers of organisms. If gonococci or chlamydia

are suspected, additional aliquots should be sent to the laboratory for smears and appropriate cultures. With

respect to pericardial, pleural, synovial, and peritoneal fluids, the inoculation of blood culture broth bottles at

the bedside or in the laboratory may be beneficial.

An additional specimen should be submitted to the laboratory for a Gram stain. The specimen in the blood

culture bottle is processed as a blood culture, facilitating the recovery of small numbers of organisms and

diluting out the effects of antibiotics. Citrate or sodium polyanetholsulfonate (SPS) may be used as an

anticoagulant.

Specimens collected by percutaneous needle aspiration (paracentesis) or at the time of surgery should be

inoculated into aerobic and anaerobic blood culture bottles immediately at the bedside. Fluid from CAPD

patients can be submitted to the laboratory in a sterile tube, urine cup, or the original bag. The bag is entered

with a sterile needle and syringe to withdraw fluid for culture. Fluid should be directly inoculated into blood

culture bottles (at least 20 mL [10 mL in each of two culture bottles]). Numerous studies indicate that in

addition to blood culture bottles, an adult Isolator tube is a sensitive and specific method for culture.

Bone:

Bone marrow is typically aspirated from the interstitium of the iliac crest. Usually, this material is not

processed for routine bacteria, because blood cultures are equally useful, and false-positive cultures for skin

bacteria (Staphylococcus epidermidis) are frequent. Some laboratories report good recovery from bone marrow

material injected into a pediatric Isolator tube (ISOLATOR 1.5 mL, Alere,

altham, MA) as a collection and transport device. The lytic agents within the Isolator tube are thought to lyse

cellular components, presumably freeing intracellular bacteria for enhanced recovery. Bone removed at surgery

or by percutaneous biopsy is sent to the laboratory in a sterile container.

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Tissue:

Tissue specimens are obtained following careful preparation of the skin. It is critical that biopsy specimens be

collected aseptically and submitted to the microbiology laboratory in a sterile container. A wide-mouthed,

screwcapped bottle or plastic container is recommended.

Anaerobic organisms survive within infected tissue long enough to be recovered from culture. A small amount

of sterile, nonbacteriostatic saline may be added to keep the specimen moist. Because homogenizing with a

tissue grinder can destroy some organisms by the shearing forces generated during grinding, it is often best to

use a sterile scissors and forceps to mince larger tissue specimens into small pieces suitable for culturing.

Note that Legionella spp. may be inhibited by saline; a section of lung should be submitted without saline for

Legionella isolation.

If anaerobic organisms are of concern, a small amount of tissue can be placed into a loosely capped, wide

mouthed plastic tube and sealed into an anaerobic pouch system, which also seals in moisture enough for

survival of organisms in tissue until the specimen is plated. The surgeon should take responsibility for seeing

that a second specimen is submitted to anatomic pathology for histologic studies. Formaldehyde-fixed tissue is

not useful for recovery of viable microorganisms, although some organisms can be recovered after very short

periods. Material from draining sinus tracts should include a portion of the tract’s wall obtained by deep

curettage. Tissue from infective endocarditis should contain a

portion of the valve and vegetation if the patient is undergoing valve replacement. In some instances,

contaminated material may be submitted for microbiologic examination. Specimens, such as tonsils or autopsy

tissue, may be surface cauterized with a heated spatula or blanched by immersing in boiling water for 5 to 10

seconds to reduce surface contamination.

The specimen may then be dissected with sterile instruments to permit culturing of the specimen’s center,

which will not be affected by the heating. Alternatively, larger tissues may be cut in half with sterile scissors or

a blade and the interior portion cultured for microbes.

Because surgical specimens are obtained at great risk and expense to the patient, and because supplementary

specimens cannot be obtained easily, it is important that the laboratory save a portion of the original tissue (if

enough material is available) in a small amount of sterile broth in the refrigerator and at –70° C (or, if

necessary, at –20° C) for at least 4 weeks in case additional studies are indicated. If the entire tissue must be

ground up for culture, a small amount of the suspension should be placed into a sterile tube and refrigerated.

Specimen processing, direct examination, and culture:

Fluids and Aspirates: Techniques for laboratory processing of sterile body fluids are similar except for those

previously discussed that are directly inoculated into blood culture bottles. Clear fluids may be concentrated by

centrifugation or filtration, whereas purulent material can be inoculated directly to media. Anybody fluid

received in the laboratory that is already clotted must be homogenized to release trapped bacteria and minced or

cut to release fungal cells. Either processing such specimens in a motorized tissue homogenizer or grinding

them manually in a mortar and pestle or glass tissue grinder allows better recovery of bacteria. Hand grinding is

often preferred, because motorized grinding can generate considerable heat and thereby kill microorganisms in

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