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the specimen. Grinding may lyse fungal elements; therefore, it is not recommended with specimens processed

for fungi. Small amounts of whole material from a clot should be aseptically cut with a scalpel and placed

directly onto media for isolation of fungi.

All fluids should be processed for direct microscopic examination. In general, if one organism is seen per oil

immersion field, at least 105 organisms per milliliter of specimen are present. In such cases, often only a few

organisms are present in normally sterile body fluids. Therefore, organisms must be concentrated in body

fluids. For microscopic examination, cytocentrifugation should be used to prepare Gram-stained

smears because organisms can be further concentrated up to 1000-fold. Body fluids should be concentrated by

either filtration or high-speed centrifugation. Once the sample is concentrated, the supernatant is aseptically

decanted or aspirated with a sterile pipette, leaving approximately 1 mL liquid in which to thoroughly mix the

sediment. Vigorous vortexing or drawing the sediment up and down into a pipette several times is required to

adequately suspend the sediment. This procedure should be done in a biologic safety cabinet. The suspension

is used to inoculate media. Direct potassium hydroxide (KOH) or calcofluor white preparations for fungi and

acid-fast stain for mycobacteria can also be performed.

 Specimens for fungi should be examined by direct wet preparation or by preparing a separate smear for

periodic acid-Schiff (PAS) staining in addition to Gram stain. Either 10% KOH or calcofluor white is

recommended for visualization of fungal elements from a wet preparation.

In addition to hyphal forms, material from the thoracic cavity may contain spherules of Coccidioides or

budding yeast cells.

Lysis of leukocytes before concentration of CAPD effluents can significantly enhance recovery of organisms.

Filtration of CAPD fluid through a 0.45-mm pore membrane filter allows a greater volume of fluid to be

processed and usually yields better results. Because the numbers of infecting organisms may be low (fewer than

1 organism per 10 mL of fluid), a large quantity of fluid must be processed. Sediment obtained from at least 50

mL of fluid has been recommended. If the specimen is filtered, the filter should be cut aseptically into three

pieces, one of which is placed on chocolate agar for incubation in 5% carbon dioxide, one on MacConkey agar,

and the other on a blood agar plate for anaerobic incubation. If fluids have been concentrated by centrifugation,

the resulting sediment should be inoculated to an enrichment broth, blood, and chocolate agars. Because

these specimens are from normally sterile sites, selective media are inadvisable because they may inhibit the

isolation of anaerobes, mycobacteria, fungi, Chlamydia spp., and viruses should be used when such cultures are

clinically indicated.

Bone: Clotted bone marrow aspirates or biopsies must be homogenized or ground to release trapped

microorganisms. Specimens are inoculated to the same media as for other sterile body fluids. A special medium

for enhancement of growth of Brucella spp. and incubation in 10% carbon dioxide may be needed. A portion of

the specimen may be inoculated directly to fungal media. Sections are also made from biopsy material (bone)

for fixation, staining, and examination (usually by anatomic pathologists) for the presence of mycobacterial,

fungal, or parasitic agents. With respect to obtaining specimens from patients suspected of having

osteomyelitis, cultures taken from open wound sites above infected bone or material taken from a draining

sinus leading to an area of osteomyelitis may not reflect the actual etiologic agent of the underlying

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osteomyelitis. Cultures of samples of bone obtained during wound debridement surgery appear to be more

useful for directing antibiotic therapy for better clinical outcome.

Diagnosis of prosthetic (artificial) joint infections is often difficult. Unfortunately, there is no universally

accepted definition for the diagnosis of infection in the absence of microbiologic evidence because clinical

symptoms such as pain do not differentiate infection from mechanical joint failure. There is no standardized

approach to the laboratory diagnosis of these infections, and published data are conflicting. Further

complicating the diagnosis is that the most common bacteria causing prosthesis infections are common skin

contaminants such as coagulase-negative staphylococci. Some studies have reported that culture is relatively

insensitive, possibly because of the organisms residing in biofilms, whereas polymerase chain reaction (PCR)

assays were able to detect a majority of prosthetic joint infections. Atkins

and colleagues recommended that five or six operative bone specimens be submitted for culture and that the

cutoff for a definite diagnosis of infection be three or more of these specimens yielding the same organism.

However, a recent study using PCR and culture using multiple media types and prolonged incubation found that

appropriate culture was adequate to exclude bacterial infection in hip prostheses and PCR did not enhance

diagnostic sensitivity for infection.

Normal bone is difficult to break up; however, most infected bone is soft and necrotic. Therefore, grinding the

specimen in a mortar and pestle may break off some pieces. Small shavings from the most necrotic-looking

areas of the bone specimen may sometimes be scraped off aseptically and inoculated to media. Pieces should be

placed directly into media for recovery of fungi. Small bits of bone can be ground with sterile broth to form a

suspension for bacteriologic and mycobacterial cultures.

If anaerobes are to be recovered, all manipulations are best performed in an anaerobic chamber. If such an

environment is unavailable, microbiologists should work quickly within a biosafety cabinet to inoculate

prereduced anaerobic plates and broth with material from the bone.

Solid Tissue:Tissue should be manipulated in a laminar flow biologicsafety cabinet. Processing tissue within

an anaerobic chamber is even better. The microbiologist should cut through the infected area (which is often

discolored) with a sterile scalpel blade. Half of the specimen can be used for fungal cultures and the other half

for bacterial cultures. Both types of microbial agents should be considered in all tissue specimens. Some

samples should also be sent to surgical pathology for histologic examination.

 Specimens should be cultured for viruses or acid-fast bacilli when requested. Material that is to be cultured

for parasites should be finely minced or teased before inoculation into broth. Direct examination of stained

tissue for parasites is often performed in the anatomic pathology lab. Imprint cultures of tissues may yield

bacteriologic results identical to homogenates and may help differentiate microbial infection within the tissue’s

center from surface colonization (growth only at the edge).

Additional media can be inoculated for incubation at lower temperatures, which may facilitate recovery of

certain systemic fungi and mycobacteria.

Tissue may also be inoculated to tissue culture cells for isolation of viruses. Brain, lung, spinal fluid, and blood

are generally good specimens for viral isolation. Tissue may be examined by immunofluorescence for the

presence of herpes simplex virus, varicella-zoster virus, cytomegalovirus, or rabies viral particles. Lung tissue

should be examined by direct fluorescent antibody test for Legionella spp.

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The tissues of all fetuses, premature infants, and babies who have died of an infectious process should be

cultured for Listeria. Specimens of the brain, spinal fluid, blood, liver, and spleen are most likely to contain the

organism.

Infections of the Lower Respiratory System:

The respiratory tract can be divided into two major areas: the upper respiratory tract consists of all structures

above the larynx, whereas the lower respiratory tract follows airflow below the larynx through the trachea to the

bronchi and bronchioles and then into the alveolar spaces where gas exchange occurs.

 The respiratory and gastrointestinal tracts are the two major connections between the interior of the body and

the outside environment. The respiratory tract is the pathway through which the body acquires fresh oxygen and

removes unneeded carbon dioxide. It begins with the nasal and oral passages, which humidify inspired air, and

extends past the nasopharynx and oropharynx to the trachea and then into the lungs.

 The trachea divides into bronchi, which subdivide into bronchioles, the smallest branches that terminate in the

alveoli. Some 300 million alveoli are estimated to be present in the lungs; these are the primary microscopic gas

exchange structures of the respiratory tract.

Familiarization with the anatomic structure of the thoracic cavity ensures proper specimen collection from

various sites in the lower respiratory tract for processing by the laboratory. The thoracic cavity, which contains

the heart and lungs, has three partitions separated from one another by pleura.

 The lungs occupy the right and left pleural cavities, whereas the mediastinum (space between the lungs) is occupied

mainly by the esophagus, trachea, large blood vessels, and heart.

Pathogenesis of the respiratory tract:

Basic concepts

Microorganisms primarily cause disease by a limited number of pathogenic mechanisms. Because these

mechanisms relate to respiratory tract infections. Encounters between the human body and microorganisms

occur many times each day. However, establishment of infection after such contact tends to be the exception

rather than the rule.

Whether an organism is successful in establishing an infection depends not only on the organism’s ability to

cause disease (pathogenicity) but also on the human host’s ability to prevent the infection.

Host Factors The human host has several mechanisms that nonspecifically protect the respiratory tract from

infection: the nasal hairs, convoluted passages, and the mucous lining of the nasal turbinates; secretory IgA and

nonspecific antibacterial substances (lysozyme) in respiratory secretions; the cilia and mucous lining of the

trachea; and reflexes such as coughing, sneezing, and swallowing. These mechanisms prevent foreign objects or

organisms from entering the bronchi and gaining access to the

lungs, which remain sterile in the healthy host. Aspiration of minor amounts of oropharyngeal material, as

occurs often during sleep, plays an important role in the pathogenesis of many types of pneumonia. Once

particles escape the mucociliary sweeping activity and enter the alveoli, alveolar macrophages ingest them and

carry them to the lymphatics. In addition to these nonspecific host defenses, normal flora of the nasopharynx

and oropharynx help prevent colonization by pathogenic organisms of the upper respiratory tract.

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Normal bacterial flora prevent the colonization by pathogens by competing for the same space and nutrients as

well as production of bacteriocins and metabolic products that are toxic to invading organisms.

Some of the bacteria that can be isolated as part of the indigenous flora of healthy hosts, as well as many

species that may cause disease under certain circumstances and are often isolated from the respiratory tracts of

healthy persons, are listed in Table(1).

 Under certain circumstances and for unknown reasons, these colonizing organisms can cause disease—

perhaps because of previous damage by a viral infection, loss of some host immunity, or physical damage to the

respiratory epithelium (e.g., from smoking).

 Differentiation of normal flora of the respiratory tract is important for determining the importance of an

isolate in the clinical laboratory. Colonization does not always represent an infection. It is important to

differentiate colonization from infection based on the specimen source, number of organisms present, and

presence or quantity of white blood cells.

(Organisms isolated from normally sterile sites in the respiratory tract by sterile methods that avoid

contamination with normal flora should be definitively identified and reported to the clinician.)

Microorganism Factors:

Organisms possess traits or produce products that promote colonization and subsequent infection in the host.

The virulence, or disease-producing capability of an organism, depends on several factors including adherence,

production of toxins, amount of growth or proliferation, tissue damage, avoiding the host immune response, and

ability to disseminate.

( Table 1) Organisms Present in the Nasopharynx and Oropharynx of Healthy Humans:

Possible Pathogens

Acinetobacter spp.