(Study type: Blood collected in bottles containing standard aerobic and anaerobic culture media, sterile body fluid [such as amniotic, cerebrospinal fluid, pericardial fluid, peritoneal fluid, pleural fluid, synovial], sputum, stool, throat/nasopharynx, urine or swab from affected area placed in transport media tube provided by laboratory. Stool sample should be a random, freshly collected specimen submitted in a clean plastic container. Urine should be collected in a sterile plastic collection container; transport tubes containing a preservative are highly recommended if urine testing will not occur within 2 hr of collection. Primary specimens such as blood, body fluids, tissue, stool, or urine (when available) are preferred over specimens collected on swabs as they will best reflect the pathological process affecting the site of interest; for this reason, specimens collected in the operating room should be submitted on primary specimens rather than on swabs whenever possible; related body system: Circulatory, digestive, immune, integumentary, nervous, reproductive, respiratory, and urinary systems.)

Optimally, specimens should be obtained before antibiotic use. The method used to culture and grow the organism depends on the suspected infectious organism. There are transport media specifically for bacterial organisms. The laboratory will select the appropriate media for suspect organisms and will initiate antibiotic sensitivity testing if indicated by test results. Sensitivity testing identifies the antibiotics to which organisms are susceptible to ensure an effective treatment plan.

The testing laboratory should be consulted for specific collection and transport instructions if molecular test methods are requested. The advent of liquid-based specimen collection has quickly led to automated specimen processing, monitoring of specimen growth, and reading of liquid cultures for most specimen types. Automated chromogenic artificial intelligence (AI) algorithms are now being developed to report results (growth or no growth) with 100% sensitivity such that no culture would be reported as negative by AI that would have been reported as positive by traditional manual reading.

Molecular technologies and mass spectrometry are other approaches to testing various specimen types; there are now numerous applications in the areas of pathogen identification, disease identification, and treatment development. Next-generation sequencing (NGS) is a category of molecular testing used to sequence DNA fragments. NGS technology is capable of sequencing DNA fragments from pathogenic organisms across a range in size from small sequences (single-nucleotide polymorphisms) to large sequences (thousands or billions).

The indications for NGS have expanded significantly, and it is now an important tool in the diagnosis/identification of pathogenic organisms. NGS also provides a means and/or the potential to perform prognostic epidemiological studies, lineage tracing (cell lines), discovery of genetic variants with drug resistance sequence variations, development of individualized therapeutic regimens, and development of preventive measures (e.g., vaccines). Multiplex assays are another type of molecular testing designed to simultaneously sequence DNA fragments from multiple pathogens to help identify mixed populations of infectious agents.

Vaccine-preventable bacterial diseases include diphtheria, Haemophilus. influenza, and pneumococcal pneumonia.

Anal and Genital Cultures

When indicated by patient history, anal and genital cultures may be performed to isolate the organism responsible for sexually transmitted infections (STIs). Chlamydia, gonorrhea, and syphilis are reportable STIs. Chlamydia is an intracellular obligate pathogen. Culture of infected epithelial cells is considered the gold standard for the identification of chlamydia because of the higher sensitivity of nucleic acid amplification or DNA probe assays relative to antibody assays. Therefore, culture should always be the test of choice in cases of suspected or known child abuse.

The CDC, American Academy of Family Physicians, American Academy of Pediatrics, American College of Nurse-Midwives, and American College of Obstetricians and Gynecologists recommend universal screening for all pregnant patients at 35 to 37 wk’ gestation to identify the presence of group B streptococcus (GBS), a significant and serious neonatal infection transmitted as the newborn passes through the birth canal of colonized patients.

GBS by NAAT can provide accurate results faster than traditional culture methods. Swabs are collected first from the lower vagina and then from the rectum. The laboratory should be consulted prior to specimen collection for proper instructions regarding transport media or other acceptable containers.

Anal and genital cultures may be performed on pregnant patients to identify the presence of GBS. Neonatal GBS is the most common cause of sepsis, pneumonia, and meningitis in newborns. The disease is classified as either early onset (first week of life) or late onset (after the first week of life).

Anal and genital cultures may also be performed on patients of any age if sexual abuse is suspected.

Blood Cultures

Pathogens can enter the bloodstream from soft-tissue infection sites, contaminated IV lines, or invasive procedures (e.g., surgery, tooth extraction, cystoscopy). Blood cultures are collected whenever bacteremia (bacterial infection of the blood) or septicemia (a condition of systemic infection caused by pathogenic organisms or their toxins) is suspected.

Although mild bacteremia is found in many infectious diseases, a persistent, continuous, or recurrent bacteremia indicates a more serious condition that may require immediate treatment. Early detection of pathogens in the blood may aid in making clinical and etiological diagnoses.

Blood cultures can detect the presence of bacteria and fungi. Organisms can be classified in a number of ways; blood culture findings use oxygen requirements to categorize findings into one of two groups. Blood culture begins with the introduction of a blood specimen into two types of culture medium. The medium is designed to promote the growth of organisms; one group of organisms requires oxygen (aerobic), and the other requires either sparing amounts to no oxygen at all (anaerobic). A blood culture may also be done with an antimicrobial removal device (ARD) if antibiotic therapy is initiated prior to specimen collection. This involves transferring some of the blood sample into a special vial containing absorbent resins that remove antibiotics from the sample before the culture is performed.

Traditional automated culture methods entail incubation of inoculated culture containers for a specific length of time, at a specific temperature, and under other conditions suitable for growth. If organisms are present, they will produce carbon dioxide as they metabolize the nutrients in the culture media. The presence of carbon dioxide in the culture is detected when the culture bottles are “read” by an instrument at specified intervals over a period of time.

There are a number of automated blood culture systems with sophisticated computerized algorithms. The complex software allows for frequent monitoring of growth throughout the day and rapid interpretation of culture findings. With these systems, as soon as a positive culture is detected, usually within 24 to 72 hr, the bottle can be removed from the system and a Gram stain performed to provide a preliminary identification of the bacteria present. This preliminary report provides an opportunity for the HCP to initiate therapy.

A sample from the positive blood culture bottle is then subcultured on the appropriate plated media for growth, isolation, and positive identification of the organism. The plated organisms are also used for sensitivity testing, if indicated. Sensitivity testing identifies the antibiotics to which the organisms are susceptible to ensure an effective treatment plan and can take several days. Negative cultures are generally removed from the automated culture system after 5 days and finalized as having “no growth.”

The subspecialty of microbiology has been revolutionized by molecular diagnostics. Molecular diagnostics involves the identification of specific sequences of DNA. The application of molecular diagnostics techniques, such as PCR, has led to the development of automated instruments that can identify a single infectious organism or multiple pathogens from a small amount of blood in less than 2 hr. The instruments can detect the presence of gram-negative bacteria, gram-positive bacteria, and yeast commonly associated with bloodstream infections. The instruments can also detect sequence variations in the genetic material of specific pathogens that code for antibiotic resistance.

Ear and Eye Cultures

Ear and eye cultures are performed to isolate the organism responsible for chronic or acute infectious disease of the ear and eye.

Skin and Soft Tissue Cultures

Skin and soft tissue samples from infected sites must be collected carefully to avoid contamination from the surrounding normal skin flora. Skin and tissue infections may be caused by both aerobic and anaerobic organisms. Therefore, a portion of the sample should be placed in aerobic and a portion in anaerobic transport media. Care must be taken to use transport media that are approved by the laboratory performing the testing.

Sputum Cultures

This test involves collecting a sputum specimen so the pathogen can be isolated and identified. The test results will reflect the type and number of organisms present in the specimen as well as the antibiotics to which the identified pathogenic organisms are susceptible. Sputum collected by expectoration or suctioning with catheters and by bronchoscopy cannot be cultured for anaerobic organisms; instead, transtracheal aspiration or lung biopsy must be used.

Sterile Fluid Cultures

Sterile fluids can be collected from the affected site. Refer to related body fluid studies (i.e., amniotic fluid, cerebrospinal fluid, pericardial fluid, peritoneal fluid, pleural fluid, synovial fluid) for specimen collection.

Stool Cultures

Stool culture involves collecting a sample of feces so that organisms present can be isolated and identified. Certain bacteria are normally found in feces. However, when overgrowth of these organisms occurs or pathological organisms are present, diarrhea or other signs and symptoms of systemic infection occur. These symptoms are the result of damage to the intestinal tissue by the pathogenic organisms.

Because there are numerous pathogenic organisms that can cause disease, routine stool culture normally screens for a small number of common pathogens associated with infections of the digestive system (e.g., food poisoning), such as Aeromonas,Bacteroides spp, Campylobacter,S. aureus,Salmonella,Shigella, and Yersinia.

Identification of other bacteria is initiated by special request or upon consultation with a microbiologist when there is knowledge of special circumstances. An example of this situation is an outbreak of Clostridioides. difficile in a long-term care facility or hospital unit where the infection can spread rapidly from one person to the next. A life-threatening C. difficile infection of the bowel may occur in patients who are immunocompromised or are receiving broad-spectrum antibiotic therapy (e.g., clindamycin, ampicillin, cephalosporins). The bacteria release a toxin that causes necrosis of the colon tissue. The toxin can be more rapidly identified from a stool sample using an immunochemical method than from a routine culture. Appropriate interventions can be quickly initiated and might include IV replacement of fluid and electrolytes, cessation of broad-spectrum antibiotic administration, and institution of vancomycin or metronidazole antibiotic therapy.

The American Society for Clinical Pathology and the American Academy of Family Physicians recommend against routine stool testing for community-acquired gastrointestinal (GI) pathogens in patients who are hospitalized and develop diarrhea after the third day of hospitalization. The rationale is that the test methods in use have been developed to identify pathogens commonly associated with community-acquired GI infections, which for the most part are self-limited upon appropriate treatment; treatment is focused on preventing and reversing dehydration.

Exceptions may include:

• Testing for C. difficile in patients over 2 yr of age if they develop diarrhea after the third day of hospitalization
• Testing for older adults and immunocompromised patients who may still harbor community-acquired pathogens and develop diarrhea after the third day of hospitalization

The laboratory will initiate antibiotic sensitivity testing if indicated by test results. Sensitivity testing identifies the antibiotics to which organisms are susceptible to ensure an effective treatment plan.

The subspecialty of microbiology has been revolutionized by molecular diagnostics. Molecular diagnostics involves the identification of specific sequences of DNA. The application of molecular diagnostics techniques, such as PCR, has led to the development of automated instruments that can identify a single infectious agent or multiple pathogens from a small amount of stool in less than 2 hr. The instruments can detect the presence of bacteria, viruses, or protozoans commonly associated with GI infections. Additional information about identification of pathogens in stool specimens (e.g., Rotavirus, C. difficile) can be found in the study titled “Fecal Analysis.”

Throat/Nasopharyngeal Cultures

There are a number of bacterial organisms responsible for pharyngitis. The routine throat culture is a commonly ordered test to screen for the presence of group A beta-hemolytic streptococci. Streptococcus pyogenes is the gram-positive organism that most commonly causes acute pharyngitis. The more dangerous sequelae of scarlet fever, rheumatic heart disease, and glomerulonephritis are less frequently seen because of the early treatment of infection at the pharyngitis stage.

Specific cultures can be set up to detect other pathogens, such as Bordetella (gram negative), Corynebacteria (gram positive), Haemophilus (gram negative), or Neisseria (gram negative) if they are suspected or by special request from the HCP. C. diphtheriae is the causative pathogen of diphtheria. N. gonorrhoeae is a sexually transmitted pathogen. In children, a positive throat culture for Neisseria usually indicates sexual abuse.

Urine Cultures

A urine culture involves collecting a urine specimen so that the organism causing disease can be isolated and identified. Urine can be collected by clean catch, urinary catheterization, or suprapubic aspiration. The severity of the infection or contamination of the specimen can be determined by knowing the type and number of organisms (colonies) present in the specimen.

Commonly detected organisms are those normally found in the genitourinary tract, including gram-negative Enterococci,E. coli,Klebsiella,Proteus, and Pseudomonas. A culture showing multiple organisms indicates a contaminated specimen.

Colony counts of 100,000/mL or more indicate urinary tract infection (UTI).

Colony counts of 1,000/mL or less suggest contamination resulting from poor collection technique.

Colony counts between 1,000 and 10,000/mL may be significant depending on a variety of factors, including patient’s age, gender, number of types of organisms present, method of specimen collection, and presence of antibiotics.

The CDC, American Academy of Family Physicians, American Academy of Pediatrics, American College of Nurse-Midwives, and American College of Obstetricians and Gynecologists recommend universal screening for all pregnant patients at 35 to 37 wk’ gestation to identify the presence of GBS, a significant and serious neonatal infection transmitted as the newborn passes through the birth canal of colonized patients.

Rapid GBS screening test kits can provide results within minutes on urine specimens. Rapid test findings should be used in conjunction with symptoms, history, and traditional methods to include culture and Gram stain or culture-enhanced molecular methods.

Urine cultures may be performed on pregnant patients to identify the presence of GBS. Pregnant patients with positive results for a GBS UTI at any time during pregnancy should receive appropriate medical treatment at the time of diagnosis and also receive intrapartum antibiotic prophylaxis to offer continued protection in the absence of complete eradication of the infection at the time of delivery. Neonatal GBS is the most common cause of sepsis, pneumonia, and meningitis in newborns. The disease is classified as either early onset (first week of life) or late onset (after the first week of life).

Wound Cultures

A wound culture involves collecting a specimen of exudates, drainage, or tissue so that the causative organism can be isolated and pathogens identified. Specimens can be obtained from superficial and deep wounds.