CHAPTER

66 SPECIMEN COLLECTION AND

HANDLING FOR DIAGNOSIS OF
INFECTIOUS DISEASES
Anna R. Plourde, Kathleen G. Beavis

GENERAL PRINCIPLES, 1352 Cerebrospinal Fluid, 1355 Vaginal Secretions, 1362

Timing of Specimen Collection, 1352 Other Body Fluids, 1356 Endocervical and Urethral
Specimen Volume, 1352 Specimens, 1362
TISSUES, 1357
Specimen Collection, 1352 Vesicles, 1362
Specimen Collection and Processing,
Specimen Transport, 1352 1357 Ulcers, 1363
Unacceptable Specimens, 1353 FECES, 1363
EYE, 1357
Standard Precautions, 1353 Viruses, 1363
Conjunctival Specimens, 1357
Referral Testing, 1353 Bacterial Pathogens, 1363
Corneal Specimens, 1357
BLOOD, 1353 Parasites, 1364
RESPIRATORY TRACT, 1358
Specimen Collection, 1353 SKIN AND SUBCUTANEOUS
Nasopharyngeal Specimens, 1358
Specimen Timing and Volume, 1353 LESIONS, 1365
Throat Specimens, 1358
Specimen Draws, 1354 Fluid-­Filled Lesions (Vesicles,
Specimen Processing, 1358
Recovery of Microorganisms, 1354 Pustules, Bullae, and Abscesses),
Sputum and Tracheal Aspirates, 1359 1365
Automated Blood Culture Systems,
1354 Bronchoscopy Specimens, 1360 Wound Infections, 1365
Detection and Notification of URINARY TRACT, 1361 Ulcers, 1366
Positive Cultures, 1354 Specimen Collection and Transport, SUMMARY, 1366
Detection of Viruses, 1355 1361
Detection of Parasites, 1355 Specimen Processing, 1361 SELECTED REFERENCES, 1366

BODY FLUIDS, 1355 GENITAL TRACT, 1362

SPECIMEN VOLUME
KEY POINTS The volume of specimen collected must be adequate for performance of
• Collect specimens from site of infection before initiating therapy. the microbiological studies requested. If insufficient volume is received,
• Collect an adequate volume of sample for testing required. the health care worker caring for the patient should be notified; either an
additional sample can be obtained, or the physician must prioritize the
•  issue, fluid, or aspirates are always superior to a swab specimen. The
T requests.
only exception is collecting culture material from a hard-­to-­reach spot Very little specimen is obtained with a swab, and much of the specimen
such as a throat or cervix. is retained within the swab tip. Swabs should not be used as collection
•  se required collection and transport materials to preserve specimen
U devices unless the specimen source is the throat, cervix, or other difficult-­
integrity. to-­reach area. If a swab is used to collect the specimen, a polyester-­tipped
• Communicate clear orders and source information.
swab on a plastic shaft is acceptable for most organisms. Calcium alginate
should be avoided for collection of samples for viral culture because it could
• E xpedite the transport of specimens to the laboratory and do not al- inactivate herpes simplex virus (HSV), cotton may be toxic to Neisseria
low them to sit in collection areas. gonorrhoeae, and wooden shafts should be avoided because the wood may
be toxic to Chlamydia trachomatis. Swabs are not optimal for detection of
anaerobes, mycobacteria, or fungi, and they should not be used when these
Appropriate specimen collection, transport, and processing are crucial pre- organisms are suspected. An actual tissue sample or fluid aspirate is always
analytical steps in the accurate diagnosis of infectious diseases. Guidelines superior to a swab specimen for the recovery of pathogenic organisms. 
for specimen handling are discussed in this chapter. General principles are
reviewed first followed by discussion of the most common types of speci-
mens submitted to the clinical microbiology laboratory for testing.
SPECIMEN COLLECTION
Specimens should be obtained from the site of infection with minimal
contamination from adjacent tissues and organ secretions, and with the
GENERAL PRINCIPLES exception of stool, should be collected in a sterile container. All specimens
should be labeled with the name and identification number of the person
TIMING OF SPECIMEN COLLECTION from whom the specimen was collected, the source of the specimen, and
For optimal detection of the pathogens responsible for an infectious the date and time it was collected. 
disease, specimens should be collected at a time when the likelihood of
recovering the suspected agent is greatest. For example, the likelihood of SPECIMEN TRANSPORT
recovering most viruses is greatest in the acute phase of the illness. Speci-
mens for recovery of bacteria should ideally be collected before antimicro- After collection, specimens should be placed in a biohazard bag and trans-
bial therapy is started.  ported to the laboratory as soon as possible. If a delay is unavoidable, urine,

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sputum and other respiratory specimens, stool, and specimens for detec- limited to no more than 40 mL. Cultures of bacteria and fungi should be
tion of C. trachomatis or viruses should be refrigerated to prevent over- grown on solid media in tubes. The cap of the primary container (tube
growth of normal flora. Cerebrospinal fluid (CSF) and other body fluids, or vial) should be sealed with waterproof tape and inserted into a second
blood, and specimens collected for recovery of N. gonorrhoeae should be container surrounded by sufficient packing material to absorb the entire
held at room temperature because refrigeration adversely affects recovery volume of the culture or specimen if the primary container were to leak
of potential pathogens from these sources.  or break. If several primary tubes are placed in a second container, they

PART 7
must be either individually wrapped or separated so as to prevent con-
tact between them, and there must be secondary packaging, which must be
UNACCEPTABLE SPECIMENS leakproof. The second container should be capped and placed in a shipping
Each laboratory director must establish criteria for rejecting specimens container made of corrugated fiberboard or hard plastic. An itemized list
unsuitable for culture. Most clinical microbiologists agree that the follow- of contents must be enclosed between the secondary and outer packag-
ing specimens should be rejected: ing. The secondary and outer containers should be of sufficient strength to
• Any specimen received in formalin maintain their integrity at temperature and air pressures to which they will
• 24-­hour sputum collections be subjected. If a specimen must be shipped on dry ice (which is considered
• Specimens in containers from which the sample has leaked to be a hazardous material), it must be marked “Dry ice, frozen medical
• Specimens that have been inoculated onto agar plates that have dried specimen.” The dry ice should be placed outside the second container with
out or are outdated the packing material in such a way that the container does not become
• Specimens contaminated with barium, chemical dyes, or oily chemicals loose inside the outer container as the dry ice evaporates. All infectious
• Foley catheter tips shipping packages must be labeled with an official label containing the
• Duplicate specimens (except blood cultures) received in a 24-­hour pe- address and contents as well as the name and telephone number of the
riod person responsible for the shipment. All laboratorians who package and
• Blood catheter tips submitted for patients without concomitant positive ship materials that are known or reasonably expected to contain a pathogen
blood culture must have documentation of training. There are several commercial and
The following specimens should be rejected for anaerobic culture: government-­based resources for training. 
• Gastric washings
• Urine other than suprapubic aspirate
• Stool (except for recovery of Clostridium difficile for epidemiologic stud-
BLOOD
ies or for diagnosis of bacteria associated with food poisoning) The rapid identification and susceptibility testing of bloodborne pathogens
• Oropharyngeal specimens except deep tissue samples obtained during a is one of the most critical functions of the microbiology laboratory. Even
surgical procedure with the increased use of molecular technologies capable of identifying
• Sputum organisms and some markers of resistance from either blood specimens
• Swabs of ileostomy or colostomy sites or positive blood culture bottles, blood cultures are still the “gold stan-
• Superficial skin specimens  dard” for identifying bacteria responsible for bacteremia, sepsis, infections
of native and prosthetic valves, suppurative thrombophlebitis, mycotic
aneurysms, and infections of vascular grafts. Blood cultures also are use-
STANDARD PRECAUTIONS ful in diagnosing invasive or disseminated infections caused by certain
Safety is the responsibility of the laboratory director and, per the Clini- fungi, especially Cryptococcus neoformans, Candida spp., Fusarium spp., and
cal Laboratory Improvement Amendments (CLIA), cannot be delegated to Histoplasma capsulatum. Serologic testing or nucleic acid amplification tests
others. The laboratory director should collaborate with infection control, (NAATs) are needed to identify two important bacterial causes of culture-­
institutional safety committees, environmental services, engineering, and negative endocarditis, Coxiella burnetii and Bartonella spp. Parasites are usu-
others within the hospital and department to ensure that there are cur- ally detected in blood by microscopic examination of peripheral smears,
rent and necessary policies and procedures, engineering controls, personal but enzyme immunoassay (EIA) and NAATs are also used.
protective equipment (PPE), and a trained work force. The laboratory
director is also responsible for making sure the policies and procedures are
followed. Specimen processing can have additional challenges because this
SPECIMEN COLLECTION
workforce often has the least amount of formal technical education. Timely detection and accurate identification of organisms in the blood
Universal Precautions were designed to protect workers from infectious depend on appropriate collection, transport, and processing of the speci-
substances in blood and body fluids. Body Substance Isolation Precautions men. In general, two to three sets of blood cultures should be collected for
were designed to protect workers from transmission of organisms from identification and susceptibility testing before antimicrobial therapy is ini-
moist body surfaces. In 1996, the US Hospital Infection Control Prac- tiated. Consensus guidelines recommend peripheral venipuncture rather
tices Advisory Committee unified these into “Standard Precautions.” As than draws from intravascular catheters. Good skin cleaning and phle-
described in Biosafety in Microbiological and Biomedical Laboratories (BMBL), botomy technique minimize the presence of skin contaminants in blood
Standard Precautions apply to (1) blood; (2) all body fluids, secretions, and cultures (Miller et al., 2018). The use of iodine tincture, or chlorhexidine
excretions except sweat regardless of whether or not they contain visible gluconate, is preferable to povidone–iodine solutions because the former
blood; (3) nonintact skin; and (4) mucous membranes (Wilson, 2009). require only about 30 seconds after application to achieve antiseptic effect;
Standard Precautions must be followed when handling all specimens. A risk the latter requires up to 2 minutes. Each set of peripheral blood cultures
assessment should be performed in each laboratory area; it should detail must be drawn from a separate venipuncture site so that if a typical con-
the particular risks with each procedure. The safety recommendations for taminating organism is present in one set and not the other, the organism
engineering controls, PPE, and work practices can be tailored to the risk can be more easily classified as a contaminant.
(Callihan et al., 2014). Appropriate barriers are used to prevent exposure Compared with venipuncture, the risk for contamination is increased
of skin and mucous membranes to the specimen. Gloves and a lab coat when blood cultures are drawn from an indwelling vascular device.
must be worn at all times when handling patient specimens, and masks, Although it might seem advantageous to save the patient the discomfort of
goggles (or working behind a plastic shield), and impermeable gowns or a venipuncture, a contaminated blood culture can cause even more diag-
aprons must be worn when there is a risk for splashes or droplet forma- nostic cultures, unnecessary antibiotics, and a prolonged stay in the hospi-
tion. Optimally, all specimen containers, but at a minimum, those contain- tal. If it is necessary to draw a culture through an intravenous line, another
ing respiratory secretions and those submitted specifically for detection blood culture should be collected simultaneously from a venipuncture to
of mycobacteria or fungi should be opened in a biological safety cabinet. aid in the interpretation of a contaminated line culture (Miller et al., 2018). 
Specimens collected for virus isolation should be handled in a biological
safety cabinet to prevent contamination of the cell cultures. 
SPECIMEN TIMING AND VOLUME
The optimal time to draw blood for cultures when bacteremia or funge-
REFERRAL TESTING mia is suspected is just before a fever or chill, but this is not predictable.
When specimens or cultures must be shipped to a reference laboratory, The single most important factor to detect bacteremia is volume of blood
they must be packaged according to dangerous goods shipping guidelines collected. In adults with bacteremia, the number of colony-­forming units
(see International Air Transport Association website, available at http:// (CFUs) per milliliter of blood is frequently low. Therefore, for adults, col-
www.iata.org/whatwedo/cargo/dgr/Pages/index.aspx). Specimens must be lecting multiple sets of blood cultures with each bottle filled to optimal

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 volume is most important. (Washington & Ilstrup, 1986). In infants and Three major automated continuously monitoring blood culture sys-
CHAPTER 66  SPECIMEN COLLECTION AND HANDLING FOR DIAGNOSIS OF INFECTIOUS DISEASES
children, the concentration of microorganisms in blood is higher, and col- tems are available commercially in the United States. With these systems,
lection of 1 to 5 mL of blood per culture is adequate. When blood cultures the usual incubation period is 5 days (Reisner & Woods, 1999). The BacT/
are drawn by personnel other than phlebotomists, it can be a challenge to ALERT 3D system (bioMerieux) is based on the colorimetric detection of
obtain sufficient volumes in the culture bottles. With the current focus carbon dioxide (CO2) produced during microbial growth. A CO2 sensor is
on detecting and treating sepsis, it behooves the pathologist to collabo- bonded to the bottom of each blood culture bottle and is separated from
rate with clinical colleagues to optimize the amount of blood collected and the broth medium by a membrane that is impermeable to most ions and
improve the ability of the blood culture to detect bacteremia (Hazen et al., to components of media and blood but freely permeable to CO2. Inocu-
2020; Khare et al., 2020).  lated bottles are placed in cells in the instrument, which provides continu-
ous rocking of both aerobic and anaerobic bottles. If bacteria are present,
they generate CO2, which is released into the broth medium; the pH then
SPECIMEN DRAWS decreases, causing the sensor to change color from green to yellow. Color
Recommendations concerning the number of blood specimens to col- changes are monitored once every 10 minutes by a colorimetric detector.
lect are based on the nature of the bacteremia: transient, intermittent, This system supports the growth of aerobic and anaerobic bacteria; bot-
or continuous. Transient bacteremia follows manipulation of a focus of tles are available to support the growth of mycobacteria. This system has
infection (e.g., an abscess, a furuncle, or cellulitis), instrumentation of a received Food and Drug Administration (FDA) clearance for monitoring
contaminated mucosal surface (as occurs during dental procedures, cys- bacterial contamination of platelets.
toscopy, urethral catheterization, suction abortion, or sigmoidoscopy), or The BACTEC continuous-­ monitoring system (BD Diagnostics) is
a surgical procedure in a contaminated site (e.g., transurethral resection based on fluorescent technology. Bonded to the base of each vial is a CO2
of the prostate, vaginal hysterectomy, colon resection, and debridement sensor that is impermeable to ions, medium components, and blood but
of infected burns). Transient bacteremia also occurs early in the course freely permeable to CO2. If organisms are present, they release CO2 into
of many systemic and localized infections such as meningitis, pneumo- the medium; it then diffuses into the sensor matrix and generates hydrogen
nia, pyogenic arthritis, and osteomyelitis. Most intermittent bacteremias ions. The subsequent decrease in pH increases the fluorescence output of
are associated with an undrained abscess, whereas continuous bacteremia the sensor, changing the signal transmitted to the optical and electronic
is the hallmark of intravascular infection, such as bacterial endocardi- components of the instrument. The computer generates growth curves,
tis, mycotic aneurysm, or an infected intravascular catheter. Continuous and data are analyzed according to growth algorithms. Inoculated bottles
bacteremia also occurs during the first few weeks of typhoid fever and are placed in individual cells of the instrument and rocked continuously.
brucellosis. Adult and pediatric aerobic and anaerobic bottles are available, as well as a
The optimal number of blood cultures for detection of bacteremia bottle for the recovery of yeasts and mycobacteria.
in patients without endocarditis is controversial. Most authorities agree The VersaTREK system (Trek Diagnostic Systems) detects growth of
that two or three 20-­mL blood samples drawn over a 24-­hour period organisms in broth by measuring gas consumption and gas production.
and equally distributed into aerobic and anaerobic blood culture bottles Each inoculated vial is fitted with a disposable connector that contains a
will detect most bloodstream infections. One investigator demonstrated recessed needle. The needle penetrates the bottle stopper and connects
that 80% of bacteremias were detected with two blood cultures and 96% the bottle headspace to the sensor probe. The sensor monitors changes
with three blood cultures. All bacteremias were detected with four blood within the headspace in the consumption and production of all gases (CO2,
cultures, but the routine collection of four blood cultures (up to 80 mL N2, and H2) by growing organisms and creates data points internally in
of blood) should be weighed against the risk for anemia (Cockerill et al., the computer. Media are available to identify aerobic and anaerobic bacte-
2004). The optimal time interval between cultures is unknown, but 30 to ria, including mycobacteria. Some clinicians continue to request extended
60 minutes for the first two sets has been suggested, with another one to incubation protocols for Brucella spp. and other bacteria known as the
two sets drawn over the remaining 24 hours if symptoms of septicemia per- HACEK group (Haemophilus, Aggregatibacter [formerly Actinobacillus], Car-
sist (Cockerill et al., 2004). However, if initiation of antimicrobial therapy diobacterium, Eikenella, Kingella spp.). These organisms needed prolonged
is deemed urgent, cultures should be collected before therapy is begun, incubation times when manual methods predominated, but they can be
from separate sites, within a few minutes. detected during the routine incubation times of the automated instruments.
Organisms such as the coagulase-­ negative staphylococci, viridans When mycobacteria or fungi are suspected, liquid medium developed
streptococci, Corynebacterium spp., Bacillus spp., and Propionibacterium spp. by the manufacturer of automated and semiautomated broth culture sys-
are frequent blood culture contaminants but may also be true pathogens. tems can be used. These blood cultures are incubated for a prolonged
Collecting two sets of blood cultures per febrile episode helps distinguish period of 4 to 8 weeks. 
probable pathogens from contaminants. If two sets are drawn from differ-
ent venipuncture sites, the odds of both sets being contaminated by skin DETECTION AND NOTIFICATION OF POSITIVE
flora are very low. If two sets are drawn at the same time and only one set
contains a skin contaminant, it is safe to assume that the culture was con-
CULTURES
taminated during collection. If only one set is drawn and a contaminant is Positive blood cultures containing commonly isolated aerobic organisms
present, it can be difficult to not treat the organism, especially if the patient are usually detected within 12 to 36 hours of incubation. Until recently,
has had recent surgery.  the initial report was limited to a Gram stain; identification and suscep-
tibility results could be expected no sooner than 24 to 48 hours after the
Gram stain report. Both the FilmArray blood culture identification panel
RECOVERY OF MICROORGANISMS (BioFire Diagnostics) and the Verigene system (Nanosphere) use molecu-
Host factors such as antibodies, complement, phagocytic white blood cells, lar methods to identify more than 90% of the organisms in the time when
and antimicrobial agents may impede recovery of microorganisms from a Gram stain would be reported. They can also identify the presence or
blood; therefore, various approaches have been used to counteract these absence of mecA and other resistance genes, allowing empiric therapy to
factors. Diluting the blood specimen in broth medium in a 1:10 ratio pro- be tailored (Bhatti et al., 2014a). The Accelerate Pheno system (Acceler-
vides optimal neutralization of the serum bactericidal activity (Washing- ate Diagnostics) can identify the organisms causing 90% of bacteremias
ton & Ilstrup, 1986). Incorporating 0.02% to 0.05% sodium polyanethol and provide minimal inhibitory concentrations of key antimicrobial agents
sulfonate in the blood culture medium inhibits coagulation, phagocytosis, within 8 hours of a positive blood culture (Charnot-­Katsikas et al., 2018).
and complement activation and inactivates aminoglycosides. Methods that As these systems evolve and continue to provide even more rapid, clinically
counteract the presence of antimicrobial agents include using antibiotic-­ actionable information, laboratories should work with clinical colleagues
adsorbent resins or the lysis-­centrifugation system.  to make sure these more expensive modalities have impact on patient care.
“Pushing” information to clinicians has been shown to decrease the time to
escalation or de-­escalation of empiric antimicrobials (Banerjee et al., 2015).
AUTOMATED BLOOD CULTURE SYSTEMS The use of matrix-­assisted laser desorption ionization–time of flight
Three commercially available automated blood culture systems, each with mass spectrometry (MALDI-­TOF MS) has shortened the time to iden-
advantages and disadvantages, are available. These continuously monitored tification. Although most laboratories allow for overnight incubation
automated detection systems have essentially replaced manual systems. All before testing mature colonies, an aliquot of the positive culture can be
systems use nutritionally enriched liquid media, which are capable of sup- centrifuged and the pellet inoculated onto prewarmed plates. About 95%
porting growth of most bacteria and many pathogenic yeasts. Tradition- of isolates can be identified with the MALDI-­TOF after 4 hours of incuba-
ally, two bottles, an aerobic and an anaerobic, are inoculated. tion (Bhatti et al., 2014b). Although more labor intensive, the reagents of

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this method are much less expensive than the molecular methods. Cultures
containing anaerobes are usually not detected for 48 to 72 hours, and iden- BODY FLUIDS
tification is not available for 3 to 4 days. Fastidious organisms, such as those
found in the HACEK group, may not be detected until 3 to 5 days.
CEREBROSPINAL FLUID
In a small percentage of cases, no organisms will be seen on a Gram CSF is collected to diagnose meningitis and, less frequently, viral enceph-
stain from a positive blood culture. It is very important to inoculate and alitis. Infectious meningitis, a medical emergency requiring early therapy

PART 7
monitor media in these cases. Brucella spp. can be difficult for an inexperi- to prevent death or serious neurologic sequelae, is divided into acute,
enced eye to note on Gram stain; the colonies are visible on solid media a subacute, and chronic clinical syndromes, based on duration of symp-
few days later. Conversely, some organisms are visible on Gram stain but toms. Potential pathogens are listed in Table 66.1. Enteroviruses are the
not on routine media. These organisms grow on chocolate agar or sheep agents most commonly responsible for meningitis, and they should be
blood agar with a Staphylococcus aureus streak.  considered first in the differential diagnosis of meningitis in a child or
adolescent during the late summer and early fall. The pyogenic bacteria
responsible for meningitis vary with the age of the affected individual
DETECTION OF VIRUSES (Table 66.2).
With regard to viruses, blood specimens are most commonly collected to
monitor response of infection with human immunodeficiency virus (HIV), Sample Collection and Transport
hepatitis C virus (HCV), hepatitis B virus (HBV), or cytomegalovirus CSF is usually obtained by lumbar spinal puncture, but sometimes it is
(CMV) to antiviral therapy by using quantitative polymerase chain reac- aspirated from the ventricles or collected from a shunt. As when collect-
tion (PCR) to measure viral load. Such assays are commercially available ing blood for culture, careful skin antisepsis is essential for collection
for each of these viruses, and in all cases, manufacturer’s guidelines for of CSF, which typically is submitted to the laboratory in three or occa-
specimen collection and transport should be followed. For HIV and HCV, sionally four tubes. The first tube filled should not be sent to Micro-
blood specimens also may be collected for genotyping (commercial assays biology because it can contain skin flora. At least 0.5 to 1.0 mL should
are available), and PCR (qualitative or quantitative PCR) generally is used be sent to Microbiology for Gram stain and bacterial culture; ideally,
to confirm an initial positive HCV antibody result. As with viral load, man- the microbiology laboratory would receive 5 to 10 mL to accommodate
ufacturer’s guidelines regarding specimen collection and transport should requests for NAAT and cultures for mycobacteria and fungi (Miller
be followed. et al., 2018). The parameters of normal CSF and the usual changes
In addition to assessing response to antiviral therapy, measuring viral that occur during meningitis caused by different organisms are listed
load in a blood specimen is useful for monitoring disease and for diagnosis in Table 66.3.
of disease in specific situations. In immunocompromised patients, espe- CSF should be transported promptly to the laboratory and processed as
cially transplant recipients but also patients with acquired immunodefi- rapidly as possible. If a brief delay in processing is unavoidable, the speci-
ciency syndrome (AIDS), determining the level of CMV deoxyribonucleic men should be held at room temperature unless viral culture is requested,
acid (DNA) in blood is used to predict those at high risk for development in which case a portion (preferably 1 mL but no less than 0.5 mL) may
of CMV disease and direct the initiation of preemptive therapy. Monitor- be refrigerated for a short time. Specimen processing differs for bacteria,
ing the level of BK virus and Epstein-­Barr virus (EBV) DNA in serum fungi, viruses, and parasites and is discussed separately for each group of
or plasma by quantitative PCR is indicated in transplant recipients. If a organisms. 
commercially available NAAT assay is used, the package insert guidelines
for specimen collection and transport should be followed. If, on the other
hand, an assay developed and validated in house is used, guidelines pub-
lished by that laboratory should be followed.    TABLE 66.1
Infectious Meningitis Syndromes
DETECTION OF PARASITES Onset or
Blood specimens are useful for diagnosis of malaria, babesiosis, trypano- Syndrome Duration Probable Pathogens
somiasis, and some filariasis (Rosenblatt, 2009). Specimens should be Acute <24 hours Pyogenic bacteria
collected in tubes with anticoagulant and transported promptly to the
Subacute 1–7 days Enteroviruses, pyogenic bacteria
laboratory. If smears must be sent to a reference laboratory, they should
follow the reference laboratory’s instructions for fixation soon after they Chronic Persisting at least 4 Mycobacterium tuberculosis
are made. The techniques used in the laboratory for detecting the afore- weeks Treponema pallidum
mentioned parasites are the same and are discussed here in order of the Brucella spp.
simplest to the most complicated. Leptospira interrogans
Standard Precautions need to be used when preparing smears and read- Borrelia burgdorferi
ing fresh (unfixed) smears. Additional fixation time in methanol could be
Cryptococcus neoformans
recommended depending on the patient’s travel history. The simplest
technique for detecting parasites in a sample of blood is the direct mount, Coccidioides immitis
prepared by placing 1 drop of blood on a glass slide, covering it with a Histoplasma capsulatum
cover glass, and examining it immediately. Direct mounts are excellent for
diagnosis of trypanosomiasis or filariasis because the trypomastigotes and
the microfilariae easily can be seen moving, often with low or medium
power. Stained smears make the definitive diagnosis.   TABLE 66.2
The thin smear, made as for hematologic work and stained in a similar Common Bacterial Causes of Acute Meningitis by Age
manner, is the standard preparation for speciating Plasmodium spp., Babe-
sia spp., Trypanosoma spp., and microfilaria. Thin smears for parasitologic Age Organisms
work are fixed and then preferably stained manually with Giemsa stain,
Neonates–3 months Group B streptococcus
but automated hematologic staining is adequate. Smears are first scanned
at low power to detect microfilariae, which are large objects (between 100 Escherichia coli
and 200 μm) and easily seen, usually at the lateral edges of the smear. After Listeria monocytogenes*
they are located, microfilariae should be studied under oil immersion for Streptococcus pneumoniae
identification. After scanning with low power, the smear is examined with 4 months–6 years† Streptococcus pneumoniae
a high dry objective, searching for trypanosomes, and finally under oil 6–45 years Neisseria meningitidis
immersion to find and identify Plasmodium, Babesia, and Trypanosoma spp.
Older than 45 years Streptococcus pneumoniae
Thick smears are useful for detecting all the parasites mentioned ear-
lier and are part of the minimum laboratory workup for their diagnosis. Listeria monocytogenes
One drop of blood is placed on a clean glass slide and, with the corner of Group B streptococcus
another slide, is gently spread to cover 1 cm square. The preparation is *May cause meningitis in immunocompromised individuals in all age groups.
allowed to dry and without fixation is stained with Giemsa stain, allowing †Incidence of meningitis caused by Haemophilus influenzae type b in the United
for its dehemoglobinization.  States has declined dramatically as a result of vaccination.

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CHAPTER 66  SPECIMEN COLLECTION AND HANDLING FOR DIAGNOSIS OF INFECTIOUS DISEASES
  TABLE 66.3
Normal Cerebrospinal Fluid Parameters and Changes in
Infectious Meningitis
Protein Glucose
Condition WBCs (cells/μL)a (mg/dL) (mg/dL)

Normal 5 (lymphocytes) 14–45 45–100

(2/3 serum)
Meningitis
Acute or sub- >500 (PMNs) ↑ ↓
acute bacterial
Chronic 200–2000 ↑ ↓
bacterial
Tuberculous, (lymphocytes) ↑ ↓
fungal
Enteroviral 200–2000 (PMNs ↑ Normal
early; lymphocytes
later) Figure 66.1 India ink preparation of cerebrospinal fluid showing encapsulated
aCell
yeast forms of Cryptococcus neoformans. (400×.)
type listed usually predominates.
↑, Increased; ↓, decreased; PMN, polymorphonuclear leukocyte; WBC, white blood
cell.
Supernatant of a centrifuged specimen or unspun CSF can be used for
these latter two tests. 

Sample Processing for Bacterial and Fungal Culture Cerebrospinal Fluid Sample Processing for Diagnosis of
Processing CSF for routine bacterial culture includes concentration (if 1 Viral and Parasite Infections
mL or more of specimen is received), preparation of a smear by cytocen- Currently, NAATs are used most often for diagnosis of viral infections of the
trifugation for staining with Gram stain, and culture. The supernatant is central nervous system. Other diagnostic methods are conventional cell culture
decanted into a sterile tube, leaving about 0.5 mL of sediment and fluid, (primarily for detection of enteroviruses, although PCR is preferred) and sero-
which is thoroughly mixed on a vortex mixer or by forcefully aspirating up logic tests for viruses that cause encephalitis (western equine, eastern equine,
and down into a sterile pipette. Venezuelan equine, St. Louis, Japanese, La Crosse, and West Nile viruses).
Diagnosis of chronic bacterial meningitis requires specific requests CSF is occasionally sent to the laboratory for diagnosis of African
because the CSF is handled differently for each entity. To diagnose brucel- trypanosomiasis (Trypanosoma gambiense and Trypanosoma rhodesiense) or
losis, the CSF is processed as described earlier for routine bacterial culture, infection with free-­living amebae (Naegleria fowleri and Acanthamoeba spp.).
but the media are incubated for 2 to 3 weeks. For leptospirosis, Leptospira When the specimen is received in the laboratory, it should be processed
interrogans may be cultured from the CSF during the first few weeks of immediately. Wet preparations are prepared directly from the specimen
illness, but the Centers for Disease Control and Prevention (CDC) can and from the sediment by first shaking the tube gently (a step necessary
test using a NAAT. The diagnosis of neurosyphilis can be challenging and because the parasites often stick to the wall of the tube) and then centrifug-
requires neurologic signs and symptoms, reactive serologic results, and ing the specimen at 250 g for 10 minutes. Preparations are examined under
CSF findings including pleocytosis, elevated protein, and a positive Vene- the microscope with the condenser in a low position to allow visualization
real Disease Research Laboratory (VDRL) test result. If the CSF-­VDRL of trophozoites or, preferably, by phase contrast microscopy.
result is negative in the presence of clinical and serologic support for neu- Cultures of free-­living amebae from CSF are done on nonnutrient agar
rosyphilis, a CSF fluorescent treponemal antibody absorption test might plates covered with a suspension of Escherichia coli or Enterobacter aerogenes.
be useful. This test is more sensitive and less specific that the CSF-­VDRL The fluid is centrifuged at 250 g for 10 minutes, the supernatant is removed
(https://www.cdc.gov/std/tg2015/syphilis.htm). The specimen should be with a sterile pipette, and the sediment is mixed with 0.5 mL of saline solu-
refrigerated until it is tested. Involvement of the central nervous system by tion and poured at the center of the plate. The culture is incubated at 37°C
Borrelia burgdorferi (Lyme disease) also is diagnosed serologically by detec- and examined for amebae daily for 10 days using a microscope under a 10×
tion of specific immunoglobulin (Ig) M and IgG antibodies in CSF and objective (Martinez & Visvesvara, 1991). 
serum.
At least 5 mL of CSF is recommended for mycobacterial culture; OTHER BODY FLUIDS
cytocentrifugation is required to make a smear and optimize the yield. A
NAAT may be useful, but a negative result does not exclude mycobacterial Fluid is collected from the pericardial, thoracic, or peritoneal cavity or
infection. from joint spaces by aspirating with a needle and syringe. A volume of 1
Processing CSF for detection of fungi is similar to that described for to 5 mL is adequate for isolating most bacteria, but 10 to 15 mL is opti-
detecting bacteria. Organisms are concentrated by filtration or by centrifu- mal for recovery of mycobacteria and fungi, which are generally present in
gation. A cytocentrifuge preparation or a smear of the sediment stained low numbers. Moreover, to diagnose peritonitis associated with chronic
with Gram or other stain is examined, and appropriate media (e.g., brain-­ ambulatory peritoneal dialysis, collection of at least 50 mL of fluid may
heart infusion or SABHI agar without antibiotics) are inoculated for improve recovery of the responsible pathogen. To transport the fluid, it is
culture.  aspirated into a sterile container and delivered promptly to the laboratory.
Alternatively, peritoneal fluid may be directly inoculated into blood cul-
Additional Diagnostic Tests ture bottles at the patient’s bedside; however, submission of fluid in blood
In addition to culture, there is a commercially available molecular panel culture bottles eliminates the possibility of direct Gram staining and delays
for meningitis including bacterial, viral, and fungal targets. This panel does the identification and susceptibility testing of any pathogens isolated. Cli-
not replace culture; the panel does not detect all CSF pathogens, and cul- nicians should be advised to send fluid in a sterile container as well as in
ture is still necessary for susceptibility results (Liesman et al., 2018; Tan- blood culture bottles to allow for the performance of a Gram stain and
sarli & Chapin, 2020). faster identification if the culture is positive.
Two types of rapid tests are available for diagnosis of meningitis caused Enteroviruses, primarily Coxsackie viruses A and B, are among the most
by C. neoformans: those specific for the capsular antigen (latex agglutina- common causes of infectious pericarditis. These viruses may be detected in
tion and enzyme-­linked immunosorbent assay [ELISA]) and the nonspe- pericardial fluid by NAAT or traditional cell culture, but because they are
cific India ink preparation, which allow visualization of encapsulated yeast not recovered in all cases, collection of throat washings and stool (which
cells (Fig. 66.1). The sensitivity of the India ink stain, performed by mixing are more likely to yield the virus), in addition to pericardial fluid, is strongly
1 drop of CSF sediment with 1 drop of India ink (available at art supply recommended for virus isolation from persons with suspected enteroviral
stores), is low, except in HIV-­infected persons. Therefore, the cryptococ- pericarditis. Other viruses (HSV, varicella zoster virus [VZV], CMV, EBV,
cal latex agglutination test or the ELISA, both of which are highly specific HBV, mumps virus, and influenza virus) are infrequent agents of pericardi-
and have sensitivities of more than 90%, is recommended for diagnosis. tis and usually are not detected in pericardial fluid.

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Sample Processing for Bacterial Culture cultures. These are best performed in concert with histopathologic exami-
Processing fluid from body cavities for detection of bacteria involves nation to assess the true extent of the infection (Miller et al., 2018). 
preparing a smear for Gram stain and inoculating appropriate media for
culture. As mentioned earlier, the sample may be inoculated into blood
culture bottles at the bedside, although this is not optimal unless additional
EYE
specimen is sent for Gram stain and inoculation onto media. In the labora- The most common ocular specimens received in clinical laboratories are

PART 7
tory, the fluid is centrifuged, and the supernatant is removed, leaving about conjunctival and corneal samples collected for the diagnosis of conjuncti-
0.5 mL of fluid in addition to the sediment, which is mixed thoroughly and vitis and keratitis, respectively. Because specimen material is often scant,
then used to prepare smears and inoculate media. Alternatively, a small processing should be strategized based on the most likely etiologic organ-
volume of noncloudy, nonviscous fluid (about 0.1 mL) may be removed isms and informed by consultation with the clinician. The necessary col-
before centrifugation and used to prepare a cytocentrifuged smear. lection materials, including culture media for direct inoculation or viral
Fluid specimens submitted for detection of mycobacteria are pro- transport media (VTM), should be provided by the laboratory. Specimens
cessed as described earlier for CSF. Fluids for fungal culture should be should be labeled with the exact anatomic site (e.g., conjunctiva, cornea) to
concentrated by centrifugation as described for bacteria. The supernatant aid appropriate microbiological workup (Miller et al., 2018).
is removed, leaving 1.5 to 2.0 mL, in which the sediment is thoroughly
mixed. A smear of the sediment is prepared for staining with Gram or Cal-
cofluor white. Ideally, 0.5 to 1.0 mL of sediment is inoculated to primary
CONJUNCTIVAL SPECIMENS
fungal planting media (as for CSF), but lesser volumes are acceptable.  Conjunctival scrapings or swab specimens are collected to determine the
etiologic agent of conjunctivitis. Bacteria are the most common etiologic
Parasitologic Examination agents of infectious conjunctivitis, and those most frequently implicated
Body fluids are rarely collected for detection of parasites; however, Ent- are Streptococcus pneumoniae and S. aureus in adults; and Haemophilus influ-
amoeba histolytica may be found in the pericardial, pleural, or peritoneal enzae, S. pneumoniae, and S. aureus in children. Trachoma, caused by the
cavity as a result of rupture of an abscess of the liver (into the peritoneal, bacterium C. trachomatis, is a leading cause of blindness worldwide. C. tra-
pleural, or pericardial cavity) or of the lungs (into the pleural or pericar- chomatis may also cause inclusion conjunctivitis in newborns and, less com-
dial cavity) or to perforation of amebic ulcers (into the peritoneal cavity). monly, in adults. Viruses are responsible for about 15% to 20% of cases of
Hydatid cysts are infrequently diagnosed by examination of body cavity acute infectious conjunctivitis, and in the United States, most epidemics of
fluid, also caused by rupture of a cyst into a viscus contiguous to the cav- viral conjunctivitis (pinkeye) are caused by adenoviruses or herpes simplex
ity in question. The fluid collected is usually clear and contains hydatid virus. Rarely, parasites are causes of conjunctivitis.
sand (see Chapter 60) but rarely is turbid because of superimposed bacte-
rial infection. Uncommonly, in individuals with a filarial infection, exam- Specimen Collection and Processing
ination of wet preparations of a body cavity fluid may demonstrate the Conjunctival cells are obtained from the superior and inferior tarsal con-
microfilariae; in patients with Strongyloides hyperinfection, larvae may be junctiva by using a swab moistened with broth or a sterile platinum spatula.
detected in body cavity fluids.  If a bacterial or fungal infection is suspected, culture media are inoculated
directly by the individual collecting the sample to maximize the quantity
and viability of organism recovered and the likelihood of detection. Media
TISSUES typically includes a chocolate agar plate for recovery of fastidious bacteria
Tissue specimens obtained surgically are procured at great expense and and fungi, with additional media as necessary based on suspected organisms
at considerable risk to the patient; therefore, it behooves the surgeon to (Das et al., 2010). Ideally, direct smears are also prepared, which may be
obtain an amount of material that is adequate for both histopathologic and useful in preliminary identification of the organism. If direct preparation
microbiological examination. Swabs are rarely adequate for this purpose of smears and inoculation of media is not possible, swab specimens may be
because of their limited capacity to hold specimen for culture. The histopa- obtained, though swabs yield minimal material. Smears should be air dried
thology of the lesion serves not only to differentiate between infection and and promptly transported, with the inoculated media, to the laboratory.
malignancy but also to distinguish between a suppurative and a granulo- The conjunctiva and the eyelids of both the involved and uninvolved eye
matous process. In some cases, special stains are helpful in establishing the are often cultured concomitantly to characterize the normal flora, useful in
cause of the process. In chronic lesions, the differential diagnosis includes assessing culture results from the involved eye.
disease caused by actinomycetes, Brucella spp., mycobacteria, and fungi, If viral conjunctivitis is suspected, a second sample (swab or scrapings)
any one of which may be present only in small numbers, again emphasizing should be collected and placed in viral transport medium. Viral conjuncti-
the need to obtain adequate samples for examination and culture. vitis is now primarily diagnosed using NAATs because of their increased
sensitivity and rapid turn-­around time but require validation because there
are no NAATs for any ocular source that are FDA approved to date. Viral
SPECIMEN COLLECTION AND PROCESSING culture remains an option if a NAAT is not available. A rapid diagnosis may
Tissue obtained surgically for culture should be placed into a sterile, also be provided by direct or indirect immunofluorescent staining of thin
wide-­mouthed, screw-­capped container. As a general rule, tissue should smears of conjunctival cells with virus-­specific antibodies.
be bisected aseptically by the surgeon in the operating room, and mate- To detect C. trachomatis, NAATs are now the test of choice but, as for
rial representative of the pathologic process should be submitted for both viral NAATs, require in-­house validation (Dize et al., 2013). Alternative
histopathologic and microbiological examination. Good communication methods include examination of direct smear and culture. Specimens col-
between the anatomic pathologist and microbiologist is important, espe- lected in viral or chlamydia transport medium are appropriate for all three
cially in cases of fever of unknown origin for which an exploratory lapa- types of methods (Miller et al., 2018). For direct examination, a smear pre-
rotomy is being performed and multiple biopsy specimens are taken. pared from conjunctival scrapings may be stained with Giemsa stain and
Tissue received in the laboratory is finely minced with sterile scissors examined for epithelial cells with basophilic intracytoplasmic inclusions
or scalpels, added to a small volume of broth, and then rendered homoge- diagnostic of C. trachomatis or preferably with monoclonal fluorescent
neous either in a tissue grinder, mortar and pestle, or Stomacher to provide antibodies, which are more sensitive and specific than Giemsa. The swab
a 20% suspension. This suspension is used to inoculate all of the necessary is rolled across the surface of the glass slide provided, the material is fixed,
culture media and is then stored under refrigeration for at least 2 weeks and the slide is transported promptly to the laboratory and held at room
before being discarded. temperature or refrigerated briefly. Slides are stained and examined with a
Soft tissue specimens can be submitted after many injuries, including fluorescent microscope for elementary bodies (see Chapter 64). Specimens
burns, animal or human bites, trauma, or surgery. The likely pathogens containing fewer than 10 columnar or metaplastic squamous cells are con-
vary with the type of injury. When soft tissue specimens are received, there sidered inadequate, and results should be reported as inconclusive, with an
should be accompanying information to guide the workup in Microbiol- explanation, and another specimen should be requested. Culture of C. tra-
ogy. Human mouth flora is typically found in human bites; bites from chomatis should be performed when a diagnosis of chlamydial conjunctivitis
animals are associated with organisms typical to the biting animal (e.g., is strongly suspected but other methods have produced negative results. 
Pasteurella canis from dogs and Pasteurella multocida from cats). Animal bites
are typically polymicrobial and contain both aerobes and anaerobes. Soft
tissue specimens after trauma can contain organisms depending on the
CORNEAL SPECIMENS
type of trauma, including organisms usually thought of as environmental Corneal scrapings and biopsy specimens are useful in determining the
contaminants. Soft tissue specimens from burns can require quantitative etiologic agent of keratitis, an infection that can cause loss of vision and

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 requires immediate attention. Bacteria account for 65% to 90% of keratitis must be sent to a reference laboratory for culture, the swab should be
CHAPTER 66  SPECIMEN COLLECTION AND HANDLING FOR DIAGNOSIS OF INFECTIOUS DISEASES
cases. In the United States, S. aureus, S. pneumoniae, Pseudomonas aerugi- inoculated into a solid transport media such as Regan-­Lowe or Bordet-­
nosa, and Moraxella spp. are most frequently implicated. Contamination of Gengou agar, incubated at 37°C for 48 hours, and then shipped at ambi-
contact lens storage cases is a predisposing factor for keratitis; outbreaks ent temperature. The direct fluorescent antibody (DFA) staining method,
have infrequently been caused by Fusarium spp. and Acanthamoeba spp. which provides a rapid diagnosis but is associated with false-­positive and
Viral keratitis is most commonly caused by recurrent HSV type 1, VZV, false-­negative results, can be performed on a smear prepared from a naso-
and EBV; the adenoviruses are less frequent causes. Postsurgical infections pharyngeal swab or washing (van der Zee et al., 2015).
can be caused S. aureus, coagulase-­negative staphylococci, and the Myco- For detection of C. trachomatis, a nasopharyngeal swab specimen is col-
bacterium chelonae–Mycobacterium abscessus group. If keratitis follows a trau- lected with a polyester-­tipped swab, which may be used for culture or for
matic event, the laboratory should culture for environmental contaminants preparation of a smear for DFA staining. Detection methods are discussed
such as Nocardia spp., fungi, and P. aeruginosa, in addition to the organisms in Chapter 62. For assessment of C. trachomatis or C. pneumoniae, the swab
that often present as normal flora, such as S. aureus, coagulase-­negative should be placed in an appropriate transport medium and transported to
staphylococci, S. pneumoniae, and Cutibacterium spp (Gray et al., 2011). the laboratory as soon as possible or refrigerated for a short time. NAAT is
recommended for detection of C. pneumoniae (Miller et al., 2018).
Specimen Collection and Transport For culture of C. diphtheriae, swabbing multiple sites within the naso-
Corneal scrapings are collected with a sterile platinum spatula and are used pharynx likely increases sensitivity. Any membranes present, as well as
for preparation of smears by direct transfer to glass slides for staining and areas from beneath membranes, should be sampled for highest yield. Swabs
for inoculation to appropriate media for culture. Media typically include should be immediately transferred to the microbiology laboratory. If delay
chocolate agar for recovery of fastidious bacteria, as well as brain–heart is unavoidable, a semisolid transport medium (e.g., Amies) helps maintain
infusion agar with 10% sheep blood and inhibitory mold agar for fungal bacterial viability (Efstratiou et al., 2000).
isolation (Leber, 2016). If evaluation for viral keratitis is requested, scrap- Carriers of MRSA may be detected within a few hours by commercial
ings should be placed directly into VTM and delivered promptly to the NAATs or after 24 to 48 hours using MRSA-­specific chromogenic agar
laboratory. Viral keratitis is now diagnosed primarily with NAATs, though media (Bocher et al., 2008; Bischof et al., 2009; Tacconelli et al., 2009).
no FDA-­approved assays are yet available. Viral culture remains an option Nasal secretions are collected from the anterior nares with a polyester-­
if a NAAT is not available. Immunofluorescent staining of thin smears tipped swab, which is placed in a tube transport system and promptly deliv-
prepared directly from scrapings may be performed using virus-­specific ered to the laboratory. If a NAAT is used, the specimen must be collected
antibodies (Azher et al., 2017). When the culture of scrapings of a suspi- with the swab recommended by the manufacturer. 
cious corneal ulcer is negative, a superficial keratectomy or corneal biopsy
specimen may be obtained by the ophthalmologist, an approach especially
useful for detection of fungi and Acanthamoeba spp. 
THROAT SPECIMENS
Throat swabs can be used to diagnose bacterial pharyngitis and Vincent’s
angina (caused by Fusobacterium necrophorum and other anaerobes). Group
RESPIRATORY TRACT A streptococcus (GAS) is the cause of pharyngitis in less than one third of
NASOPHARYNGEAL SPECIMENS cases, but GAS needs to be identified and treated to prevent acute rheu-
matic fever, suppurative sequelae, transmission to others, and worsening
Nasopharyngeal aspirates, washings, and swab specimens are collected of signs and symptoms. C. diphtheriae and N. gonorrhoeae are the other
predominantly for diagnosis of viral respiratory infections, now typically bacterial causes of pharyngitis for which antimicrobial therapy has been
diagnosed using rapid molecular methods (see Chapter 64). Nasopharyn- demonstrated to be effective. Other streptococci (group C and group G)
geal specimens may also be submitted for workup of bacterial pneumonia also cause pharyngitis, but the utility of identifying them from culture is
caused by Bordetella pertussis, C. trachomatis, Chlamydia pneumoniae, or rarely controversial. Antibiotics have been given to lessen the duration of symp-
Corynebacterium diphtheriae or to identify carriers of methicillin-­resistant S. toms, but controlled studies demonstrating evidence of a clinical response
aureus (MRSA). Endoscopically or surgically collected specimens from the are lacking (Shulman et al., 2012).
nasal sinuses are submitted for workup of sinusitis, typically in chronic or Throat washings or swab specimens are also useful for detection of
complicated cases, which are often associated with S. aureus, gram-­negative viruses shed in oral secretions without causing pharyngitis (HSV, CMV,
bacilli, Streptococcus spp., anaerobes, or fungi (Miller et al., 2018). or enteroviruses).
Because of the risk for respiratory obstruction, throat swabs are contra-
Specimen Collection, Transport, and Processing indicated for workup of epiglottitis (Rabinowitz, 1978).
Nasopharyngeal aspirates and washings are superior to swabs for viral
recovery, but swabs are frequently submitted because they are more conve- Specimen Collection and Transport
nient. Whereas washings or swab specimens are collected for detection of Throat swab specimens are collected by depressing the tongue with a
B. pertussis, swabs can be used for detection of C. pneumoniae. A swab is the tongue blade, introducing the swab between the tonsillar pillars and
preferred specimen for C. trachomatis and C. diphtheriae. behind the uvula without touching the lateral walls of the buccal cavity, and
An aspirate is collected with a plastic tube (e.g., one used to feed pre- swabbing back and forth across the posterior pharynx. Swab specimens col-
mature infants) attached to a 1-­mL syringe or a suction catheter with a lected for detection of viruses should be placed in a viral transport medium,
mucus trap. A wash is obtained with a rubber suction bulb by instilling and those for detection of bacteria should be placed in a tube transport sys-
and withdrawing 3 to 7 mL of sterile phosphate-­buffered saline. Swabs are tem containing modified Stuart’s medium. For detection of N. gonorrhoeae,
obtained by removing all mucus from the nasal cavity, inserting a small use of calcium alginate swabs should be avoided because they are toxic
flexible nasopharyngeal swab along the nasal septum to the posterior phar- to the organism and can inhibit PCR (Lauer & Masters, 1988). Dacron-­,
ynx, and rotating against the mucosa several times. It is important to note rayon-­, polyurethane-­, and nylon-­tipped swabs are acceptable alternatives.
that whereas cotton-­tipped swabs and calcium alginate swabs inhibit PCR Specimens should be inoculated immediately to a selective medium, such
reactions, cotton-­tipped swabs are toxic to B. pertussis (Cloud et al., 2002). as modified Thayer-­Martin agar, or placed into charcoal-­containing or
Dacron or nylon swabs are preferable alternatives. other specific medium (Graver & Wade, 2004). Throat washings for diag-
To detect viruses, nasopharyngeal specimens are placed into an appro- nosis of viral infections are obtained by gargling with 5 mL of viral trans-
priate transport medium, with or without antibiotics, and transported port medium containing antibiotics. Throat washings and swab specimens
promptly to the laboratory or stored briefly in the refrigerator and packed should be delivered promptly to the laboratory or refrigerated for a short
in ice for transport as soon as possible. If a NAAT is used, the specimen time if a delay in transport is unavoidable. 
must be collected as recommended by the manufacturer. Viral detection
methods are discussed in more detail in Chapter 64.
The most sensitive method for detecting B. pertussis is PCR; a num-
SPECIMEN PROCESSING
ber of FDA-­approved assays are commercially available (van der Zee et al., For diagnosis of GAS pharyngitis, culture is most sensitive; however, this
2015; US FDA, 2020). If the sample must be transported to a reference requires overnight incubation. GAS grows well on horse and sheep blood
laboratory for PCR, the swab should be shipped dry or in saline. To detect agar, producing complete hemolysis (beta-­hemolysis) of the agar. For rapid
B. pertussis by culture, inoculation of washings or swab specimens at the diagnosis of GAS, there are many commercially available point-­of-­care
bedside is optimal to preserve organism viability. If this is not possible, the tests. These tests have a high specificity, so a throat culture does not need
sample is placed into sterile Casamino Acids broth, transported promptly to follow a positive result. However, their sensitivity is lower. A commer-
to the laboratory, and processed within 2 hours for culture. If the sample cial direct probe is the most sensitive of the rapid tests, but this test is not

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usually used as a point-­of-­care test (Shulman et al., 2012). Other rapid,
direct tests for GAS (several are commercially available) are less sensitive
(as low as 70%); therefore, if one of these is used, two throat swab speci-
mens should be collected. If the direct test result is positive, the second
swab may be discarded, but if the direct test result is negative in children, a
confirmatory culture must be performed using the second swab. In adults,

PART 7
because the incidence of streptococcal infection and the risk for rheumatic
fever are low, confirmatory culture is optional (Shulman et al., 2012).
To detect N. gonorrhoeae in the throat, the swab specimen should be
inoculated as soon as possible onto a selective medium, such as modified
Thayer-­Martin agar, to maximize yield. For diagnosis of diphtheria, both
nasopharyngeal and throat swab specimens are collected and transported
to the laboratory immediately. If laboratory personnel are not experienced
in the recovery and identification of C. diphtheriae, the specimens should
be sent in a semisolid transport media (e.g., Amies) to a reference labora-
tory. A differential inhibitory medium containing potassium tellurite, such
as Tinsdale medium, is optimal for cultivating C. diphtheriae. However,
this medium is expensive, has a short shelf-­life, and is difficult to obtain
from commercial vendors; therefore, it is seldom used in clinical laborato-
ries. Colistin–nalidixic acid blood agar (CNA) is an acceptable alternative
for the cultivation of C. diphtheriae, but because CNA is not a differential Figure 66.2  Gram stain of a sputum specimen demonstrating greater than 10 squa-
medium, all diphtheroid colony types must be evaluated to exclude C. diph- mous epithelial cells per low-­power field, which is unacceptable for culture. (10×.)
theriae when this agent is suspected. In addition, a sheep blood agar plate
should be inoculated and examined for GAS.
Vincent’s angina is an acute necrotizing ulcerative tonsillitis that may
be caused by F. necrophorum and other anaerobes. A presumptive diagnosis
may be made if gram-­negative fusiform bacilli and spirochetes are seen
in gram-­stained smears prepared from a swab specimen of the ulcerated
lesion. Cultures of the involved area are not usually helpful because many
species of anaerobes are present in the oral cavity. However, blood cultures
should be collected because the illness is commonly accompanied by sepsis. 

SPUTUM AND TRACHEAL ASPIRATES

Microbiological studies of sputum (expectorated and induced) and tracheal
aspirate specimens are done primarily to determine the etiologic agents of
pneumonia and to assess mycobacterial infection. The Infectious Diseases
Society of America and the American Thoracic Society have published
guidelines for the diagnosis of community-­acquired pneumonia (Metlay
et al., 2019). Although routine sputum cultures are of questionable util-
ity in many cases of community-­acquired pneumonia, they should be per-
formed if an unusual pathogen is suspected that would alter antimicrobial
management. Clinical factors such as severe pneumonia requiring hospi-
talization or high risk for MRSA pneumonia, among others, support the
utility of sputum culture (Metlay et al., 2019). Tracheal aspirates represent
Figure 66.3 Gram stain of a sputum specimen showing encapsulated, lancet-­
lower respiratory secretions collected in a Lukens trap from patients with shaped, gram-­positive diplococci, consistent with Streptococcus pneumoniae. (100×.)
tracheostomies. Patients with tracheostomies rapidly become colonized
with gram-­negative bacteria and other potential nosocomial pathogens
and because bacteria colonizing the respiratory tract cannot be differenti- Specimen Processing
ated from bacteria causing invasive disease by culture of tracheal aspirates, Sputum and tracheal aspirates for routine bacterial culture should be
interpretation of routine culture results is difficult. Culture for Legionella screened before they are plated to determine whether they are repre-
spp., mycobacteria, and fungi must be requested separately from rou- sentative of lower respiratory secretions or of saliva. A smear prepared
tine culture because each of these requires special media for cultivation. from a portion of the specimen consisting of purulent material is stained
Pneumocystis jirovecii cannot be cultured and is instead typically diagnosed with the Gram stain. In general, specimens with more than 10 epithelial
using DFA staining; therefore, testing of P. jirovecii testing must also be cells per low-­power field (Fig. 66.2) are considered to have significant
requested separately (Miller et al., 2018). contamination with saliva and should be rejected. Specimens with fewer
than 25 epithelial cells and more than 25 neutrophils per low-­power field
Specimen Collection and Transport are probably acceptable (Murray & Washington, 1975). The number of
Optimally, expectorated sputum is collected early in the morning before neutrophils is not usually considered as a sole criterion when determin-
eating. The individual should rinse his or her mouth with water and then ing specimen quality because the individual from whom the sputum was
expectorate a specimen, preferably 5 to 10 mL, resulting from a deep collected may be neutropenic. Screening the quality of sputum samples
cough. If mycobacteria are suspected, collection of three samples, at least submitted for detection of M. pneumoniae, Legionella spp., and mycobac-
one of which is an early morning specimen, is recommended. Additionally, teria is not generally required (Ingram & Plouffe, 1994; Havlik, 1995;
the patient should not rinse his or her mouth before producing a speci- McCarter & Robinson, 1996). The gram-­stained smears prepared from
men for mycobacterial workup because environmental mycobacteria that specimens that are acceptable for culture are examined under oil immer-
can be found in tap water such as Mycobacterium gordonae or M. avium-­ sion to determine the relative amounts of organisms. The quantity of
intracellulare could be introduced into the specimen. For persons with a organisms (rare, few, moderate, or many) is estimated for each kind of
nonproductive cough, a specimen may be induced by allowing the individ- bacterium (e.g., gram-­positive cocci in pairs [Fig. 66.3], chains, or clus-
ual to breathe aerosolized droplets of a solution of 3% sodium chloride for ters; gram-­positive bacilli; gram-­negative diplococci; and gram-­negative
about 20 minutes or until a cough reflex is initiated (Leber, 2016). Sputum rods), noting whether or not they are intracellular. Tracheal aspirates for
and tracheal aspirate specimens should be collected into a sterile container which no organisms are observed in the gram-­stained smear should prob-
and delivered promptly to the laboratory or refrigerated for a short time if ably be rejected (Morris et al., 1993). Portions of acceptable specimens
a delay is unavoidable. If Mycoplasma is suspected, the specimen should be containing purulent material are inoculated as outlined in Chapter 62. For
inoculated into liquid mycoplasmal growth media at the bedside if possible specimens from persons with cystic fibrosis, also inoculating a medium
because the organism is very sensitive to environmental conditions such as selective for Burkholderia cepacia is recommended. The most sensitive
drying (Leber, 2016).  method for detecting B. pertussis is PCR; a number of FDA-­approved

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 assays are commercially available (van der Zee et al., 2015; US FDA,
CHAPTER 66  SPECIMEN COLLECTION AND HANDLING FOR DIAGNOSIS OF INFECTIOUS DISEASES
2020). If PCR is not possible, B. pertussis culture should be performed as
described above for nasopharyngeal samples.
When Legionnaires’ disease is suspected, both Legionella culture and
a rapid, direct test (e.g., fluorescent antibody on a respiratory specimen
or Legionella antigen on a urine specimen) are recommended. DFA stain-
ing, which provides rapid results but is less sensitive than culture, can be
used to supplement culture. Urinary antigen testing also provides rapid
results but only detects L. pneumophila serotype 1 (Shimada et al., 2009).
PCR can be performed but is considerably more expensive than culture,
DFA, or urine antigen testing, and no FDA-­approved assays are avail-
able yet (Benitez & Winchell, 2013). Culture, the most sensitive of these
methods, can detect all Legionella spp. and serotypes and should always be
performed. Decontaminating the specimens with acid (KCl-­HCl buffer)
before plating may enhance recovery of legionellae from sputum (Mur-
doch, 2003). Several drops of the specimen should then be inoculated
onto each selective and nonselective buffered charcoal yeast extract agar
plate. Use of the selective agar inhibits the growth of most other respira-
tory flora; however, some strains of Legionella spp. are susceptible to the Figure 66.4  Cytologic preparation of bronchoalveolar lavage fluid shows an en-
medium’s inhibitory agents. Thus, a nonselective plate should always be larged cell with intranuclear and intracytoplasmic inclusions, consistent with cyto-
included. megalovirus. (Papanicolaou stain, 250×.) (Courtesy of Vicki J. Schnadig, MD, De-
For optimal detection of mycobacteria in sputum, the specimen must partment of Pathology, University of Texas Medical Branch, Galveston, TX.)
be decontaminated to prevent the normal respiratory flora from overgrow-
ing the slower-­growing mycobacteria. This process and detection methods
are discussed in Chapter 61. All specimens submitted for mycobacterial as possible. If a delay is unavoidable, the specimen should be stored in the
workup should be processed in a biological safety cabinet, preferably in refrigerator.
an isolated room with negative air pressure (Biosafety Level 3 laboratory). To collect bronchoalveolar fluid, the tip of a bronchoscope is carefully
All specimens submitted for fungal culture should also be handled as wedged into an airway lumen. A volume of saline (usually >140 mL) in
described for mycobacteria. The quality of the specimens should be deter- three to four aliquots is injected through the lumen, sampling an estimated
mined by screening with smears stained with Gram stain (as described ear- 1 million alveoli. The total volume returned varies based on the volume
lier for bacteria). Acceptable expectorated sputum, induced sputum, and instilled but is typically 10 to 100 mL. The specimen should be promptly
tracheal aspirate specimens should be inoculated onto culture media for transported to the laboratory (in <30 minutes) and processed as soon as
recovery of fungi. In general, to culture fungi, media with and without possible because saline may be toxic to some respiratory pathogens (Leber,
blood enrichment and media containing antimicrobial agents should be 2016). If a delay cannot be avoided, the fluid should be stored in the refrig-
used. However, when making media selection, the laboratory director also erator. A number of FDA-­approved NAATs for detection of respiratory
should consider cost and the types of fungus usually encountered in the viruses are available (US FDA, 2020). If a bronchoscopy specimen is being
patient population served by the laboratory. collected for testing by NAAT, the manufacturer’s instructions for collec-
If viscous, sputum specimens for P. jirovecii detection by DFA stain- tion and processing should be followed. 
ing are first treated with a mucolytic agent (e.g., N-­acetyl-­L-­cysteine or
dithiothreitol) and vigorously vortexed until the specimen is almost com- Specimen Processing
pletely liquefied. After centrifugation, the sediment is smeared on glass To process the protected brush specimen, the fluid in which the brush is
slides and fixed, with fixation technique dependent on the stain manufac- suspended is agitated on a vortex mixer, and the resulting suspension is
turer’s instructions. Slides are then stained and examined microscopically used for a cytospin preparation and for culture inoculum. The BAL sample
(Leber, 2016).  should be examined for small pieces of tissue; if present, they should be
placed in a sterile container, kept moist with sterile saline, and processed
in addition to the fluid (Sharp et al, 2004). If the specimen is viscous and
BRONCHOSCOPY SPECIMENS fungi (including P. jirovecii) are suspected, treatment of the sample with
Bronchoalveolar lavage (BAL) fluid and protected brush specimens are a mucolytic agent (e.g., N-­acetyl-­L-­cysteine or dithiothreitol) and sub-
useful for diagnosis of acute bacterial pneumonia, bacterial pneumo- sequent centrifugation should be considered to concentrate contents and
nia in ventilated patients who have not received antimicrobial therapy improve fungal recovery. The centrifuged specimen can then be used to
and for detection of opportunistic pathogens in immunocompromised prepare slides for staining (as discussed later) or inoculation to appropriate
patients with pneumonia (Baselski & Wunderink, 1994; Carroll, 2002). fungal culture media (outlined in Chapter 60).
BAL specimens may also be collected for suspected mycobacterial infec- Cytocentrifuge preparations can be useful for assessment of multiple
tion in patients who are unable to produce sputum (Holani et al., 2014). types of respiratory pathogens. For bacterial detection, staining cytospin
Although many have advocated quantitative cultures to improve the preparations with Gram stain is recommended because visualizing one or
specificity of cultures from the lower respiratory tract, recent reviews more bacteria per oil immersion field (in the absence of squamous epi-
have not shown an improvement in outcome for intubated patients thelial cells) strongly suggests acute bacterial pneumonia (Kahn & Jones,
compared with qualitative cultures (Caliendo et al., 2013). Only pro- 1987; Baselski & Wunderink, 1994). Examination of cytocentrifuge prepa-
tected brush specimens are suitable for anaerobic culture (Baselski & rations stained with Papanicolaou stain allows detection of cytopathic
Wunderink, 1994). For culture of Legionella spp., sputum specimens changes, especially useful for diagnosis of CMV pneumonia (Fig. 66.4)
are preferable because BAL samples are diluted with saline and may (Woods et al., 1990). Cytospin preparations may also be stained with an
contain small amounts of the anesthetic used locally, which inhibits the acid-­fast stain for detection of mycobacteria; with specific antibodies, such
organism. as those for detection of Legionella species or P. jirovecii; or with nonspe-
cific stains (e.g., silver stain, Calcofluor white, or Giemsa) for detection of
Specimen Collection and Transport P. jirovecii or other fungi.
A protected brush sample is collected with a small brush that holds 0.001 For bacterial culture, the laboratory may choose to inoculate plates
to 0.01 mL of secretions, placed within a double-­cannula enclosed cath- quantitatively either by the serial dilution method or the calibrated loop
eter. The outer cannula has a displaceable polyethylene glycol plug at the method (Leber, 2016); however, the use of quantitative cultures is contro-
tip. To obtain a specimen, the cannula is inserted to the desired area via versial, and pathologists should review their use with the clinical staff. The
bronchoscopy; the inner cannula is pushed out, dislodging the protective intent of this type of culture is to improve the specificity of the culture,
plug (water-­soluble); and the brush is extended beyond the inner cannula. with colony counts above a particular threshold appearing to correlate with
After the sample is taken, the brush is pulled back into the inner cannula, infection.
and both the brush and inner cannula are pulled into the outer cannula to For detection of mycobacteria, the specimen should be decontaminated
prevent contamination of the brush when the catheter is removed. The and handled as described in Chapter 61. Viral detection methods from
brush is then placed into 1 mL of sterile saline or broth. The specimen BAL specimens include NAAT and conventional cell culture, each further
should be transported immediately to the laboratory and processed as soon described in Chapter 64. 

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URINARY TRACT
The urinary tract above the urethra is sterile in healthy humans, but the
urethra is normally colonized with many different bacteria, so urine speci-
mens collected by a noninvasive method (e.g., clean-­ catch, midstream
specimen) become contaminated during their passage. Growth of a pre-

PART 7
dominant uropathogen in the quantity of 105 CFU/mL of bacteria or
greater of urine has been considered highly indicative of infection. Quan-
titative cultures have been used to help differentiate between contami-
nants and presumed pathogens, but the entity of asymptomatic bacteriuria
continues to confound culturing practices in the microbiology laboratory.
Asymptomatic bacteriuria is the presence of bacteria in urine, with or with-
out white cells, in patients (often women) who have no symptoms of uri-
nary tract infection. This has led to the unnecessary use of antimicrobials.
Some institutions have attempted to “screen” urine specimens, culturing
only specimens meeting predetermined criteria often involving numbers of
leukocytes or the presence of leukocyte esterase. Contemporary guidelines,
bolstered by the Choose Wisely campaign to reduce inappropriate ordering
of laboratory tests, instead focus on not culturing urine from nonpregnant,
asymptomatic women (Nicolle, 2019). Growth of 105 CFU/mL of bacte-
ria or greater of urine has been considered highly indicative of infection,
but lower thresholds are relevant in certain populations. For example, in
young, sexually active women with the acute urethral syndrome (dysuria,
frequency, and urgency), as few as 102 CFU/mL is considered significant
in the presence of concomitant pyuria (Stamm et al., 1982). True uri-
nary infections associated with fewer than 105 CFU/mL may also occur
in infants and children; in older men (Schaeffer & Nicolle, 2016); and in
persons who are catheterized, were recently treated with antimicrobial Figure 66.5 Technique for inoculating urine onto agar plates. (Redrawn from
agents, drink large amounts of fluids (which dilutes urine), have symptoms Woods GL, Gutierrez Y, editors: Diagnostic pathology of infectious diseases, Philadel-
and concomitant pyuria, have urinary obstruction, or have pyelonephritis phia, 1993, Lea & Febiger, p. 602.)
acquired from hematogenous spread (especially infections caused by yeast
and S. aureus). Consequently, proper interpretation of urine culture results
requires communication between clinicians and laboratory personnel. A 0.001-­mL loop is used to inoculate all urine specimens except those col-
lected from women with suspected acute urethral syndrome and supra-
pubic aspirates. Both the latter are inoculated with a 0.01-­mL loop. The
SPECIMEN COLLECTION AND TRANSPORT appropriate loop is inserted vertically into the well-­mixed urine sample,
Acceptable methods of urine collection include midstream clean catch and the loopful of urine removed is spread over the surface of the agar
(preferably a first-­voided morning specimen), catheterization, and supra- plate as illustrated in Figure 66.5. Without reflaming, the loop is again
pubic aspiration. In general, 24-­hour urine specimens should be rejected inserted vertically into the urine, and the removed sample is inoculated
except when detection of Schistosoma haematobium is requested specifically. onto a second plate.
Most commonly, the midstream flow of a clean-­catch urine is collected. Some bacteria are not detected by routine culture of urine; when these
For women, the periurethral area and perineum should be cleaned before pathogens are suspected, specific tests must be requested. For example,
the specimen is collected (Nicolle, 2019). The first few milliliters of urine urine is an acceptable specimen for detection of N. gonorrhoeae and C. tra-
are passed into the toilet bowel or a bedpan to flush out bacteria normally chomatis by NAAT. The manufacturer’s directions for urine collection and
colonizing the urethra, and the midstream portion is collected in a sterile processing must be followed. Leptospira interrogans may be detected in urine
container with a wide mouth and tightly fitting lid. after the first week of illness and for several months thereafter. To detect L.
Catheterization is associated with the risk for inducing a nosocomial interrogans in urine, the specimen should be processed as soon as possible
infection and should therefore be restricted to persons who are unable to after collection because acidity may harm the organisms. One or 2 drops of
produce a midstream sample, for example, individuals with an altered sen- undiluted urine and urine diluted 1:10 in broth are inoculated to 5 mL of
sorium or those unable to void for neurologic or urologic reasons. Using Fletcher’s medium or Ellinghausen-­McCullough-­Johnson-­Harris medium
strict aseptic technique, the catheter is inserted into the urethra, the first containing 5-­fluorouracil. Culture of urine for mycobacteria is discussed in
few milliliters of urine passed are discarded to clear organisms that may Chapter 61. Yeasts may be recovered from urine on the media plated for
have entered the tip of the catheter during placement, and the midpor- routine bacterial culture, but if fungal culture is specifically requested, the
tion of the sample is obtained for culture. Urine may be collected from sediment of a centrifuged urine specimen should be plated onto media such
an indwelling catheter by aspirating with a 28-­gauge needle and syringe as inhibitory mold or SABHI agar containing antibacterial agents.
through the rubber connector between the catheter and the collecting tub- Urine specimens collected for culture of viruses should be submitted
ing, taking care to first disinfect the puncture site. Urine should not be col- in liquid media containing antibiotics (e.g., penicillin, gentamicin, and
lected from catheter bags, and Foley catheter tips should not be accepted amphotericin B), or antibiotics should be added to the sample when it is
for culture because they almost always are contaminated with urethral received in the laboratory to minimize bacterial contamination of cell cul-
organisms. tures. PCR is more typically used than viral culture to identify viruses such
Suprapubic aspiration is used primarily for neonates. The procedure as CMV, adenovirus, and HSV. Detection of BK virus in urine may be
requires a full bladder; the overlying skin is disinfected, the bladder is requested; however, PCR, rather than culture, is recommended. Because
punctured above the symphysis pubis with a 22-­gauge needle on a syringe, BK virus PCR is offered primarily by reference laboratories, it is best to
and about 10 mL of urine is aspirated. contact that laboratory for collection and shipping requirements.
All urine specimens should be transported promptly to the laboratory More than half of urine specimens submitted to the clinical laboratory
and should be processed within 2 hours after collection. If a delay in trans- for culture yield no growth or have bacterial counts below levels considered
port or processing cannot be avoided, specimens may be refrigerated up to clinically significant. As discussed above, samples from asymptomatic, non-
24 hours. Collection kits containing preservatives to maintain the bacte- pregnant patients should not be cultured or screened for culture. Screening
rial population stable for 24 hours at room temperature are commercially tests are used to quickly identify urine samples which are more likely to
available and advantageous if refrigeration cannot be guaranteed.  have “positive” culture results and to provide rapid results, eliminate nega-
tive specimens, and allow more time for positive specimens, improving
efficiency and cost. In general, urine screen and culture results correlate
SPECIMEN PROCESSING well when 105 CFU/mL or greater is the reference, but they compare less
Quantitative bacterial culture of a urine specimen is done by inoculat- favorably in the presence of lower colony counts. Commercial automated
ing appropriate media with a measured amount of urine, most commonly screening methods have been developed but are either no longer avail-
with a plastic or wire-­calibrated loop designed to deliver a known volume. able or not actively marketed. Screening urine specimens by staining with

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 ENDOCERVICAL AND URETHRAL SPECIMENS
CHAPTER 66  SPECIMEN COLLECTION AND HANDLING FOR DIAGNOSIS OF INFECTIOUS DISEASES

Endocervical specimens are collected to determine the etiologic agents of

cervicitis and to identify asymptomatic persons infected with an organism
that cause sexually transmitted disease (e.g., C. trachomatis, N. gonorrhoeae).
Endocervical specimens are obtained after the cervix is visualized with the
aid of a speculum moistened only with warm water because lubricants may
contain antibacterial agents. If a Papanicolaou smear is indicated, that sam-
ple should be collected first.
Specimens for microbiological studies are generally collected with a
swab. Using a polyester-­tipped swab with a plastic shaft is recommended.
If a test kit is used for organism detection, the specimen must be collected
with the swab specified by the manufacturer. Before collecting specimens
for detection of N. gonorrhoeae and C. trachomatis, all discharge must be
removed from the cervical os. The swab or brush then is inserted 1 to 2 cm
into the endocervical canal (past the squamocolumnar junction), rotated
firmly against the wall for 10 to 30 seconds, withdrawn without touching
the surface of the vagina, and placed in the appropriate transport medium
Figure 66.6 “Clue cells” in a smear of vaginal discharge. (Papanicolaou stain,
or tube system used to prepare a slide for DFA staining (for C. trachomatis)
400×.) (Courtesy of Vicki J. Schnadig, MD, Department of Pathology, University of or used to immediately inoculate an agar medium for recovery of N. gonor-
Texas Medical Branch, Galveston, TX.) rhoeae. Specimen handling varies based on the organism sought.
NAAT is recommended for detection of N. gonorrhoeae and C. trachoma-
tis; several FDA-­approved assays are available for simultaneous detection of
Gram stain is economical, but because examination of smears is tedious and both organisms (US FDA, 2020). If molecular methods are not available,
time consuming, this approach rarely is adopted in the clinical laboratory. culture may be performed, though sensitivity is lower (CDC, 2014). To
The commercial dipstick test that combines nitrate reductase (an enzyme isolate N. gonorrhoeae, direct inoculation of a selective agar medium, such
present in most of the gram-­negative bacilli that cause urinary tract infec- as modified Thayer-­Martin, within a container to which a CO2-­generating
tions) and leukocyte esterase (an enzyme produced by neutrophils) is rapid, tablet is added, is optimal. Alternatively, the swab specimen can be placed
inexpensive, and simple to perform and can suggest that a urinary tract in a tube transport system and delivered to the laboratory within 2 hours.
infection is absent if both markers are negative, especially in symptomatic If a delay in transport cannot be avoided, the swab should be left at room
patients (Deville et al., 2004; St. John et al., 2006).  temperature and never refrigerated. Note that the viability of N. gonor-
rhoeae in Amies or modified Stuart medium substantially decreases after
several hours (Miller et al., 2018). For chlamydial culture, the specimen
GENITAL TRACT should be transported in a transport medium containing antibiotics (e.g.,
M4 medium) to inhibit overgrowth of bacteria and fungi. To maintain the
VAGINAL SECRETIONS viability of Chlamydia organisms, the specimen should be transported to the
Vaginal secretions are useful in determining the etiologic agent of vulvo- laboratory and processed immediately. If delays in transport or processing
vaginitis and bacterial vaginosis (BV). In women, the most common cause are unavoidable, specimens should be stored in the refrigerator if they can
of BV is overgrowth of Gardnerella vaginalis (in association with a variety be processed within 48 hours. Immunofluorescent staining (for chlamydial
of anaerobes), whereas vulvovaginitis is most commonly caused by Candida elementary bodies) and EIA are also available for chlamydial testing; the
spp. and Trichomonas vaginalis. manufacturer’s instructions for specimen handling must be followed.
Regardless of the testing method, vaginal secretions should first be col- To detect C. trachomatis and N. gonorrhoeae in men, a urethral swab
lected from the mucosal membrane of the vaginal wall using a sterile swab, specimen or first-­voided urine sample, depending on the detection method
with use of a manufacturer-­provided swab if a testing kit is utilized. used, should be obtained. Optimally, urethral swab specimens are collected
Molecular assays are generally most sensitive for diagnosis of vulvo- at least 2 hours after the patient has voided. Samples for N. gonorrhoeae are
vaginitis and BV. Three assays are currently available for simultaneous obtained first, which maximizes the bacterial load and thus the likelihood
detection of Candida spp., T. vaginalis, and G. vaginalis: a DNA probe assay of isolation (CDC, 2014). The conditions concerning types of swab and
(Affirm VPIII microbial identification system, Becton Dickinson), a quan- specimen transport are the same as those described for endocervical swab
titative multiplex PCR assay (NuSwab, Laboratory Corporation of Amer- specimens, although the smaller urogenital swabs are used. The swab is
ica Holdings), and an FDA-­approved microbiome-­based multiplex PCR inserted into the urethra for 2 to 4 cm, rotated in one direction for 5 sec-
assay (BD Max Vaginal Panel, Becton Dickinson) (Coleman & Gaydos, onds, withdrawn, and placed in the appropriate transport medium or used
2018). When testing by molecular methods, the manufacturer’s protocol to prepare smears for DFA staining to detect C. trachomatis or for Gram
for specimen collection, transport, and processing should be followed. staining to diagnose gonorrhea. Detection of intracellular gram-­negative
If BV is suspected, a Gram stain of vaginal secretions should also be diplococci in a smear of urethral discharge from symptomatic men pro-
considered. When interpreted with a standardized scoring system (e.g., the vides a presumptive diagnosis.
Nugent score), the Gram stain is the most specific test for diagnosis of BV For optimal detection of HSV, NAAT is recommended, with specimen
(Nugent et al., 1991). Culture is not recommended for diagnosis because handling as per the test manufacturer or reference laboratory to which the
G. vaginalis can be found in healthy women (Janulaitiene et al., 2017). sample is being sent. Alternatively, viral culture may be performed. The
Although several rapid point-­of-­care tests exist for diagnosis of BV swab specimen is placed in viral transport medium such as M4 and trans-
and vulvovaginitis (e.g., wet-­mount preparations, vaginal pH test, whiff ported as soon as possible to the laboratory. If immediate delivery is not
test), the sensitivity and specificity of these methods vary widely, and feasible, the specimen should be stored in the refrigerator. For patients
they are considered suboptimal (Miller et al., 2018). A wet mount is who have visible lesions on the cervix, a DFA may be performed on smears
performed by placing a swab of the discharge in a tube containing about prepared from scrapings of the lesion base, though sensitivity is only about
1 mL of normal saline, removing the swab from the saline, and press- 70% compared with NAAT (Miller et al., 2018).
ing the tip against a glass slide to express cellular material. The slide is Genital tract infection with human papillomavirus (HPV) is diagnosed
coverslipped and examined for “clue cells” (epithelial cells covered with using nonculture methods; culturing the virus in vitro is not possible.
small coccobacillary bacteria) (Fig. 66.6), consistent with the diagnosis Molecular techniques are most sensitive, with several available FDA-­
of nonspecific vaginosis; pseudohyphae, suggestive of candidiasis; and approved assays for testing of HPV genotypes associated with high risk for
motile trichomonads. Other point-­of-­care tests include the vaginal pH cervical cancer (US FDA, 2020). HPV-­induced squamous cell changes may
test and the whiff test. The vaginal pH is usually about 4.5 in women also be visualized on exfoliated cell samples (Papanicolaou smear). 
with vulvovaginal candidiasis but is above 4.5 in those with bacterial
vaginosis or trichomoniasis. A positive whiff test is the generation of
a pungent, fishy odor after addition of 10% potassium hydroxide to 1
VESICLES
drop of vaginal discharge placed on a slide or on the speculum. This Vesicular genital lesions are sampled to confirm HSV infection or (in chil-
is associated predominantly with bacterial vaginosis but occasionally dren) atypical VZV infection. The vesicle fluid is aspirated with a small-­
occurs with trichomoniasis.  gauge needle and syringe or capillary pipette. If only a small vesicle is

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 Lymphogranuloma venereum, caused by serovars L1, L2, or L3
of C. trachomatis, is typically diagnosed clinically. Serologic testing by
microimmunofluorescence-­IgG or complement fixation may be performed
adjunctively (Stoner & Cohen, 2015). Although cell culture on biopsy or
cellular material is possible, sensitivity is only 30%. Any overlying exudate
is first removed from the lesion, and then a swab is firmly rotated against

PART 7
its base. Specimen transport and processing are the same as discussed for
endocervical swab specimens.
K. granulomatis infection, which causes granuloma inguinale, is also
typically diagnosed clinically. For laboratory examination, a scraping from
an area of active granulation is taken, placed into formalin, and transported
to the laboratory. The scrapings are then stained by Giemsa or Wright’s
stain for visualization of Donovan bodies, which appear as blue rods with
prominent polar granules. 

FECES
Feces and, in some cases, rectal swab specimens are useful for determining
Figure 66.7  Multinucleate giant cell with intranuclear inclusions consistent with the etiologic agent of infectious diarrhea or food poisoning, confirming the
herpes simplex virus in a smear of endocervical cells. (Papanicolaou stain, 400×.) diagnosis of diarrhea caused by C. difficile or botulinum, detecting certain
(Courtesy of Vicki J. Schnadig, MD, Department of Pathology, University of Texas sexually transmitted pathogens (e.g., C. trachomatis, N. gonorrhoeae), iden-
Medical Branch, Galveston, TX.) tifying carriers of bacterial pathogens for infection control purposes (e.g.,
vancomycin-­resistant Enterococcus spp.), and in some instances, diagnosing
helminths of the respiratory and biliary tracts. Collection, transport, and
present, it is unroofed, and the base is firmly scraped with a Dacron swab to processing of specimens are different for viruses, bacteria, and parasites
ensure collection of cells. Vesicle fluid or swab specimens should be placed and are discussed separately for each group. Just as molecular and mass
in a viral transport medium. Testing by NAAT is optimal because it is most spectrometry methods have revolutionized the diagnosis of blood and
sensitive and can provide HSV typing information (Miller et al., 2018). respiratory specimens, they are similarly changing stool specimen diag-
HSV and VZV may also be detected in clinical specimens by viral cell nosis. There are numerous FDA-­approved multiplex molecular methods
culture or direct staining of smears, but these methods are less sensitive, for the rapid identification of bacterial, protozoal, and viral causes of diar-
and a negative result does not exclude the diagnosis. To make a smear, cells rhea, as well as assays for identification of C. difficile and its toxin(s) (US
collected with a Dacron swab are spread on a glass slide, and the material is FDA, 2020). Theoretically, this could eliminate routine stool cultures
fixed in acetone for 5 to 10 minutes. The smear may be stained with Papa- and ova and parasite examinations. However, as laboratories convert from
nicolaou, Wright’s, or Giemsa stain for detection of cytopathic changes culture-­based to culture-­independent methods, they must be aware of local
(HSV and VZV have an identical appearance) (Fig. 66.7) or with specific regulations regarding submission of specimens or isolates of public health
monoclonal antibodies.  significance to public health laboratories and the need to perform suscep-
tibility testing of certain pathogens (e.g., Shigella spp. and Salmonella spp.
ULCERS recovered from select age groups). Many jurisdictions ask that laboratories
continue to send the patient’s specimen for epidemiologic analysis.
Material from genital ulcers is collected to identify the responsible patho-
gen; HSV, Haemophilus ducreyi, Treponema pallidum, C. trachomatis (serovars VIRUSES
L1, L2, L3), and Klebsiella granulomatis should be considered.
For detection of HSV, specimen collection, transport and processing Specimen Collection and Transport
are identical to the protocol discussed for vesicles. In general, recovery Stool is preferred for detection of enteroviruses and the viruses responsible
of the virus and sensitivity of the tests previously described (i.e., NAATs, for gastroenteritis (e.g., astrovirus, calicivirus, enteric adenovirus, rotavi-
antigen detection, cell culture) are lower in ulcerative lesions compared to rus). Specimens should be collected in a clean container with a tight lid,
vesicular lesions. or in VTM, depending on the type of testing to be performed. If feces
Infection with H. ducreyi is typically diagnosed clinically (Workowski cannot be obtained, a swab is inserted beyond the anal sphincter, rotated,
et al., 2015). If laboratory testing is deemed necessary, NAATs are gener- withdrawn, and placed in VTM. Specimens should be delivered promptly
ally most sensitive; several strategies have been developed for direct testing to the laboratory; if this is not possible, fresh stool specimens can be stored
of clinical specimens, but there are currently no FDA-­approved molecular at 4°C for up to 3 days and transported on wet ice. If the specimen must be
assays for diagnosis of chancroid (West et al., 1995; Lewis, 2000; Chen & mailed to a reference laboratory, it should be stored at –70°C and shipped
Ballard, 2012). Gram stain and culture are also possible but are less sensi- on dry ice. 
tive and not recommended unless the laboratory is experienced in these
methods (Miller et al., 2018). For culture, a Dacron swab should be used to Specimen Processing
collect material from the base of the ulcer and directly inoculate a choco- NAAT is recommended for detection of viral causes of gastroenteritis and
late agar plate at the bedside. If bedside inoculation is not possible, the enteroviruses because of their speed and accuracy of viral detection. A vari-
swab should be transported in modified Stuart’s medium to the labora- ety of laboratory-­developed and commercial assays exist (US FDA, 2020).
tory and held at room temperature until processed. A swab specimen may Viral culture remains an alternative method for adenovirus and enterovi-
also be smeared onto a glass slide, Gram stained, and examined for small rus/parechovirus diagnosis, while EIA is an option for detection of rotavi-
pleomorphic gram-­negative coccobacilli in groups of chains resembling a rus and adenovirus 40 and 41 (Miller et al., 2018). Specimens are processed
“school of fish.” according to the manufacturer’s directions. 
T. pallidum, the causative agent of syphilis, is usually diagnosed
serologically. Spirochetes may be detected in genital or other lesions BACTERIAL PATHOGENS
by darkfield microscopy or DFA, but these methods are unavailable
in most laboratories. To collect specimens for spirochete detection, Collection and Transport
the surface of the lesion (if multiple lesions are present, the youngest The use of culture-­independent diagnostic tests for bacterial stool patho-
should be selected) is cleaned with saline and blotted dry, and crusts gens is rapidly increasing because of their rapid turnaround time and
are removed if present. The lesion is superficially abraded until slight improved sensitivity relative to culture (Shane et al., 2017). Many FDA-­
bleeding occurs, and gentle pressure is applied to its base. The clear approved NAAT-­based single-­or multiplex assays are available (US FDA,
serum exudate from the subsurface is collected by directly touching the 2020). Regardless of the testing method, stool is preferred for detection of
fluid with a glass slide or by aspiration with a 26-­gauge needle (followed pathogenic bacteria. Rectal swab specimens are an alternative but are less
by drawing up 1 drop of saline into the syringe) with transfer of the fluid sensitive for culture in adults (Miller et al., 2018). Swabs should generally
to a glass slide. A coverslip is placed on the fluid, and the specimen is be reserved for detection of N. gonorrhoeae, C. trachomatis, and Shigella spp.
examined immediately by darkfield microscopy for motile spirochetes and for assessment of vancomycin-­resistant Enterococcus (VRE) carriage
or stained for DFA testing. (Leber, 2016). Stool specimens should be collected in a clean container

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 with a tight lid, and the specimen should not be contaminated with urine, Stool specimens or gastric contents collected from persons with short-­
CHAPTER 66  SPECIMEN COLLECTION AND HANDLING FOR DIAGNOSIS OF INFECTIOUS DISEASES
barium, or toilet paper because these substances can affect pathogen via- incubation food poisoning should be evaluated for toxins from S. aureus
bility. Rectal swab specimens are obtained by inserting a swab past the and Bacillus cereus, and specimens from patients with long-­incubation food
anal sphincter (deep enough to become visibly stained with stool), gen- poisoning should be evaluated for Clostridium perfringens. This testing is
tly rotated, withdrawn, and placed in a tube transport system containing normally conducted by experts at public health laboratories, who should
appropriate transport media (e.g., Cary-­ Blair medium, manufacturer’s be contacted regarding appropriate specimen collection and transport.
transport medium for NAAT). All specimen types should be transported The clinical diagnoses of foodborne botulism and infant botulism may be
to the laboratory within 2 hours and quickly processed because the drop confirmed by detecting botulinal toxin, Clostridium botulinum, or both in
in pH that occurs as the stool cools may inhibit the growth of some patho- feces. Most clinical laboratories are not properly equipped to process speci-
gens, especially Shigella spp. If a delay in processing is unavoidable or if the mens from persons with suspected botulism. In the United States, when a
specimen must be sent to a reference laboratory, transport of an aliquot of case of botulism is identified, investigators at the CDC should be notified
the specimen in transport media such as Cary-­Blair is recommended. to ensure appropriate specimen collection and transport, diagnosis, treat-
Clinicians may consider testing a second stool specimen in adult ment, and investigation of the potential outbreak.
patients who initially test negative for bacterial pathogens, but this is rarely Diseases associated with C. difficile infection (CDI), such as pseudo-
necessary in pediatric patients. In adults, an enteric bacterial pathogen was membranous colitis and antibiotic-­associated diarrhea, are caused by the
detected in 87% of initial stool specimens, with a second specimen increas- toxins produced by the organism. In conjunction with clinical symptoms,
ing this rate to 98% (Rohner et al., 1997). However, in pediatric patients, several laboratory tests are available as diagnostic aids for CDI. These
98% of initial specimens were positive for a bacterial pathogen (Church include toxigenic culture (TC); the cell cytotoxicity neutralization assay
et al., 1995). (CCNA); EIAs and immunochromatographic assays for detection of glu-
Stool cultures for patients with diarrhea who have been hospitalized for tamate dehydrogenase (GDH) antigen, toxin A or B, or both toxins; and
more than 3 days are inappropriate unless the patient has been consum- NAATs. TC and the CCNA serve as two reference methods for C. difficile
ing food brought into the hospital. Offering C. difficile testing for health diagnosis; however, both are slow and labor intensive. A testing algorithm
care–associated diarrhea may be considered instead. For diagnosis of C. that uses NAAT alone or in combination with GDH or toxin detection
difficile disease, 20 to 50 mL of liquid stool should be submitted in a sterile by immunoassay is recommended as an alternative approach for C. dif-
container. The stool specimen is stable for 2 days refrigerated or 1 week ficile detection (Kraft et al., 2019). NAAT-­only testing has been shown to
frozen. Testing should be performed only on patients with three or more be highly sensitive and specific for detection of the C. difficile toxin genes
unformed stools in 24 hours and who have not recently received laxatives. A and B (Kraft et al., 2019). However, a NAAT-­only approach may be
Unless the patient has ileus, testing of formed stool should be avoided cost prohibitive for laboratories, especially when testing volumes are high.
because up to 15% of individuals are asymptomatic carriers, and testing Although immunoassays for GDH have low specificity for CDI because
may result in misdiagnosis and unnecessary treatment. Routine testing of they do not distinguish isolates that produce toxin from those that do not,
children younger than 2 years old is not recommended because toxigenic C. they have high sensitivity and a rapid turnaround time (15–45 minutes).
difficile colonization is common in this age group (McDonald et al., 2018).  Therefore, some have adopted a multistep algorithm by which samples
are first screened with a GDH assay. Specimens yielding negative results
Specimen Processing require no further testing, whereas GDH-­positive specimens are tested for
Processing stool or rectal swab specimens for detection of bacteria is based toxin(s) using EIA or NAAT (Leber, 2016).
on the organism or group of organisms expected to be present. Specimens For epidemiologic studies, C. difficile may be isolated from stool or from
received for “routine” bacterial culture should be processed to allow recov- rectal swab specimens placed in an anaerobic transport system. Because
ery of Shigella spp., Salmonella spp., and Campylobacter jejuni/coli by plating to many bacteria are present in stool, procedures that select for C. difficile
appropriate differential, inhibitory, and noninhibitory media. The microbi- must be used. The most effective medium for this purpose is cycloserine
ology laboratory director also might consider routinely examining stool for cefoxitin fructose egg yolk agar (CCFA), with or without horse blood. The
Shiga toxin–producing E. coli (STEC) and Aeromonas spp., depending on the organism grows more quickly and luxuriantly on formulations containing
prevalence of disease caused by these bacteria. The prevalence of gastroen- horse blood. The medium is incubated anaerobically for 48 hours, and the
teritis caused by Yersinia enterocolitica, Vibrio cholerae, or other Vibrio spp. or plates are examined for colonies that have a peripheral fringe and a “horse
Plesiomonas shigelloides is low in most parts of the United States; therefore, stable” odor. Alternatively, C. difficile may be isolated by using an alco-
specific requests for their detection are most cost effective. The patient pop- hol spore selection procedure, in which 1 mL of the original specimen is
ulation, season, and locale cause different laboratories to look for different mixed with 1 mL of absolute ethanol. The mixture is allowed to stand for 1
pathogens. For this reason, every report from a stool culture should list the hour at room temperature followed by subculture to CCFA and incubation
organisms that were identified or ruled out by the laboratory’s protocol. anaerobically.
To detect STEC, Shiga toxin EIA or NAAT is preferred because they Swabs for assessment of VRE carriage can be inoculated to selective
are more sensitive than culture and detect all serotypes of the organism media containing vancomycin or VRE chromogenic agar, with subsequent
(Pulz et al., 2003; Gavin et al., 2004). If culture is the only available method, NAAT-­based testing to differentiate vanA-­versus vanB-­containing isolates
the stool specimen is inoculated onto sorbitol-­MacConkey agar (contain- (Leber, 2016).
ing 1% d-­sorbitol instead of lactose), a medium that differentiates isolates With regard to mycobacterial culture, stool specimens usually are
of STEC, which do not ferment sorbitol, from almost all other E. coli, submitted for isolation of Mycobacterium avium complex (primarily from
which are sorbitol positive. When isolation of Y. enterocolitica is requested, patients with AIDS), but M. tuberculosis complex and other Mycobacterium
cefsulodin-irgasan-novobiocin (CIN) agar is inoculated and incubated at spp. also may be recovered. Processing the specimen (1–2 g of formed
room temperature. The organism also can be recovered by inoculating stool or 5 mL of liquid stool) involves decontamination and concentration,
media typically used for routine bacterial culture. A MacConkey plate may preparation of smears, and inoculation of media as discussed in Chapter 58. 
be incubated at room temperature for 48 hours. Colonies of Y. enteroco-
litica are purple and pinhead in size. Vibrio spp. frequently grow on the
media used for routine stool culture, but for their optimal recovery, thio-
PARASITES
sulfate citrate bile salts sucrose agar is inoculated. Plesiomonas shigelloides The backbone of diagnostic parasitology in clinical laboratories is exami-
also grows on media used for routine culture, but because up to 30% of P. nation of stool samples for parasitic protozoa and helminth eggs or larvae.
shigelloides isolates ferment lactose, their colonies do not appear sufficiently Laboratories performing such tests should have adequate facilities for han-
distinct to be recognized on these media, and screening all colonies for dling stool samples and a good microscope with a calibrated micrometer to
Plesiomonas is not cost effective. For this reason, culture of stool or rectal measure the organisms found. Staining of fecal smears is also required for
swab specimens for P. shigelloides should be requested specifically. Use of identification of intestinal protozoa. In addition to morphologic examina-
the selective-­differential medium inositol brilliant-­green bile salts agar has tion, several antigen-­or NAAT-­based commercial assays have been devel-
been suggested but is not essential. oped for diagnosis of intestinal parasites (e.g., Giardia spp., Cryptosporidium
Rectal swab specimens submitted for detection of C. trachomatis and N. spp.) as part of a gastrointestinal pathogen panel (US FDA, 2020).
gonorrhoeae are placed in transport medium and delivered promptly to the
laboratory or are refrigerated for a short time. NAATs are recommended Specimen Collection and Transport
for detection of these organisms because they have superior sensitivity to The specimen (usually collected by the patient) can be collected in a clean,
culture and are highly specific (Schachter et al., 2008). Although NAATs dry, wide-­mouthed container. A portion of the sample is then aliquoted
are not FDA approved for extragenital site testing, they can be validated into two vials containing preservatives, one with modified polyvinyl alco-
for this purpose. hol (PVA) and the other with 10% formalin. Fecal specimens should not be

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collected from the toilet bowl and should not be contaminated with urine, evaluation are described in detail in Chapter 60. Also discussed in that
water, mineral or castor oil, antidiarrheal compounds, or radiologic con- chapter are special stains required for detection of Cryptosporidium spp.,
trast medium because these can destroy organisms or interfere with their microsporidia, and Cyclospora spp. 
detection (Garcia, 2016). After being collected and preserved, the sample
should be delivered to the laboratory at the patient’s convenience. The use SKIN AND SUBCUTANEOUS LESIONS
of fresh stool is optimal for examination for motile trophozoites; however,

PART 7
it is usually very difficult to transport a fresh specimen to the laboratory FLUID-­FILLED LESIONS (VESICLES, PUSTULES,
and into the hands of the microbiologist within the 30 to 60 minutes after
collection required to visualize motile trophozoites. Although it is rarely
BULLAE, AND ABSCESSES)
possible to perform wet mounts on fresh stools for motile trophozoites, Fluid-­containing lesions can be from viral, bacterial, or fungal causes; the
they can still be identified on stained preparations, and the use of preserved appearance and distribution of the lesions suggest the most likely cause
specimens for examination minimizes the exposure of laboratory personnel and should guide the specimen collection strategy. Vesicles are often viral,
to live organisms. with HSV and VZV being common culprits. However, a bacterial cause
Several kits are commercially available for collection and transport of should be considered if vesicles are limited to the face or extremities, as in
fecal samples; the choice depends on testing to be performed (e.g., spe- nonbullous impetigo. Pustules are usually bacterial. Bullae that are gener-
cial stains, immunoassays, NAAT) and the test manufacturer’s recom- alized over the body are also typically associated with bacteria, especially
mendations for specimen fixative (Miller et al., 2018). The question of Staphylococcus spp., whereas bullae limited to the foot are usually fungal.
how many fecal specimens are required for identification of all individuals Finally, abscesses can be either bacterial or fungal, with anaerobic bacteria
with intestinal protozoa or helminths is still without a definitive answer. especially likely in the case of a closed abscess.
It has been advised that a minimum of three specimens be examined, with
one specimen collected every other day (Garcia et al., 2003; Clinical and Specimen Collection and Transport
Laboratory Standards Institute, 2005). For cost containment, the clinician Regardless of the cause, aspirated fluid is the optimal specimen for test-
should request examination of only one specimen because about 90% of all ing fluid-­filled lesions because this maximizes the concentration of viable
infections are diagnosed in the first sample (Montessori & Bischoff, 1987). pathogen while limiting contamination by extraneous microbes. Before
If a parasite is not detected in the first sample, a second or third should fluid collection for bacterial or fungal testing, the overlying skin should
be requested. If two or more specimens collected on different days are be disinfected and allowed to dry completely. However, if viral testing is
received in the laboratory at the same time, it has been suggested they be desired, skin disinfection should be avoided because antiseptic agents may
pooled and evaluated as one (Peters et al., 1988). This practice, however, inactivate viruses (Williamson et al., 2017).
is controversial. Stool examinations for parasites in patients who have been Fluid can be aspirated using a needle and syringe. If viral testing is to be
hospitalized for more than 3 days are inappropriate unless they are immu- performed, the fluid should be immediately rinsed into VTM. Otherwise,
nocompromised (Siegel et al., 1990).  the needle can be removed from the syringe and the syringe capped for
transport to the laboratory.
Specimen Processing If fluid aspiration is not possible, swabs may be used for sampling.
The examination of stool samples for parasites includes preparation of Flocked swabs are preferable to nonflocked swabs because they yield
saline solution and of iodine (Lugol’s)-­stained wet mounts of freshly col- greater organism recovery (Van Horn et al., 2008). Any overlying crust
lected samples (i.e., those that can be examined within a few hours of col- should first be gently removed and the lesion unroofed if necessary. One
lection). This is followed by concentration of cysts and helminth eggs and swab should then be used to absorb any fluid present, and a second swab
finally by preparation of smears for staining with the trichrome stain. The should be used to vigorously rub the base and margins of the lesion. A
wet preparations, if performed, should be made and examined before doing minimum of two swab specimens should be collected: one for culture and
the concentration and trichrome staining. Even if a parasite is seen in the the other for preparation of smears for staining. However, if more than
wet mount, examination of a concentrated specimen is recommended one group of organisms (e.g., bacteria and fungi, fungi and viruses) is to be
because specimens submitted for ova and parasite examination may con- tested, collecting at least three swab specimens is recommended.
tain multiple parasites. In particular, helminth eggs are often only visible Swabs for bacterial or fungal testing may be placed into a commercially
in the concentrate. available swab transport system, with use of an anaerobic transport system
The standard fecal smear is made from a fresh fecal sample as follows: if anaerobes are being considered. If viral causes are suspected, at least one
1 drop of saline solution is placed on a clear glass slide. With an applicator swab should be placed into VTM. In addition, viral immunofluorescent
stick, a small amount of feces is picked up and with circular movements is staining (e.g., for HSV and VZV) may be helpful; slides should be prepared
mixed thoroughly with the saline (until enough sample is dissolved), and at the bedside by rolling the entire surface of the swab over a glass slide
the mixture is covered with a 22-­mm square cover glass. A good wet smear and allowing the material to air dry. All specimens should be promptly
prepared as described, if placed on a paper with small print, should allow transported to the laboratory to limit loss of fastidious organisms and over-
the print to be read through the smear. The smear stained with iodine is growth of contaminants. 
prepared in the same manner. Both the saline solution and the iodine solu-
tion wet preparations can be made on the same glass slide, at the same Specimen Processing
time mixing the feces with the saline solution first and then with the iodine Processing specimens for detection of bacteria or fungi involves prepara-
solution. The saline solution smear will show trophozoites and cysts of pro- tion of a smear for staining with Gram stain (for bacteria) or Calcofluor
tozoa, plus all the helminth eggs and larvae. In addition, the saline solution white (for fungi) and inoculation of appropriate media for culture. Addi-
shows movement of trophozoites, which is useful for their identification. tionally, if fungi are suspected, the fluid should be examined for grains or
The smear stained with iodine does not show trophozoites because they granules with a dissecting microscope because their presence indicates a
are destroyed by the iodine unless the sample was previously fixed. The mycetoma (Walsh et al., 2018). If present, grains and granules should be
main advantage of the iodine stain is that it allows better visualization of teased out of the specimen, washed in sterile solution, and examined for
some morphologic characteristics of cysts. To prepare wet mounts from color (Leber, 2016). One portion should be crushed between two glass
formalin-­fixed material, the contents are mixed well, and 1 drop is placed slides for examination for hyphae, and a second portion should be crushed
directly onto the glass slide and onto 1 drop of iodine solution. and inoculated directly onto appropriate media.
Examination of the saline solution smear is carried out with medium Methods for viral detection include NAATs, antigen detection, and cell
power (10× objective) at first. Beginning at the upper-­left corner of the culture, any of which can be used to test fluid or swabs in VTM. Swab spec-
cover slide, the slide is moved horizontally from right to left. The operation imens received in VTM should be vigorously agitated on a vortex mixer,
is repeated until the entire 22-­mm square cover glass is examined. All hel- with removal and disposal of the swab. Specimens can be briefly refriger-
minth eggs or larvae and all protozoal cysts should be noted. Examination ated until they are processed. 
with the high-­power objective is done next for detection and identification
of protozoa, looking randomly for about 5 to 10 minutes per preparation. WOUND INFECTIONS
Note that pathogenic E. histolytica and nonpathogenic Entamoeba dispar
cannot be distinguished based on morphology alone; an immunoassay or Specimen Collection and Transport
NAAT is needed for species-­level identification (Tanyuksel & Petri, 2003). Optimal wound sampling methods include aspiration (collected and trans-
The concentration technique, which can be done both on fresh and ported as discussed earlier for fluid-­filled lesions), tissue biopsy, and curet-
fixed specimens, is particularly useful because it allows detection of organ- tage. Swabs are far less helpful because they often are contaminated by skin
isms present in low numbers. The concentration methods used for routine commensal bacteria, leading to diagnostic confusion (Lipsky et al., 2012;

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 Nelson et al., 2018). All specimens should be delivered promptly to the The diagnosis of the ulceroglandular form of tularemia, caused by F.
CHAPTER 66  SPECIMEN COLLECTION AND HANDLING FOR DIAGNOSIS OF INFECTIOUS DISEASES
laboratory. If transport will be delayed, inject a portion of the aspirate into tularensis, requires collection of a swab specimen from the ulcer base. The
an anaerobic transport tube. Tissue samples are transported in a sterile cup swab should be processed in a biological safety cabinet for routine bacterial
with moist sterile gauze to prevent desiccation. If a delay is unavoidable, culture, and the plates should be held for seven days because F. tularen-
specimens may be stored in the refrigerator, except those for anaerobe sis is a slowly growing bacterium that produces pinpoint colonies in 3 to
recovery, which should be maintained at room temperature.  5 days on chocolate agar but does not grow on sheep blood agar. Suspi-
cious isolates should be referred immediately to the nearest public health
Specimen Processing laboratory.
Specimens should be Gram stained and inoculated to appropriate media Mycobacteria most commonly isolated from cutaneous ulcers include
for culture, as discussed in Chapters 59 and 62.  Mycobacterium fortuitum, Mycobacterium abscessus, Mycobacterium chelonae,
Mycobacterium marinum, and Mycobacterium haemophilum. Mycobacterium
ulcerans is a common cause of skin lesions in some parts of the world but
ULCERS is difficult to culture. Exudate aspirated with a needle and syringe or a
Ulcers may be primarily infected (e.g., those caused by viruses, Bacillus tissue sample is optimal for recovery of mycobacteria. Swab samples are
anthracis, C. diphtheriae, Francisella tularensis, P. aeruginosa, mycobacteria, not acceptable because mycobacteria become entrapped in the fibers of the
or fungi) or secondarily colonized with aerobic and anaerobic bacteria, as swab and are difficult to dislodge. Transport of specimens is identical to
is typical for chronic ulcers. that described earlier for wound infections. Specimens may be refrigerated
Specimens for viral detection from cutaneous ulcers are collected and for a short time until they are processed. Because some species of mycobac-
transported identically to those described above for fluid-­filled lesions. For teria infecting the skin and extremities grow better at lower temperatures
bacterial, mycobacterial, or fungal isolation, handling of specimens differs or with additional growth factors, it is important that personnel processing
by suspected cause. specimens are aware of the specimen source.
B. anthracis is the cause of cutaneous anthrax, a rare disease in the For fungal detection, an aspirate of the exudate (handled as described
United States that occurs among persons working with raw wool and other earlier for aspirates of fluid-­filled lesions) or tissue sample from the active
animal products contaminated with spores. For optimal diagnosis, speci- margin of the ulcer is optimal. A swab specimen of the exudate is subop-
mens should be sent to a public health laboratory where NAAT can be timal. Processing the specimen for detection of fungi involves preparing a
performed. The public health laboratory’s instructions for specimen col- smear for direct microscopic examination (potassium hydroxide or Calco-
lection and transport should be followed. If specimens are processed in the fluor white preparation) and inoculation of appropriate media for culture.
clinical laboratory, they must be handled in a biological safety cabinet. The Chronic skin ulcers are often colonized by aerobic, facultative, or
swab is inoculated onto sheep blood agar and incubated in ambient air. anaerobic bacteria. To identify the responsible organisms, cultures of deep
C. diphtheriae causes cutaneous diphtheria, an ulcerative lesion covered tissue biopsy (after thorough debridement) or deep aspiration of purulent
with a layer of necrotic debris resembling a membrane. For optimal diag- material by needle and syringe are most useful. Surface cultures of such
nosis, a smear for staining with methylene blue is prepared from material ulcers are not valuable because they usually represent colonizing bacteria
from the edge of the membrane, and two swab specimens from the mem- rather than the causative agent (Miller et al., 2018). Transport and process-
brane itself are collected. One swab is used for routine bacterial culture and ing of tissue and aspirated material are identical to those described earlier
the other for inoculation of media selective for C. diphtheriae (e.g., cysteine for wound infections.
tellurite agar) if available.
Ecthyma gangrenosum is an ulcerative cutaneous lesion that almost
always occurs during bacteremia with P. aeruginosa. Ideally, two swab spec-
ACKNOWLEDGMENT
imens are collected from the ulcer base; one is used to prepare a smear for We gratefully acknowledge the contributions of Ann C. Croft and Gail L.
Gram staining and the other for culture. Woods, who authored this chapter in previous editions.

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