Online Medical Reference

The Microbiology Laboratory
and the Internist

Anthony K. Leung

Gary W. Procop

Published: August 2010

General Principles

Clinical laboratories play a vital part in health care in the United States. Traditionally, clinical laboratories are comprised of the following sections: blood banking, chemistry, hematology, and microbiology. In a university or academic setting, these laboratories are usually under the department of clinical pathology, with the supervision of a board-certified pathologist or microbiologist. Many independent commercial laboratories are now available to provide timely and reliable services to clinicians and hospitals when such services are not locally available. With the advent of molecular and genomic medicine, newer divisions and subspecialties have been created within the arena of clinical pathology. For example, some larger academic and research institutions have divisions that specialize in incorporating molecular methods into their practice.

The primary role of a microbiology laboratory in patient care is to aid clinicians in identifying causative agents of various infectious diseases and help determine the antimicrobial susceptibility profiles, when appropriate.1 The information provided by the microbiology laboratory enables the clinicians to initiate or modify therapy, which will have a direct impact on patient outcome. The role of the laboratory begins when patient specimens are received in the laboratory; the laboratory should process and handle the specimens appropriately and in a timely fashion—that is, in a manner that ensures that the integrity of the specimen will not be compromised. Depending on the type of specimens and tests requested, the specimens will be worked up accordingly by performing different testing procedures, and accurate and reproducible results will be generated. Clinicians will then use the information obtained to make clinical decisions and initiate treatment options best suited for that particular patient. To facilitate the process, it is of the utmost importance that clear communication exists between the clinician placing the order and the laboratory professionals performing the tests. This communication is critical because it facilitates efficient use of laboratory resources, as well as improving turnaround time for test results.

Ideally, the microbiology laboratory should be under the supervision of a physician or laboratory scientist with expertise in both infectious diseases and microbiology. The insight provided by such a laboratory-based professional helps ensure that quality and clinically relevant information will be provided to health care providers in a timely manner. The microbiology laboratory should be in compliance with the rules and regulations established by the College of American Pathologists (CAP), Clinical Laboratory Improvement Amendments (CLIA) and the Joint Commission (formerly called the Joint Commission on Accreditation of Healthcare Organizations). An accredited microbiology laboratory should be routinely inspected by the CAP or the Joint Commission, to ensure that all standardized laboratory methods and procedures are being followed. Many safety aspects of operating a laboratory will also be evaluated during such an inspection.

CLIA-88 was passed in Congress in 1988 and the final regulations were published in 1992. The purpose of this legislation was to establish that all laboratories that perform clinical testing should provide timely, reliable, and accurate results to clinicians and patients, regardless of where the test was performed. This act has defined that laboratory tests fall under three categories—waived complexity, moderate complexity, and high complexity. Tests that require moderate and high complexity must be performed in a certified laboratory by trained medical technologists. Some of the tests currently available in the marketplace are CLIA waived, which means that testing can be performed outside a laboratory setting. Some examples of these include home pregnancy tests, which may be purchased directly by the consumer (i.e., sold over the counter) and rapid HIV-1 and HIV-2 antibody tests, which can be performed in an outpatient clinic or emergency room.

Medical technologists are an integral part of a microbiology laboratory. These technologists usually possess a baccalaureate degree and have received additional training in accredited medical technology programs in hospitals, colleges, or universities. On completion of this training, they must take the certifying examination offered by the American Society of Clinical Pathologists (ASCP). Only after they have achieved the appropriate certification can the technologist start working in an accredited laboratory. Some technologists choose to be a generalist, meaning that they can rotate to different sections of the laboratory, such as chemistry and microbiology, and perform different assays; conversely, they may elect to specialize in one particular area to gain further experience and expertise.

Communication between the microbiology laboratory and clinicians remains the most important aspect of patient care. The role of the laboratory is to provide advice and guidance to clinicians to optimize specimen collection. Therefore, the laboratory should provide easy access of such information to clinicians and also clearly state the rejection criteria for various tests. With the advent of communication tools such as the Internet, critical clinical information can be provided to clinicians efficiently and in a timely fashion.

It is also prudent to remember that the role of the microbiology laboratory is to identify the causative agent from various specimens, which in turn aids the clinicians to make or confirm a diagnosis, or both. However, it should also be emphasized that the laboratory is providing clinically relevant and useful information. Otherwise, if the information is misleading or inaccurate, inappropriate therapy may be prescribed to the patient. This would increase health care costs and expose patients to unwarranted treatment; unnecessary and overused antimicrobial therapy is an important cause of the emergence of antimicrobial resistance.

The following example illustrates this point. A patient with a chronic indwelling catheter for total parenteral nutrition (TPN) administration is admitted to the hospital for general malaise and tiredness. However, no fever is reported. The patient is seen in the emergency department and a single sample of blood is drawn from the catheter for culture while the patient is en route to being admitted to the hospital. Three days later, that single sample indicates growth and a preliminary Gram stain reveals gram-positive cocci in clusters. The diagnosis of catheter-related infection is made and vancomycin therapy is initiated. Subsequently, the isolate from the blood culture is identified as a coagulase-negative Staphylococcus and the clinician requests to have antimicrobial susceptibility testing performed on that isolate. Clinically, the patient is doing better after hydration, and repeated blood culture fails to demonstrate any evidence of catheter infection. The positive predictive value of a single sample for blood culture in this setting for a true infection is low; this isolate likely represents a contaminant, given that the isolated organism is a coagulase-negative Staphylococcus species. By providing the requested susceptibility, one assumes that the clinician has determined that this isolate is the cause of an infection and that the patient should be treated with an antimicrobial agent. However, in this scenario, no therapy is warranted and the patient improved after proper hydration and adjustment to the TPN formulation and discharged home. In such a case, vancomycin could have been discontinued on speciation of the blood isolate and antimicrobial susceptibility testing was unnecessary; both added to the health care costs, but also implied that the isolate was associated with a bloodstream infection. The usefulness of and issues regarding blood culture will be discussed in more detail.

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Specimen Requirements

Regardless of the specimen type, it is important for the laboratory to receive an adequate amount of specimen for testing. Unfortunately, it is not uncommon for the laboratory to receive a single swab with requests for viral, aerobic and anaerobic bacterial, fungal, and mycobacterial cultures. The yield of a culture is directly proportional to the amount of specimen initially inoculated onto the primary recover medium. Therefore, to obtain optimal results, a sufficient amount of specimen should be submitted. For example, when submitting an aspirate of an abscess, the clinician should submit more than a few drops of fluid or a swab for culture. Insufficient quantity of specimen will affect the yield of final culture and produce false-negative culture results. The quantity issue is particularly significant if the suspected pathogen is a mycobacterium or filamentous fungus, for which swabs are unacceptable and usually a larger quantity of tissue, fluid, or aspirate is needed.

When the clinician submits specimens to the laboratory for testing, all relevant information, such as the date and time of collection, type of specimen, and brief clinical scenario should be provided on the requisition form. The information is helpful for the laboratory to determine which growth medium to use to optimize growth. In certain cases in which a particularly virulent organism is suspected, the form should clearly indicate this to alert the laboratory technologist to handle the specimens with special safety measures and precautions, such as using a biologic safety cabinet. For example, this should be done if Brucella or Francisella is suspected clinically, because Petri plates from wound and blood cultures are usually handled on the bench, rather than in a biologic safety cabinet. For some diseases, such as tularemia, caused by Francisella tularensis), serologic tests may be the preferred method of diagnosis rather than culture, given the highly contagious nature of this organism and the fastidious growth requirements in vitro. In addition, lymph nodes can be submitted for histopathologic examination, although at times it is difficult to separate tularemia from other granulomatous diseases such as tuberculosis and cat-scratch disease. Serology usually is the preferred method for diagnosing tularemia.

In addition to the issue of quantity, when submitting a specimen, it should be of a quality acceptable for testing. The microbiology laboratory should therefore clarify which types of specimens are acceptable for microbiologic testing. These rejection criteria should be written and be readily available to those collecting the specimens, as well as to the technicians responsible for their processing. A poor-quality specimen is of no use to anyone and may yield unreliable results that could potentially affect patient care. Poor-quality specimens may produce false-negative results, because true pathogens may be overgrown by normal flora, and false-positive results; also, a commensal potential pathogen may be mistaken for the true causative agent of disease.

Blood

Blood culture is the cornerstone of diagnosing many infectious diseases, and is of particular importance for determining the causative agent of endocarditis and other endovascular infections.2 Different methods and commercial systems are now available for detecting microorganisms. Generally, the blood culture system uses a broth-based medium that supports the growth of aerobic and anaerobic bacteria, depending on the bottle type used, and also some fungi, predominantly yeasts.

A major reason for failing to detect microorganisms in the bloodstream is an inadequate sampling of blood volume. When the recommended blood volume is inoculated (10 mL/bottle for an adult) into the blood culture bottles, most of the bacteremic or fungemic episodes can be recovered from two or three sets of blood cultures.2 The number of bottles used per draw and the number of draws per blood culture order can be confusing. A single blood culture order consists of two blood culture draws from different anatomic sites, preferably not through an IV catheter. Each of these draws, in turn, consists of filling two blood culture bottles with 10 mL of blood each; usually one aerobic and one anaerobic bottle comprise a blood culture set. It should be emphasized that the recommended volume of 20 to 30 mL is for each set of blood cultures, and that at least two sets are used for each blood culture order (i.e., four bottles total).

Further discussion is warranted regarding how many sets of blood cultures should be drawn to detect bacteremia, fungemia, or both in septic patients. A single set of blood cultures is inappropriate for detecting such septic episodes. Some of these are characterized by intermittent bacteremia, and a single set would not be sensitive enough to isolate the causative agent consistently. The clinical objective is to send enough blood to evaluate for microorganisms adequately, without sending so many sets as to produce iatrogenic anemia, which is a real risk in hospitalized patients undergoing numerous blood draws for various tests.

There is little controversy when highly pathogenic microorganisms such as Staphylococcus aureus or Pseudomonas aeruginosa are isolated from the bloodstream. However, a single set of blood cultures with coagulase-negative staphylococci, Corynebacterium, or other gram-positive bacteria consistent with skin flora could represent blood culture contaminants or a true infection (i.e., the positive predictive value of a single blood culture draw is low). In other words, it is difficult to determine the clinical significance of such blood culture findings, because these organisms are part of the normal human skin flora. Clarity may come from assessing the clinical findings in such patients. For example, the patient may have clear evidence of infection associated with an indwelling catheter site. In such a case, clinicians should have a high index of suspicion of an active case of infection, even if it is a single set of blood cultures. It would then be necessary to combine the clinical findings with other laboratory findings (e.g., an elevated neutrophil count) to help interpret the findings of the single blood culture set that turned positive with a common skin commensal.

It is recommended that what would truly be helpful here is an additional blood culture set. Drawing two blood cultures from different sites not only increases the volume sampled, but also helps diagnose infections caused by common skin commensals. The likelihood of a true infection being caused by one of these organisms is increased if more than one blood culture set is positive for the same type of organism—for example, two of two blood culture sets are all found to contain coagulase-negative staphylococci. This is especially true for patients with catheters; the isolation of coagulase-negative staphylococci could be a clue to a catheter-related infection, because its course is usually indolent. Thus, a good rule of thumb is that two or three sets of blood cultures with adequate blood volume should be used and can detect most cases of bacteremia and fungemia.

Prolonged incubation of blood cultures for the detection of fastidious or slow-growing bacteria is controversial. This was a common practice before the use of highly sensitive automated blood culture systems. The concept is to provide more time for the more fastidious organisms to grow, thereby increasing the chances of recovery. This was classically used to detect the HACEK organisms (Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae), which were well-described causes of “culture-negative” endocarditis. However, the newer blood culture system technology and improvements in blood culture media have resulted in recovery rates for these organisms that are almost as good as those for more common pyogenic bacteria, such as Staphylococcus aureus.3 Nevertheless, some will still extend incubation or perform terminal subcultures if a fastidious pathogen is a suspected cause of endocarditis. In our experience, we have seen culture and biopsy-proven cases of Propionibacterium acnes endocarditis that grew only after extended incubation.

This matter is controversial because it is also well known that the longer a blood culture is incubated, the higher the likelihood that the organisms that grow will be a contaminant, particularly if it is part of the normal skin microbiota. The issue is that even small numbers of contaminating bacteria such as diphtheroids and coagulase-negative staphylococci will grow in the nutrient-rich environment of the blood culture bottle and, given enough time (i.e., extended incubation) will yield positive results. In such cases, these contaminants might be treated as true pathogens, thus exposing patients to unnecessary antibiotics and potentially increasing the length of hospital stay.

Sputum and Other Respiratory Tract Specimens

Diagnosing lower respiratory tract infections and pneumonia remains complicated and requires a combination of clinical and laboratory findings for optimal diagnosis. Expectorated sputum, even with its own limitations, remains the most common and easiest specimen to obtain for diagnosis. However, when a sputum sample is submitted to the microbiology laboratory for routine bacterial culture and stain, clinicians should be familiar with some of the issues regarding this type of specimen.4 The specimen is first smeared onto a glass slide and Gram staining is performed as a screen. A high-quality purulent sputum specimen should contain many neutrophils and only relatively few epithelial cells. If many epithelial cells are seen—specifically if more than 25 epithelial cells are seen per low-power field—the specimen is contaminated with upper oropharyngeal flora (i.e., saliva) and may not even be from the lower respiratory tract. In such a case, even if microorganisms are seen on Gram staining, it cannot be ascertained whether the organism in question is a pathogen or simply part of the normal upper respiratory tract flora.

It is important to remember that many organisms in the upper respiratory tract (e.g., Streptococcus pneumoniae) are also causes of pneumonia. Thus, the positive predictive value of Gram staining and the culture is low for predicting the cause of pneumonia if the sputum specimen is contaminated (i.e., not representative of the lower respiratory tract). Therefore, sputum specimens contaminated with upper respiratory tract contents, as indicated by the presence of excessive squamous epithelial cells, should be rejected and another specimen should be obtained if clinically indicated. It is often helpful to use a respiratory therapist to obtain an induced sputum specimen using hypertonic saline.

If the diagnosis cannot otherwise be confirmed, it may be necessary to perform a bronchoalveolar lavage (BAL) for better assessment of the lower respiratory tract. BAL lavage fluid is the best specimen for diagnosing lower tract infections. This type of specimen is obtained by performing a bronchoscopy, a procedure that requires a physician’s time, partial anesthesia, and some degree of risk. When properly done, the chance of contamination by upper respiratory flora is minimized. In addition to sampling the lower respiratory tract for culture and cytologic testing, this procedure affords tissue sampling—transbronchial biopsy—if indicated. More recently, mini-BAL aspirates from endotracheal or tracheostomy tubes have been submitted instead of BAL for patients on mechanical ventilation. These specimens are easier to obtain than those obtained by traditional BAL and are often used as a surrogate specimen to diagnose lower respiratory tract infections, especially ventilator-associated pneumonia (VAP). Although helpful, it should be emphasized that VAP is difficult to diagnose by respiratory culture alone.5 These specimens, like sputa, may be contaminated with upper respiratory tract flora and therefore have similar limitations. The specificity of diagnosing VAP may be increased by combining several findings, such as respiratory culture, fever, new infiltrate, and leukocytosis. Unfortunately, many specimens obtained from tracheal aspirates contain a mixture of both gram-positive and gram-negative bacteria. The positive predictive value for such specimens is also low and the presence of these organisms might or might not be associated with lower respiratory tract infections. Thus, interpreting culture results is difficult; historical and clinical information should also be included to help determine the presence or absence of infections.

Finally, it is prudent to remember that Gram staining is only a screening tool to aid clinicians in diagnosis. The classic causes of atypical pneumonia, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella species, do not stain (M. pneumoniae and C. pneumoniae) or do not stain well (Legionella) with Gram staining. The diagnosis of infections by these agents uses other methodologies such as serology testing, antigen detection, polymerase chain reaction (PCR) assay, or a combination of these. An astute clinician should have a high index of suspicion for pneumonia if the history, physical examination, and radiograph are suggestive of such an infection, even if the Gram staining result is nondiagnostic or negative for microorganisms, and appropriate empirical antimicrobial treatment should be given.

Stool

Patients with diarrheal illness could have a spectrum of clinical presentations, ranging from mild, self-limited, loose stools, with minimal to moderate abdominal discomfort, to severe abdominal pain, with or without bloody stool. Many bacterial, viral, and parasitic organisms are capable of causing gastrointestinal (GI) infections. It is beyond the scope of this chapter to cover this exhaustive list and many excellent texts and references are available to discuss these pathogens more fully. However, some key concepts should be emphasized to help the clinician obtain maximal laboratory data to make an accurate diagnosis and treat patients appropriately. The laboratory, at the same time, is challenged to perform cost-effective and efficient assays while helping clinicians achieve their diagnostic goals.

Diarrheal illness generally can be divided anatomically into those that predominantly affect the small intestine and those that affect the large intestine. Pathogens that affect the small intestine usually cause a malabsorptive type of symptoms, with loose watery stools. Some classic examples include Giardia lamblia and Cryptosporidium parvum infections. Infections that affect the large intestine usually manifest with diarrhea caused by colitis that may be bloody; these patients may be toxic or even develop septic shock if the infection is severe enough. Some large intestinal pathogens include Salmonella, Shigella, Campylobacter, and Clostridium difficile.

To investigate patients with diarrhea in a hospital setting, a former practice (no longer supported) was to order stool culture and ova and parasite testing (three times) as an initial investigation. However, studies have suggested that there is low predictive value to these tests for patients admitted to the hospital for longer than 3 days, unless the clinical history, epidemiology, and examination suggest an infectious origin.6 Therefore, stool cultures for typical enteric bacterial pathogens and examinations for parasitic causes of diarrhea should be limited to outpatients with diarrhea, patients with diarrhea on admission, or those who develop it within the first few days of admission. Thereafter, the likelihood of diarrhea being caused by these agents is extremely remote.

It is important to note that most cases of diarrhea develop because of noninfectious causes, and may be medication related. C. difficile nonetheless deserves special attention for hospitalized patients. It has been shown that C. difficile–associated pseudomembranous enterocolitis is the main cause of hospital-acquired diarrhea, and clinicians should maintain a high index of suspicion for this disease under the appropriate clinical circumstances. Patients with leukocytosis, fever, loose watery stools, and abdominal pain should undergo further investigation and C. difficile should be ruled out. The absence of these signs, however, does not specifically diagnose or exclude this disease; the diagnosis is confirmed by testing the stool, usually for toxins produced by this organism. Ideally, the stool should be loose and free-flowing.

The definitive diagnosis of C. difficile infection is made by the detection of the toxins—namely, toxins A and B in the stools of patients suspected of having this disease. The toxins can be detected by enzyme-linked immunoassay (EIA) but, traditionally, the tissue culture method is used for the detection of toxin B. With this method, a cytopathic effect (CPE) is produced that may be blocked using antitoxin. In many laboratories, combined toxins A and B EIAs have replaced tissue culture, generally because they have a more rapid turnaround time and are less labor- and resource-intensive. However, it should be noted that EIA has a sensitivity of only 75% to 85% and a strong clinical suspicion should be entertained, even if there is a negative C. difficile toxin EIA result.

Recently, it has been suggested that in addition to toxins A and B detected by EIA, there is also a binary toxin-producing strain of C. difficile.7 Strains of C. difficile that produce this toxin usually produce a more fulminant and severe colitis in infected patients. Moreover, strains of C. difficile that possess this toxin are also hyperproducers of toxin A or B, or both. Because it is a hyperproducer of toxin, this newly recognized “epidemic” strain of C. difficile will be readily detected by commonly used assays, but its specific type will not be detected.

A question that arises is in regard to how many C. difficile tests should be ordered to confirm the diagnosis. It has become common practice among house officers to order “C. difficile times three” to rule out such infection. This practice was extrapolated from a study by Manabe and colleagues.8 They found that the sensitivity of EIA is 72% with the first specimen, 84% with the second, and 93% with the third. However, the number of patients required to have three specimens submitted to confirm the diagnosis was small. Moreover, it was found that the negative predictive value with the first stool was 97%. Increasing the number of stool specimens will not increase the sensitivity of detecting C. difficile toxin that was not detected in the first two specimens. Therefore, the routine practice of sending stool for C. difficile three times should be discouraged. Each C. difficile order should be made uniquely by an astute physician who is considering the clinical and laboratory findings, and the inherent limitations of each.

Wound, Wound Drainage, and Cerebrospinal Fluid

Management of wound culture presents some difficult challenges to the microbiology laboratory. Many issues need to be addressed in regard to these types of cultures:

  • What is the appropriate method to collect specimens?
  • Do swabs have any roles in bacterial cultures?
  • What is the clinical significance of cultures that contain mixed flora?
  • Should wound drainage (e.g., Jackson-Pratt drain) be cultured routinely to rule out active infections?

These are some questions that should be carefully considered before submitting specimens to the laboratory.

The most effective way to treat an abscess or infected fluid collection is to drain the abscess in conjunction with antimicrobial therapy. Rarely, an abscess can be cured just by medical treatment. Occasionally, fluid to be collected is located in a deep structure in which percutaneous or even surgical drainage is deemed too risky for the patient. In such cases, prolonged antimicrobial therapy is the only viable option and these cases should be followed closely to assess clinical response. In some other cases, a radical surgical procedure might be the best option for managing chronic infection. For example, patients with chronic empyema who have undergone multiple surgical procedures for decortication may be best managed using a Clagett open-window thoracostomy for drainage.

When obtaining specimens from a wound, it is advisable to submit the aspirated fluid in a syringe after removing the needle to help ensure the survival of anaerobic bacteria; an anaerobic transport vial is another alternative. A wound swab is not an appropriate method to collect specimens to assess for anaerobes. Quantity is always an important issue, because the more material one submits, the higher the chance of recovering the causative agent of disease. That is, swabs only hold a limited amount of fluid and anaerobic bacteria may die in transit; the submission of fluid is a superior technique. When submitting a piece of tissue, it should be placed in a sterile container and transported to the laboratory as soon as possible. removing potentially infected tissue and then swabbing it is inappropriate and will yield suboptimal results. The label should clearly contain the name and identification number for the patient and include the type of specimen, source, and a brief clinical description. The requisition, paper or electronic, must denote the microbiologic studies to be performed. This allows for timely, efficient, and accurate processing of specimens.

As noted, swabs generally are the least desirable way to collect a specimen for a wound culture. Organisms isolated from a wound culture obtained by swabbing the wound surface reflect the colonization of that wound (i.e., these do not reflect the organism that may be causing a deeper infection). This is particularly true for a chronic wound, which usually harbors multiple organisms, such as Pseudomonas aeruginosa, Enterococcus species, and Staphylococcus aureus, all of which could be true pathogens in another (deeper) location. In such a case, it is impossible to predict whether the isolated organisms are true pathogens or simply superficial colonizers; that is, there is a poor positive predictive value for a poor wound culture. In this era of health care cost containment one has to ask, “Why then even do it?” To determine the cause of a wound infection definitively, it first needs to be determined clinically whether a wound is thought to be infected. Thereafter, a surgical approach is desired in which the superficial material is débrided and a biopsy of the wound base is submitted for culture. Some clinicians have used screening criteria, which examines the proportion of squamous epithelial cells and neutrophils, to determine the usefulness of subsequent culture. Such approaches may be helpful, particularly in settings in which a high percentage of wound cultures simply reflect superficial colonization.

It has become common practice in many institutions to submit fluid from a surgical drain for culture, even if there is no evidence of infection. It is thought—again, this is not supported—that this is a surrogate marker to diagnose occult infections. However, this practice has been shown to have almost no clinical value for the above-mentioned purposes. Moreover, it may mislead clinicians and result in the institution of inappropriate therapy. Everts and colleagues9 have shown that there is poor correlation between fluids obtained from a drainage catheter versus those directly aspirated from the collection. Moreover, this group has shown that radiologic studies such as computed tomography (CT) or magnetic resonance imaging (MRI) are the best modalities for diagnosing deep tissue infections.

The appropriate assessment of cerebrospinal fluid (CSF) is absolutely crucial in managing patients with suspected cases of meningitis or encephalitis. The basic tenet for collecting CSF is similar to that for other specimens, but particular attention should be given to the aseptic technique used during collection. Before performing a lumbar puncture, the skin should be thoroughly cleansed with iodophor or chlorhexidine. The patient should preferably be lying in a recumbent position; occasionally, for obese patients it might be easier to obtain the fluid by asking the patient to sit upright and hunch forward to better accentuate the L4-L5 interspace. However, in such a position, the opening pressure cannot be accurately measured. In certain central nervous system infections, an assessment of the initial opening pressure is absolutely crucial for managing the patient. For example, a patient with Cryptococcus neoformans meningitis may have an elevated intracranial pressure (ICP), with few or subtle clinical signs and symptoms. Knowing the patient’s baseline ICP will assist clinicians and neurosurgeons regarding the performance of shunting, if necessary.

The quantity of CSF is especially crucial and as much fluid as possible should be obtained, because multiple tests are usually ordered and some tests, such as CSF for cytology, require a large amount of sample. For certain pathogens, such as fungi and mycobacterial species, the microbial recovery is directly proportional to the volume of sample used to inoculate the cultures. Therefore, at least 10 to 20 mL of CSF should be submitted to the laboratory for testing. The clinical findings should dictate the type of testing to be performed.

Viral Cultures

Viral diseases are caused by a heterogeneous mixture of viruses and, depending on each virus’s unique pathogenesis, the clinician needs to submit the appropriate specimen for testing. Many methodologies can be used for diagnosing viral diseases, such as serologic (antibody and antigen testing), viral culture, and molecular methods, such as the PCR assay and related techniques. Diagnostic virology will not be discussed in detail here; this section will focus specifically on viral cultures.

Molecular methods such as PCR have gained popularity and acceptance in recent years. This methodology is indispensable, especially in the field of clinical virology, One major drawback is the cost of running a molecular assay. Typically, tests using molecular methods are more expensive than conventional viral cultures. Consequently, viral cultures are still being used in many laboratories, especially small hospitals and laboratories in which the volume of testing might be low, and cost containment is important.

When submitting specimens for viral cultures, the principles of aseptic technique, volume, and timely submission of the specimens still apply. However, it should be noted that the specimens are usually stored in a viral transport medium (e.g., M4 medium) to keep the viruses viable. Moreover, submitting specimens with swabs made from calcium alginate can affect the recovery of herpes simplex and varicella zoster virus.10 If the specimens cannot be processed in a timely fashion, the general rule for keeping the virus viable is to refrigerate the specimens. The clinician should check with the laboratory regarding the specific types of virus collection, transport media, and storage options before obtaining specimens.

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Conclusion

The clinician and the microbiology laboratory work synergistically to provide optimal care to patients. Open communication between them enables proper collection of specimens and allows appropriate testing to be done. This enables cost-effective and efficient reporting of microbiologic results, which in turn enables the clinician to treat the patient effectively and in a timely fashion. The clinician should also supply adequate clinical information to the laboratory to facilitate proper specimen processing and testing.

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Summary

  • The clinical microbiology laboratory is an integral part of managing patients with various infectious diseases.
  • Clinicians and laboratory personnel should maintain frequent communication so that accurate and clinically relevant test results are available in a timely manner.
  • Clinicians should be aware of the limitations and understand the concepts of sensitivity, specificity, and positive or negative predictive value of different microbiology-related assays.
  • When submitting specimens to the clinical microbiology laboratory, clinicians should recognize the importance of the quality and quantity of specimens required for that specific assay.

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Suggested Readings

  • American Thoracic Society; Infectious Diseases Society of America: Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005;171:388-416.
  • Carroll KC: Laboratory diagnosis of lower respiratory tract infections: Controversy and conundrums. J Clin Microbiol 2002;40:3115-3120.
  • Everts RJ, Heneghan JP, Adholla PO, Reller LB: Validity of cultures of fluid collected through drainage catheters versus those obtained by direct aspiration. J Clin Microbiol 2001;39:66-68.
  • Manabe YC, Vinetz JM, Moore RD, et al: Clostridium difficile colitis: An efficient clinical approach to diagnosis. Ann Intern Med 1995;123:835-840.
  • McDonald LC, Killgore GE, Thompson A, et al: An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 2005;353:2433-2441.
  • Mead PB: Proper methods of culturing herpes simplex virus. J Reprod Med 1986;31: 390-394.
  • Peterson LR, Hamilton JD, Baron EJ, et al: Role of clinical microbiology laboratories in the management and control of infectious diseases and the delivery of health care. Clin Infect Dis 2001;32:605-611.
  • Washington JA II: Blood cultures: Principles and techniques. Mayo Clin Proc 1975;50: 91-98.
  • Weinstein MP: Emerging data indicating that extended incubation of blood cultures has little clinical value. Clin Infect Dis 2005;41:1681-1682.
  • Yannelli B, Gurevich I, Schoch PE, Cunha BA: Yield of stool cultures, ova and parasite tests, and Clostridium difficile determinations in nosocomial diarrheas. Am J Infect Control 1988;16:246-249.

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References

  1. Peterson LR, Hamilton JD, Baron EJ, et al: Role of clinical microbiology laboratories in the management and control of infectious diseases and the delivery of health care. Clin Infect Dis 2001;32:605-611.
  2. Washington JA II: Blood cultures: Principles and techniques. Mayo Clin Proc 1975;50:91-98.
  3. Weinstein MP: Emerging data indicating that extended incubation of blood cultures has little clinical value. Clin Infect Dis 2005;41:1681-1682.
  4. Carroll KC: Laboratory diagnosis of lower respiratory tract infections: Controversy and conundrums. J Clin Microbiol 2002;40:3115-3120.
  5. American Thoracic Society; Infectious Diseases Society of America: Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005;171:388-416.
  6. Yannelli B, Gurevich I, Schoch PE, Cunha BA: Yield of stool cultures, ova and parasite tests, and Clostridium difficile determinations in nosocomial diarrheas. Am J Infect Control 1988;16:246-249.
  7. McDonald LC, Killgore GE, Thompson A, et al: An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 2005;353:2433-2441.
  8. Manabe YC, Vinetz JM, Moore RD, et al: Clostridium difficile colitis: An efficient clinical approach to diagnosis. Ann Intern Med 1995;123:835-840.
  9. Everts RJ, Heneghan JP, Adholla PO, Reller LB: Validity of cultures of fluid collected through drainage catheters versus those obtained by direct aspiration. J Clin Microbiol 2001;39:66-68.
  10. Mead PB: Proper methods of culturing herpes simplex virus. J Reprod Med 1986;31:390-394.

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