Review of the patient's history and symptoms combined with knowledge of the microorganisms that cause infection at specific sites (e.g., E. coli is the most common cause of urinary tract infection in young women, whereas S. pneumoniae is the most common cause of pneumonia at all ages) allows one to order the appropriate investigations to document the site of infection and the infecting microorganism.
Nonspecific Methods
Symptoms and physical signs are frequently supportive of a diagnosis of infection but rarely are pathognomonic. For example, the activation of the acute inflammatory response is the most common way in which the clinical manifestations of infection become apparent. However, noninfectious conditions may also activate the same inflammatory mechanisms; therefore, the symptoms and signs of inflammation are by no means specific for infection.
Granulocytosis, the appearance of immature neutrophilic forms in the circulation and the presence of toxic granulation are typical of moderate-to-severe acute bacterial infections. This type of inflammatory response is related to the organism that is causing infection, the bone marrow reserves of the host, and other features of the host. In contrast, neutropenia may occur in any patient with overwhelming gram-negative bacterial sepsis or in certain patients (particularly alcoholics) with severe bacterial pneumonia caused by gram-positive organisms. Neutrophilic leukocytosis or leukopenia may be seen during the active phases of vasculitis, systemic lupus erythematosus, or acute drug reactions, all of which can mimic the host response to infections. Tests that attempt to measure the quality of granulocyte response to bacterial infection by incubating blood with nitroblue tetrazolium dye (NBT test) and counting the percentage of cells with reduced dye are not reliable. At present, the major value of the NBT test is in the screening of selected subjects for chronic granulomatous disease of childhood or one of its variants. Similar comments can be applied to the appearance of transformed (atypical) lymphocytes in the circulation during fever. Initially thought to be specific for infectious mononucleosis, this finding is now known to appear with regularity in patients with other disorders, including cytomegalovirus infection, acute human immunodeficiency virus infection, adenovirus infection, and occasionally toxoplasmosis. Such lymphocytes also may be difficult to differentiate from the immature forms of cells seen in patients with acute lymphocytic leukemia.
Principles
A single nonspecific laboratory test (e.g., examination of peripheral white blood cells) that is simply a marker of the activation of the acute inflammatory response is a poor way to evaluate the presence or absence of infection.
Certain laboratory tests add information by helping to define the presence of abnormalities in the organ systems of the infected host. Since many microorganisms have tissue tropism, these nonspecific methods of evaluating organ function may narrow the search for the site of infection. An abnormality in biochemical tests usually related to liver function (e.g., elevated concentrations of transaminases) and a tender liver on physical examination strongly suggest hepatitis but obviously do not differentiate among the many causes of inflammation of the liver. When the spectrum of noninfectious disorders that can mimic infections is included in these considerations, the possibilities for misdiagnosis and misapplied therapy become legion. Therefore, more specific methods are required to document infection so that rational therapy can be planned. Appropriate specimens should be collected (to make an etiological diagnosis) before instituting therapy. A “diagnostic therapeutic trial” of treatment is the least reliable way of revealing the specific nature of infections and is often synonymous with therapeutic misadventure.
| Table Source and Biologic Properties of Cytokines | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Principles
Antimicrobial therapy for infectious diseases should not be chosen on the basis of nonspecific methods of diagnosis and “probabilities” alone. Such treatment may cause more harm than good.
Specific Methods
The sampling of host tissues and body fluids for biochemical, histologic, and microbiologic testing remains the cornerstone for the diagnosis of a specific infection. Performance of such studies provides a data base to direct rational therapy and is mandatory in the seriously ill patient. The lack of adequate sampling of appropriate sites and the failure to adequately transport specimens to the laboratory are the most frequent and unrecognized reasons for failure to document the etiologic agents of an infectious disease. For example, an improperly collected urine sample (not placed in a sterile container and delayed in delivery to the laboratory) often will be contaminated by large numbers of bacteria bearing little relationship to the presence or number of the true urinary tract pathogen.
Samples for microbiologic identification
The host site to be sampled for the microbiologic identification of infecting agents is critical. Since many epithelial surfaces have their own commensal flora, enumeration of organisms per sample volume and weight may be important in differentiating the commensals from pathogens. For example, the presence of 105 and 103 colony-forming units per milliliter of clean catch midstream-voided urine is widely recognized as representing significant bacteriuria in women and men, respectively. However, fewer bacteria in urine can still represent an infection. Studies have conclusively demonstrated that acute dysuria in women with cystitis can be caused by as few as 102 colony-forming units enteric gram-negative bacilli per milliliter of urine. Therefore, it is always important for the clinician to correlate the microbiologic data with the patient's symptoms and physical signs. If dissemination of infection has taken place, samples taken from distant sites may be helpful. Thus, pneumococcal pneumonia may be documented by sampling respiratory secretions, but the strength of the diagnosis is increased by the finding of pneumococci in blood cultures.
| Table Common Infectious Syndromes Associated with Immune Deficiency | ||||||||||||
|
||||||||||||
| Table Evans's Five Realities | ||||||||||||
|
Blood cultures are frequently submitted when any evidence of dissemination of infection is suggested by systemic signs and symptoms. Any abnormal collections of fluid associated with the signs of infection should be sampled for culture. Tissue biopsy may be indicated for certain types of infections. This is particularly important in identifying the pathogens in infected immunocompromised patients (e.g., patients with acquired immunodeficiency syndrome or neoplastic disease), since the number of possible pathogens in these patients is much greater than in immunocompetent patients. The necessity for these biopsies is also influenced by weighing the seriousness of the disease against the risk of the biopsy procedure. Histologic examination of tissue samples, obtained by biopsy, with special staining techniques augments the classic microbiologic laboratory testing.
Proper transport of specimens to the laboratory requires prompt delivery under appropriate conditions to protect and allow survival of the organisms for laboratory growth or identification. Although many organisms require only the minimum moisture and nutrients of “routine” transport media, fastidious organisms may require complicated special media for transfer.
Anaerobic microorganisms may be sufficiently fragile to require oxygen-free transport systems to ensure their survival. Viral agents may require special solutions containing antibiotics to suppress bacterial growth, and ultralow temperatures for transfer and storage before inoculation into susceptible cell systems for identification. The clinician must be aware of the requirements for collecting and transporting clinical specimens correctly so that the best chances of a definitive diagnosis of infection are achieved. Discussion with laboratory personnel provides the most efficient method for ensuring that the most appropriate steps are taken.
| Table Suggested Specimens to Be Submitted for a Bacteriologic and Mycologic Diagnosis | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Table Suggested Specimens to Be Submitted for Viral Diagnosis | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Histologic examinations of stained smears remain the most rapid, inexpensive, and useful method of preliminary recognition of classes of infectious agents. The classic Gram stain allows differentiation of microorganisms into certain groups by size (small, large), form (cocci or bacilli), and staining (gram-positive or gram-negative) characteristics. This rough classification combined with other nonspecific data may provide sufficient information to allow appropriate empiric therapy before a definitive diagnosis (culture) is confirmed. Other commonly used, inexpensive, and simple rapid-staining techniques include acid-fast staining for mycobacteria, methylene blue stains and KOH or Calcofluor White preparations for fungi, and India ink preparations for the recognition of cryptococci. Dark-field microscopic examination for spirochetes, wet preparations for motile organisms, and phase-contrast examination are other convenient methods that take advantage of distinctive biologic characteristics of living microorganisms for their identification.
Cytologic examination, utilizing standard stains of infected cells scraped from body surfaces, may identify intracellular pathogens (e.g., Chlamydia trachomatis) and inclusion bodies that indicate infection by selected intracellular microorganisms. Cytomegalovirus and herpesvirus are among those agents that commonly cause formation of inclusion bodies. In addition, the use of fluorescent-labeled antibodies that bind to antigens of specific pathogenic microorganisms provides a rapid, definitive histologic test to confirm infection by a specific etiologic agent including herpesviruses, Legionella species, and C. trachomatis.
There has been increasing interest in developing new laboratory tests for the diagnosis of infectious diseases. This rapidly evolving field is based on the availability of new reagents derived from molecular biology and on the ability to automate testing. Ultimately, cost effectiveness will dictate whether these new diagnostic tests will be commonly employed. However, molecular biologic techniques have enabled investigators to produce monoclonal antibodies and genetic probes for a number of fastidious or difficult-to-detect microorganisms. Diagnostic kits that employ these new reagents may be superior to the current conventional tests, especially when these tests incorporate improved antibody-antigen detection and genetic amplification (e.g., the polymerase chain reaction [polymerase chain reaction] techniques).
Principles
The rational choice of effective treatment for infectious diseases depends on diagnostic accuracy. The physician must be certain that the appropriate specimens have been obtained and that they have been properly transported and processed to ensure accuracy and reliability of test results.
Serologic approaches to documentation of infection
Cultures of selected microorganisms may be unavailable because the methodology is not available or adequate (e.g., viral hepatitis), is unsafe for laboratory personnel (e.g., rickettsiae), or is impractical (e.g., Chlamydia species or certain viruses). A common reason for negative cultures is the use of antimicrobial agents before the culture was taken. Under these circumstances, a serologic approach to documentation of infection might be diagnostically or epidemiologically important. The host mounts nonspecific and specific defenses against various infective agents (e.g., humoral antibodies).
There are many nonspecific (e.g., VDRL for syphilis) and specific (e.g., streptolysin O for group A streptococci) antibody tests that are important diagnostic tools. It is not within the scope of this section to list the many available specific serologic tests. In general, the presence of specific IgM antibodies in high titer indicates a recent or current infection and has been particularly useful in diagnosing some viral infections and toxoplasmosis. Demonstration of a fourfold rise in specific immunoglobulin G antibody between acute and convalescent serum samples is also a useful indicator of recent infection. Sera should be obtained at an interval of 2 to 3 weeks to permit adequate time to lapse for the formation of detectable amounts of immunoglobulin G antibodies. The presence of immunoglobulin G antibody in a single serum specimen, although it indicates prior exposure to the agent, is usually of little assistance in diagnosing a current illness. Such tests are not necessary for the majority of viral infections when the natural history of disease is self-limiting and short. Serologic tests are particularly important in patients suspected of having syphilis, human immunodeficiency virus infection, hepatitis, rickettsial diseases, invasive parasitic diseases, and fungal diseases (e.g., coccidioidomycosis).
Diagnosis of human immunodeficiency virus infection and monitoring of response to treatment
The conventional algorithm for the laboratory diagnosis of human immunodeficiency virus infection in Western countries includes an initial screening test of serum or plasma. The test is repeated if initially reactive, followed by a confirmatory test, usually a Western blot. For more than a decade, the screening test of choice for the qualitative detection of antibody to human immunodeficiency virus-1 and human immunodeficiency virus-2 has been based on enzyme immunoassays (EIA) that are commercially available and licensed for use in the diagnostic laboratory. Generally, these EIAs are packaged in kit format and designed for large-volume testing. The test kit consists of a solid support such as a polystyrene 96-well microtiter plate coated with recombinant and/or synthetic proteins as antigens derived from known regions of the human immunodeficiency virus-1 and human immunodeficiency virus-2 genome. All necessary test reagents including reactive (positive) and nonreactive (negative) controls are included in the test kit. The assays are performed either manually or, usually, with the aid of semiautomated or fully automated instruments. The results are interpreted according to the manufacturer's instructions using absorbance (optical density) reading of appropriate internal controls to establish a cutoff value for nonreactive results. These EIAs generally have a sensitivity of 99.71 to 100% and a specificity of 99.83 to 99.94%.
Likewise, human immunodeficiency virus-1 and human immunodeficiency virus-2 Western blot test kits are commercially available to confirm sera found to be repeatedly EIA-reactive. Because of the relatively low prevalence of human immunodeficiency virus-2 infection, most diagnostic laboratories tend to use human immunodeficiency virus-1 Western blot test as the only confirmatory test, whereas reference laboratories provide assistance to confirm suspected human immunodeficiency virus-2. In the Western blot assay, nitrocellulose paper strips blotted electrophoretically with individual proteins of an human immunodeficiency virus-1 lysate are reacted with test serum samples. Antibodies to any of the major human immunodeficiency virus-1 proteins (p) or glycoproteins (glycoprotein) can be visualized as colored bands on the strips. Strongly positive, weakly positive, and negative controls are provided in the commercial Western blot test kit to serve as guides in the interpretation of test results. Recommended criteria have been proposed for interpretation of Western blot results (CDC 1989a; Consortium for Retrovirus Serology Standardization 1990). A negative Western blot result requires the
absence of any and all bands — not just viral bands. All other patterns are regarded as indeterminate.
| Table Major Genes and Gene Products of human immunodeficiency virus-1 | |||||||||||||||||||||||
|
|||||||||||||||||||||||
| Table Criteria for Positive Interpretation of Western Blot Results | |||||||||||||||||||||||
|
|||||||||||||||||||||||
Laboratories generally report the human immunodeficiency virus testing results as one of the following:
- human immunodeficiency virus-1 and human immunodeficiency virus-2 EIA-negative
- human immunodeficiency virus-1 and human immunodeficiency virus-2 EIA-positive and Western blot human immunodeficiency virus-1 positive
- human immunodeficiency virus-1 and human immunodeficiency virus-2 EIA-positive and Western blot human immunodeficiency virus-1 indeterminate
- human immunodeficiency virus-I and human immunodeficiency virus-2 EIA-positive and Western blot human immunodeficiency virus-1 negative
Individuals having Western blot-indeterminate testing results are usually advised to be retested, usually at 3 months and 6 months for those at low risk, and sooner for those at moderate or high risk for human immunodeficiency virus infection. Retesting is necessary as indeterminant results may be obtained in persons who have been recently infected and are in the process of seroconverting. In most cases, however, indeterminate results arise because the serum of uninfected individuals contains antibodies that will cross-react with tissue antigens present in the Western blot test strip. This occurrence has been reported in 20 to 30% of human immunodeficiency virus-negative blood samples. In patients without risk factors, almost all indeterminant results will fall into this group. If the Western blot test does not become positive in retesting at the 3-month intervals, the individual should be considered human immunodeficiency virus-negative by the physician despite persistent indeterminate results.
The course of human immunodeficiency virus-1 infection leading to the development of the acquired immunodeficiency syndrome varies considerably among infected individuals. Some infected persons rapidly progress to acquired immunodeficiency syndrome in less than 5 years, whereas others remain asymptomatic without evidence of immunological decline for more than 6 years. The median interval from infection to acquired immunodeficiency syndrome in adults is 10 to 11 years. This variable course of human immunodeficiency virus infection has created some degree of uncertainty among clinicians in the clinical management of human immunodeficiency virus-1–infected persons. Clinicians have used many clinical and laboratory markers to predict disease progression and to assess the efficacy of therapeutic drug regimens. These surrogate markers include human immunodeficiency virus-related symptoms, depletion of CD4 receptor-positive (CD4+ T cells, human immunodeficiency virus-1 p24 (core) antigenemia, serum neopterin and B2-microglobulin levels. These markers are indirect and have limitations in sensitivity and specificity.
Although the percentage or absolute number of circulating CD4+ T cells has been the best-known and most commonly used surrogate markers for acquired immunodeficiency syndrome, these numbers do not always correlate with the disease state. Some human immunodeficiency virus-1–infected persons with very low CD4+ T cell counts (so-called CD4 counts) remain healthy, whereas others with comparatively high CD4 counts experience fulminant disease. Another complicating factor is the varied sources that can lead to variability in CD4 count determination. Known factors contributing to CD4+ T-cell count variability in the circulating blood have been described: diurnal cycle, the use of tobacco, consumption of caffeine and alcohol, and a variety of physiological and nonphysiological stresses. As with many other laboratory tests,
CD4+ T-cell count determination is subject to technical variability within and between laboratories. Therefore, there is a need for alternative markers that can be used for rapid and reliable assessment of prognosis and therapeutic outcome.
Earlier observations based on the quantitation of infectious human immunodeficiency virus-1 virus in peripheral blood mononuclear cells and freshly isolated plasma by culture techniques have shown that increasing virus titers were associated with CD4+ T-cell decline and disease progression, whereas virus titers decline in response to effective therapy. These findings draw attention especially to the potential usefulness of human immunodeficiency virus-1 viral load measurement as a laboratory marker for the in vivo antiretroviral activity of various therapeutic regimens. Unfortunately, the culture techniques that measure human immunodeficiency virus-1 virus load in peripheral blood mononuclear cells or plasma are laborious and time-consuming and suffer as well from laboratory-to-laboratory variation due to inherent difficulty in standardization. Attempts using molecularly based technologies such as polymerase chain reaction to quantify human immunodeficiency virus-1 RNA in plasma have also shown that reduction in viral load was associated with increased CD4+ T-cell counts and prolonged acquired immunodeficiency syndrome-free survival.
These encouraging observations have provided the impetus to the diagnostic industry to make available viral load measurement systems in kit format that are molecularly based and are readily adaptable to diagnostic laboratories. At present, three commercial molecularly based assays are licensed by the Federal Drug Administration in the United States for human immunodeficiency virus RNA plasma viral load quantitation. All three assays are currently being used to measure viral burden in relation to disease progression and response to antiretroviral therapy. The International acquired immunodeficiency syndrome Society–USA convened an ad hoc panel of investigators and clinicians to make recommendations for the use of these assays in clinical practice.
Recent studies evaluating the ability of these three commercial assays to measure viremia levels accurately in clinical samples have shown that all three assays produced similar results. The coefficient of variation of these assays is less than 30%. A detection limit lowering from the current level to a range of 20 to 40 RNA copies or equivalents per milliliter of plasma is currently under evaluation for all three assays. The standardization of these assays for accuracy and reproducibility is paramount if they are used routinely for patient management. Assay variability has been described for blood collection, processing, and storage. The type of anticoagulant used in blood collection is a complicating factor for molecularly based methods. The most noticeable is the anticoagulant heparin, which is known for its inhibitory activity in polymerase chain reaction. Thus, plasma prepared from blood using heparin as anticoagulant cannot be used for the Amplicor monitor assay. Blood collected using ACD (acid citric dextran) as anticoagulant can be used for the Amplicor monitor and nucleic-acid-sequence-based amplification (NASBA) assays. Generally, blood collected using EDTA (ethylenediaminetetraacetic acid) as anticoagulant is the most suitable for all three assays.
Measuring antiretroviral resistance in human immunodeficiency virus infection
Incomplete inhibition of human immunodeficiency virus-1 replication may arise because of poor drug absorption, patient noncompliance with therapy, or infection with drug resistant virus variants. This incomplete inhibition may result in the emergence of drug-resistant human immunodeficiency virus-1 variants and is an important cause of therapy failure. An assessment of drug resistance may be helpful in selecting subsequent antiretroviral therapy, but this has not been proved rigorously. Drug susceptibility is determined phenotypically by determining the susceptibility of the virus isolate or genotypically by assessing resistance-conferring mutations. There are two phenotypic approaches: The first approach tests the sensitivity of the proviral population present in the PMBCs of the patient; the second approach generates recombinant fragments of the virus that contain polymerase or protease genes obtained from the plasma- or serum-associated virus or from the cell-associated provirus. There are several genotypic approaches: DNA sequencing of the entire viral population or clones; selective polymerase chain reaction assay; determination of point mutations; enzyme-immunoassay modification of the oligoligase detection reaction assay. Genotypic changes may not correlate with changes in drug susceptibility of the clinical isolate. Much still needs to be learned about the genotype and phenotype correlation in patients who receive combination antiretroviral therapy before these techniques can be applied effectively in the clinic. In the meantime, it is generally recommended that a patient's response to therapy be monitored by using the viral RNA level and CD4 count.
Measurements of cell-mediated immunity
The appearance of specific cell-mediated immunity is a commonly measured specific change in a host's response to infection. Although there are several sophisticated measurements of T-lymphocyte function to document prior exposure to an antigen, the intradermal skin test remains the simplest, cheapest, and most-used measurement of this aspect of immune function. It evaluates specific T cells that mediate delayed-type hypersensitivity reactions. Properly performed, this test provides an indication of prior exposure to (or current infection with) the antigen injected. However, intradermal skin tests of delayed-type hypersensitivity provide no indication of current activity of infection by an agent. A change from a negative to a positive test usually indicates new exposure during the interval between tests and may be correlated with active infection.
The tuberculin test with purified protein derivative is the prototype of this type of test. It has proven utility in detecting exposure to Mycobacterium tuberculosis. There are other commonly employed skin tests for screening for histoplasmosis, blastomycosis, coccidioidomycosis, and candidiasis antigenic exposure. However, results from these tests have not been clinically useful. The primary reasons for this failure are the high percentage of positive responders who do not have clinical disease and the ubiquitous nature of exposure to these antigens by persons living in zones where hyperendemic rates of these diseases occur. The dose of antigen and the size of cutaneous reaction are critical in avoiding misinterpretation of cross-reactivity with other related agents (e.g., atypical mycobacteria). False-negative tests (i.e., negative reactions to skin tests in the presence of true infection) may occur because of defects in any of the afferent or efferent arms of the cell-mediated immune system or faulty preparation or application of the antigen. False-negative tests have been seen in overwhelming tuberculous infection, intercurrent viral infections including those recently immunized with live-virus vaccines, immunosuppressant therapy, malnutrition, sarcoidosis, or various cancers and leukemias that suppress immune function. False-negative tuberculin tests may occur in the elderly because of waning immunity. The two-step test (if the Mantoux test is negative, it is repeated 2 weeks later; if it is still negative, it is a true negative result) avoids the problem of waning immunity.
Principles
Positive serologic and delayed-type hypersensitivity reactions indicate prior exposure to selected antigens. These tests have limited value in diagnosing acute infection. There is no substitute for culture or detection of the organism in clinical specimens to document the presence of infection.