important infectious diseases in the world, malaria, which is caused by protozoans within the genus Plasmodium. The various developmental stages of these parasites are detected in red blood cells. Other, less frequently encountered parasites seen in a peripheral blood smear include Babesia species, trypanosomes, and the microfilariae.
Bacterial and fungal pathogens may be seen in peripheral smears on occasion. The most likely of these is Histoplasma capsulatum, which is seen as small, intracellular yeasts in peripheral white blood cells. Ehrlichia and Anaplasma can produce characteristic inclusions (morulae), which can be seen in peripheral mononuclear cells and granulocytic cells, respectively.
Examination of tissue by the anatomical pathologist is an important technique for detecting certain infectious agents. Tissue cysts due to toxoplasmosis can be detected in brain biopsy material from patients with encephalitis. The diagnosis of Creutzfeldt-Jakob disease is based on the finding of typical lesions on brain biopsy. The finding of hyphal elements in lung tissue is an important tool in the diagnosis of invasive aspergillosis and pulmonary zygomycosis. The observation of ribbon-like elements in a sinus biopsy is pathognomonic for the diagnosis of rhinocerebral zygomycosis, a potentially fatal disease most frequently seen in diabetic patients.
Antigen detection
Visual examination of a clinical specimen is not the only means by which an infectious agent can be directly detected. A variety of tests have been developed that, like DFA, are dependent on the availability of highly specific antibodies to detect antigens of specific bacteria, fungi, viruses, and parasites. The most widely used antigen detection tests are various formats of the enzyme immunoassay or the latex agglutination assay. These tests take anywhere from 10 minutes to 2 hours. The test most widely used is a 10- to 15-minute enzyme immunoassay for the detection of group A streptococci. The sensitivity of these various formats has been reported to be 80 to 90%, with specificity usually greater than 95%. In the United States, there are more than 50 different test formats marketed for the detection of this organism. The test is done in a wide variety of laboratories, clinics, and physicians’ offices. Antigen detection tests are widely used in the United States to detect a variety of infectious agents, including Cryptococcus neoformans, Clostridium difficile toxin, respiratory syncytial virus, rotavirus, influenza virus, and Giardia and Cryptosporidium spp. It should be noted, however, that as more molecular tests become commercially available and are used as reference methods, the sensitivities of many of the rapid antigen tests deteriorate. For example, published sensitivities for rapid antigen tests for influenza are as low as 10% and those for respiratory syncytial virus are as low as 59%.
MOLECULAR DIAGNOSTICS
In addition to standard methods of culturing and identifying pathogenic microorganisms, there are now a number of molecular methods available that are able to detect the presence of the specific nucleic acid of these organisms. These methods are used in demonstrating the presence of the organism in patient specimens as well as in determining the identification of an isolated organism. In some cases, these methods are able to determine the quantity of the nucleic acid.
As an example, bacteria of a particular species will have a chromosomal nucleic acid sequence significantly different from that of another bacterial species. On the other hand, the nucleic acid sequence within a given species has regions that are highly conserved. For example, the base sequence of the Mycobacterium tuberculosis rRNA differs significantly from the base sequence in the Mycobacterium avium complex rRNA, yet the sequence of bases in this region among members of the M. tuberculosis complex is highly conserved. These properties form the basis for the use of genetic probes to identify bacteria to the species level. There are a number of commercially available genetic probes that can detect specific sequences in bacteria, mycobacteria, and fungi.
Nucleic acid hybridization is a method by which there is the in vitro association of two complementary nucleic acid strands to form a hybrid strand. The hybrid can be a DNA-RNA hybrid, a DNA-DNA hybrid, or, less commonly, an RNA-RNA hybrid. To do this, one denatures the two strands of a DNA molecule by heating to a temperature above which the complementary base pairs that hold the two DNA strands together are disrupted and the helix rapidly dissociates into two single strands. A second nucleic acid sequence is introduced that will bind to regions that are complementary to its sequence. The stringency, or specificity, of the reaction can be varied by reaction conditions such as the temperature.
In addition to the direct demonstration of a nucleic acid sequence by hybridization, amplification assays (the process of making additional copies of the specific sequence of interest) are of increasing importance in clinical microbiology. The most commonly used amplification assay is PCR (Fig. 8). PCR uses a DNA polymerase that is stable at high temperatures that would denature and inactivate most enzymes. This thermostable DNA polymerase most often is isolated from the bacterium Thermus aquaticus. Its stability at high temperature enables the enzyme to be used without the need for replacement after the high-temperature conditions of the DNA denaturation step that occurs during each cycle of PCR:
Figure 8 PCR. (A) In the first cycle, a double-stranded DNA target sequence is used as a template. (B) These two strands are separated by heat denaturation, and the synthetic oligonucleotide primers (solid bars) anneal to their respective recognition sequences in the 5′ → 3′ orientation. Note that the 3′ ends of the primers are facing each other. (C) A thermostable DNA polymerase initiates synthesis at the 3′ ends of the primers. Extension of the primer via DNA synthesis results in new primer-binding sites. The net result after one round of synthesis is two “ragged” copies of the original target DNA molecule. (D) In the second cycle, each of the four DNA strands in panel C anneals to primers (present in excess) to initiate a new round of DNA synthesis. Of the eight single-stranded products, two are of a length defined by the distance between and including the primer-annealing sites; these “short products” accumulate exponentially in subsequent cycles. (Reprinted from Manual of Clinical Microbiology, 7th ed, ©1999 ASM Press, with permission.)
1 1. The target DNA sequence is heated to a high temperature that causes the double-stranded DNA to denature into single strands.
2 2. An annealing step follows, at a lower temperature than the denaturation step above, during which sets of primers, with sequences designed specifically for the PCR target sequences, bind to these target sequences.
3 3. Last is an extension step, during which the DNA polymerase completes the target sequence between the two primers.
Assuming 100% efficiency, the above three steps generate two copies of the target sequence. Multiple cycles (such as 30) in a thermal cycler result in a tremendous amplification of the number of sequences, so that the sequence is readily detectable using any of a variety of methods—gel electrophoretic, colorimetric, chemiluminescent, or fluorescent.
When the specific target nucleic acid is RNA rather than DNA, a cDNA sequence is made with the enzyme reverse transcriptase (RT) before PCR amplification in a procedure known as RT-PCR. Examples of pathogens for which RT-PCR is used include the RNA-containing viruses HIV-1 and hepatitis C virus (HCV).
An additional feature of PCR is that the amplified nucleic acid products can be directly sequenced. These sequences can be compared with sequences found in publicly accessible databases. This allows, for example, the identification of a bacterial organism to the level of species on the basis of a sequence of hundreds of bases in the rRNA or, if the sequence is less closely related to sequences within the database, to the level of genus. In some cases, the organism may be an entirely new one. This method
