of the product for the purposes of bacterial identification is being used in clinical microbiology for the identification of slow-growing or difficult-to-identify organisms such as Mycobacterium spp., Nocardia spp., and anaerobic organisms. However, mass spectrometry has recently entered clinical microbiology and will likely replace ribosomal gene sequencing as the method of choice for these organisms, as well as all other bacteria and fungi. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) allows the identification of organisms by their protein spectra. Although initial instrumentation is expensive, identifications can be performed for less than $1 and in at little as 20 minutes. Many clinical laboratories are already using MALDI-TOF as the primary method for identifying bacteria.
After the invention of PCR, a number of other amplification assays were developed, some of which have entered the clinical microbiology laboratory. Transcription-mediated amplification (TMA), which does not require a thermal cycler, relies on the formation of cDNA from a target single-stranded RNA sequence, the destruction of the RNA in the RNA-DNA hybrid by RNase H, and the formation of double-stranded cDNA (which can serve as transcription templates for T7 RNA polymerase). A similar procedure occurs during the nucleic acid sequence-based amplification (NASBA) assay. Strand-displacement amplification (SDA) does not require a thermal cycler and has two phases in its cycle: a target generation phase during which a double-stranded DNA sequence is heat denatured, resulting in two single-stranded DNA copies; and an exponential amplification phase in which a specific primer binds to each strand at the cDNA sequence. DNA polymerase extends the primer, forming a double-stranded DNA segment that contains a specific restriction endonuclease recognition site, to which a restriction enzyme binds, cleaving one strand of the double-stranded sequence and forming a nick, followed by extension and displacement of new DNA strands by DNA polymerase.
All of these assays—PCR, TMA/NASBA, and SDA—have one thing in common: they amplify the target nucleic acid sequence, making many, many copies of the sequence. As you might imagine, there is the possibility that small quantities of the billions of amplified target nucleic acid sequences can contaminate a sample that will then undergo amplification testing, resulting in false-positive results. Steps are taken to minimize contamination, including physical separation of specimen preparation and amplification areas, positive displacement pipettes, and both enzymatic (in PCR) and nonenzymatic methods to destroy the amplified products.
An alternative method of demonstrating the presence of a specific nucleic acid sequence that does not require the amplification of the target is by amplification of the signal. One example is branched DNA (bDNA) technology (Fig. 9), which is used particularly in quantitative assays, such as HIV and HCV viral load determinations. In this assay, specific oligonucleotides hybridize to the sequence of interest and capture it onto a solid surface. In addition, a set of synthetic enzyme-conjugated branched oligonucleotides hybridize to the target sequence. When an appropriate substrate is added, light emission is measured and compared with a standard curve. This permits quantitation of the target sequence. As there is no amplified sequence to be concerned about, the risk of contamination is dramatically reduced. Another example that is widely used is a hybrid capture test for human papillomavirus (HPV) detection. In this test, HPV DNA is denatured and bound to complementary RNA probes. This hybrid is then “captured” by immobilized anti-hybrid antibodies. A chemiluminescent reaction allows for the detection of DNA-RNA hybrids and therefore HPV DNA in the sample.
Figure 9 bDNA-based signal amplification. Target nucleic acid is released by disruption and is captured onto a solid surface via multiple contiguous capture probes. Contiguous extended probes hybridize with adjacent target sequences and contain additional sequences homologous to the branched amplification multimer. Enzyme-labeled oligonucleotides bind to the bDNA by homologous base pairing, and the enzyme-probe complex is measured by detection of chemiluminescence. All hybridization reactions occur simultaneously. (Reprinted from Manual of Clinical Microbiology, 7th ed, ©1999 ASM Press, with permission.)
There are several commercially available molecular diagnostic assays for Chlamydia trachomatis and Neisseria gonorrhoeae. Although first-generation molecular tests included direct hybridization assays, nucleic acid amplification tests are now the laboratory standard due to their increased sensitivity. Depending on the manufacturer of the tests, specimens of cervical, vaginal, and urethral swabs and urine are acceptable. Because N. gonorrhoeae is a fastidious organism that may not survive specimen transport, nucleic acid amplification tests are of particular benefit in settings in which there may be a delay in the transport of the specimen to the laboratory; i.e., the viability of the organisms is not required to detect the presence of its nucleic acid. Similarly, the previous gold standard for the detection of C. trachomatis—tissue culture—was labor-intensive, required the use of living cell lines, and required rapid specimen transport on wet ice to ensure the viability of the organisms in the specimen. In almost all clinical laboratories, C. trachomatis tissue culture has been replaced by amplification technologies, which have been shown to be significantly more sensitive. As you might imagine, since these assays do not require the presence of living organisms, patients who have been treated with appropriate antibiotics may continue to have a positive assay for some time because of the presence of dead, and therefore noninfectious, organisms that contain the target nucleic acid.
Quantitative assays are now available for several different pathogens. These include tests that determine the level of HIV RNA in patients with HIV infection and are now recognized as one component of the standard clinical management of these patients. With the availability of highly active antiretroviral therapy but the potential for antiviral drug resistance, it is important to be able to closely monitor the plasma level of HIV RNA, also known as the viral load. A clinical response to antiretroviral therapy can be demonstrated by a decrease in the viral load. Similarly, an increase in the viral load may indicate either the development of viral resistance to one or more of the antiviral agents being used to treat the patient or merely patient noncompliance with therapy. Modification of therapy may be made on the basis of a rising HIV viral load and the results of HIV genotyping studies.
HIV genotyping is a test that determines the specific nucleic acid sequence present in the virus infecting a patient. There are a number of ways that this test can be performed, and direct sequencing of amplified cDNA (using RT-PCR) is one example. These results are routinely compared with a database that contains nucleic acid sequences from viral strains that are known to be both sensitive and resistant to specific antiretroviral medications. This comparison permits the clinician to note what, if any, mutations are present in the virus infecting the patient and to predict with some reasonable degree of probability whether the viral isolate is resistant to antiretroviral medications, including those being taken by the patient. These data can help the physician make a rational choice of an antiretroviral regimen in a patient whose therapy is failing. One difficulty with this test is that patients are often infected with a mixture of different HIV viral populations, both because of the high frequency of mutation that occurs with HIV and because of the selection of resistant subpopulations while the patient receives antiretroviral therapy. As a result, there may be resistant subpopulations that are below the level of detection of the standard HIV genotyping assay and that could become clinically relevant under the selective pressure of continued antiretroviral therapy.
Detection of HCV RNA using RT-PCR can be used both diagnostically and for following the effectiveness of therapy. The PCR product generated during the HCV RNA assay can be used for genotyping using a variety of hybridization assays in which specific nucleic acid sequences associated with specific genotypes are detected. Genotype 1 is more refractory to therapy than genotypes 2 and 3. Therefore, therapy is much more prolonged (48 versus 24 weeks) for genotype 1 than for 2 and 3. Further, treatment with the newer HCV protease inhibitors is currently only available for patients with genotype 1.
CULTURE
