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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 of PCR and sequencing 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.