Melissa B. Miller - Cases in Medical Microbiology and Infectious Diseases

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 In almost all clinical laboratories, C. trachomatis tissue culture has been replaced by amplification technologies, which have been shown to be significantly more sensitive.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 on manufactured medium is the most commonly used technique for detecting bacteria and fungi in clinical specimens. Although not as rapid as direct examination, it is more sensitive and much more specific. For the majority of human pathogens, culture requires only 1 to 2 days of incubation. For particularly slow-growing organisms, such as M. tuberculosis and some fungi, the incubation period may last for weeks. By growing the organism, it is available for further phenotypic and genotypic analysis, such as antimicrobial susceptibility testing, serotyping, virulence factor detection, and molecular epidemiology studies.

Certain basic strategies are used to recover bacterial and fungal pathogens. These strategies are dependent on the phenotypic characteristics of the organisms to be isolated and the presence of competing microbiota in a patient’s clinical specimen. Most human pathogens grow best at 37°C, human body temperature. Most bacterial and fungal cultures are performed, at least initially, at this temperature. Certain skin pathogens, such as dermatophytes and some Mycobacterium spp., grow better at 30°C. When seeking these organisms, cultures may be done at this lower temperature. A few clinically significant microorganisms will grow at low temperatures (4°C), while others prefer higher temperatures (42°C). These incubation temperatures may be used when attempting to recover a specific organism from specimens with a resident microbiota, such as feces, as few organisms other than the target organism can grow at these temperature extremes.

Another important characteristic of human bacterial and fungal pathogens is the impact of the presence of oxygen on the growth of these organisms. Microbes can be divided into three major groups based on their ability to grow in the presence of oxygen. Organisms that can only grow in the presence of oxygen are called aerobes. Fungi and many bacteria are aerobicorganisms. Organisms that can only grow in the absence of oxygen are called anaerobes. The majority of bacteria that make up the resident microbiota of the gastrointestinal and female genital tracts are anaerobic organisms. Some bacteria can grow either in the presence or in the absence of oxygen. These organisms are called facultativeorganisms. A subgroup of facultative organisms is called microaerophiles. These organisms grow best in an atmosphere with reduced levels of oxygen. Campylobacter spp. and Helicobacter spp. are examples of microaerophiles.

Besides temperature and oxygen, nutrients are an important third factor in the growth of microbes. Many bacteria have very simple growth requirements. They require an energy and carbon source, such as glucose; a nitrogen source, which may be ammonium salts or amino acids; and trace amounts of salts and minerals, especially iron. Some human pathogens have much more complex growth requirements, needing certain vitamins or less well-defined nutrients such as animal serum. Organisms with highly complex growth requirements are often referred to as being fastidious. A fastidious bacterium that is frequently encountered clinically is Haemophilus influenzae . This bacterium requires both hemin, an iron-containing molecule, and NAD for growth.