Histology. Some infectious agents can be seen in hematoxylin and eosin (H&E)–stained sections (e.g., the inclusion bodies formed by CMV and herpes simplex virus (HSV); bacterial clumps, which usually stain blue; Candida and Mucor among the fungi; most protozoans; all helminths). Many infectious agents, however, are better visualized by special stains that identify organisms on the basis of particular characteristics of their cell wall or coat—Gram, acid-fast, silver, mucicarmine, and Giemsa stains—or after labeling with specific antibodies (
Table 9.4 ). Organisms are usually best visualized at the advancing edge of a lesion rather than at its center, particularly if there is necrosis.
Serology. Acute infections can be diagnosed serologically by detecting pathogen-specific antibodies in the serum. The presence of specific immunoglobulin M (IgM) antibody shortly after the onset of symptoms is often diagnostic. Alternatively, specific antibody titers http://ebooksmedicine.net
Table 9.4 Techniques for Identifying Infectious Agents
Acid-faststainMycobacteria,nocardiae(modified)
SilverstainFungi,legionellae,Pneumocystis
Periodicacid–SchiffstainFungi,amebae
GiemsastainLeishmaniae,Plasmodium
Proteomicmethods/massBacteria,mycobacteria,fungispectrometry can be measured early (“acute”) and again at 4 to 6 weeks (“convalescent”) after infection; a 4-fold rise in titer usually is considered diagnostic. Assays for serum antibodies are very useful for the diagnosis of viral hepatitis. Antibodies that are not pathogen-specific are produced by patients with syphilis or infectious mononucleosis, and assays for these cross-reacting antibodies are used in diagnosis.
Molecular diagnostics. Nucleic acid amplification techniques, such as polymerase chain reaction (PCR) and transcription-mediated amplification, are used for diagnosis of gonorrhea, chlamydial infection, tuberculosis, and herpes encephalitis. Molecular assays are much more sensitive than conventional testing for some pathogens. PCR testing of cerebrospinal fluid (CSF) for HSV encephalitis has a sensitivity of about 80%, whereas viral culture of CSF has a sensitivity of less than 10%. Similarly, nucleic acid tests for genital Chlamydia detect 10% to 30% more cases than conventional Chlamydia culture does. For other infections, such as gonorrhea, the sensitivity of nucleic acid testing is similar to that of culture. Quantitative PCR for BK virus, CMV, and Epstein-Barr virus (EBV) is used to assess viral loads in transplant recipients. Molecular panels for detection of 20 or more pathogens are now routinely used to diagnose respiratory bacterial and viral infections, as well as gastrointestinal bacterial, viral and parasitic infections. Quantitative assays for viral nucleic acids are used to guide the medical management of patients infected with human immunodeficiency virus (HIV), HBV, and hepatitis C virus (HCV). Next-generation sequencing, with or without initial PCR amplification, is being used for the detection of novel or rare pathogens, and for epidemiologic investigations.
Proteomics. Mass spectrometry can be used to identify microorganisms based on protein content and has been introduced into routine clinical laboratories. It has the advantage of rapid identification of the bacterial species but is not useful for antibiotic sensitivity. That still requires culture.