Rapid antigen testing is routine for diagnosis of group A streptococcal pharyngitis. Although rapid antigen tests offer less than 100% sensitivity, their wide availability at the point of care (POC) allows practitioners to diagnose and treat this common childhood illness in a single office visit in most cases, reserving culture for antigen-negative patients (Bisno et al., 2002).

The value of detection of Streptococcus pneumoniae antigen in urine for the diagnosis of pneumonia is limited by the positive results obtained in patients with mere oropharyngeal colonization, occurring especially in children, and by sensitivities of only 50-85%. The role of this test in management of patients with community-acquired pneumonia is still evolving (Smith et al., 2003; Roson et al., 2004).

Antigen detection in urine is a major diagnostic procedure for Legionella infections. Although available tests detect only 80-90% of the serotypes associated with human disease, the method is sensitive and specific for those serotypes and is much more rapid than culture. Urinary antigen can remain positive for days to weeks after therapy is begun and thus can be performed on treated patients. Direct fluorescent antibody (DFA) testing of respiratory specimens for Legionella is insensitive, even relative to culture, and requires a skilled reader to limit false-positives. Monoclonal reagents are more specific than polyclonal reagents, but both have been described to cross-react with non-Legionella species. The true sensitivity and specificity of antigen detection in Legionella infections is difficult to determine, because culture itself is insensitive, and molecular methods are still in development (Waterer et al., 2001).

For diagnosis of enterocolitis due to Clostridium difficile toxins, there is no gold standard. Rather, a variety of diagnostic techniques are employed, including toxigenic culture, tissue culture cytotoxicity with antibody neutralization, and both rapid and conventional toxin EIAs. The various EIA methods are the most widely employed because of their modest technical requirements and rapid time-to-result; newer tests that detect both toxin A and toxin B are more sensitive than methods that detect only toxin A. Older latex agglutination and membrane EIA tests that detect C. difficile glutamate dehydrogenase (a.k.a. "common antigen") do not distinguish between toxigenic and nontoxigenic strains and lack specificity but may be used as screening tests to select specimens for further, definitive testing (Wilkins and Lyerly, 2003).

Antigen testing of stool for Helicobacter pylori has recently become an option to the urea breath test and serology. It may be particularly useful in children, where the urea breath test may be difficult to perform, and in patients in whom serologic testing is likely to be problematic, such as steroid-treated or HIV-infected patients (Versalovic, 2003).

Table 3.2. Application of techniques to detection of specific pathogens.






Streptococcus group A

Streptococcus pneumoniae

Legionella spp.

Agglutination, rapid EIA, OIA Rapid EIA

Throat swab




Clostridium difficile Agglutination, rapid EIA, ELISA, OIA Helicobacter pylori ELISA

Chlamydia trachomatis


Urine Stool

Stool Genital

Meningitis panel (H. Agglutination CSF, urine influenzae, N. meningitidis, S. pneumoniae, group B Streptococcus)



Often performed at POC. Negatives must be evaluated by culture.

94% Clinical role still evolving. Provides adjunct, but not definitive, diagnostic information in patients at risk for S. pneumoniae disease.

90%+ Requires FA microscope. Cross-reactions with some other bacteria, especially with polyclonal reagents. No gold standard for comparison.

99% Test characteristics well-established only for L.

pneumophilia group 1.

88-100% Measured sensitivity and specificity are relative to tissue culture cytotoxicity. Tests detecting Toxin A + B are more sensitive than those detecting Toxin A only.

90-94% Used as an alternative to serology and urea breath testing.

97% Being phased out, but POC versions might be valuable if sensitivity improves. No single-test format available. Not useful for screening low-prevalence populations due to poor specificity.

Inadequate sensitivity/specificity for routine clinical use. Empirical therapy given for CSF neutrophilia covers these pathogens, until culture results available. Positive predictive value of antigen tests is very low in patients without CSF leukocytosis.



Pneumocystis jiroveci (formerly P. carinii)




Entamoeba histolytica/dispar group

Trichomonas vaginalis

Plasmodium falciparum

Lymphatic filariases

Agglutination, CSF, serum 99%+ ELISA

IF Respiratory Variable

IF, ELISA, rapid Stool EIA

Higher than microscopy

IF, ELISA, rapid Stool EIA


Higher than microscopy Higher than microscopy


Rapid EIA and other rapid formats ELISA, rapid EIA



Similar to microscopy

Equivalent to microscopy; similar to or higher than concentration methods

Very high if heat or pronase pretreatment used High

Sensitivity may exceed culture. Cross-reactivity with (very rare) systemic Trichosporon infections. Prozone is a problem in high-level infections.

Requires fluorescence microscope. Sensitivity is highest for antibodies that detect antigens present in trophozoites and cysts. No significant sensitivity or specificity advantages over conventional and Calcifluor white stains.

100% No gold standard available for comparison. Specimen treatment (e.g., fixed, unfixed, or frozen) varies with different tests.

93-100% No gold standard available for comparison. Some kits detect both Giardia and Cryptosporidium.

>95% Not widely used. Reagents are available to distinguish between E. histolytica and E. dispar.

High Alternatives include wet prep (60% sensitivity relative to culture), culture, molecular detection. Wet prep is limited by specimen stability.

High Three dipstick-format rapid tests available. Cost limits use in endemic areas.

No gold standard. Cost limits use in endemic areas.


Table 3.2 (Continued)






Respiratory syncytial

Influenza A and B


ELISA, rapid EIA, lateral flow IA, IC, OIA, EVEA

NP swab or aspirate, BAL, sputum NP swab or aspirate, BAL, sputum NP swab or aspirate, BAL, sputum, throat swab


NP swab, NP aspirate, nasal wash, BAL


NP swab or aspirate, BAL, sputum


NP swab or aspirate, BAL, sputum



97-99% Very sensitive in young infants who shed high titers of virus. Mucoid samples may not disperse properly and may give rise to erroneous results.

>99% More sensitive than culture or other antigen tests. Can be multiplexed with other antibodies. IF allows assessment of sample quality.

95-99% Sensitivity higher in children and with NP aspirates and washes. Some kits require use of special swab. Many tests do not differentiate between influenza A and B. Some new rapid tests less specific than older methods. Simpler rapid tests suitable for POC. Mucoid samples may not disperse properly and may give rise to erroneous results.

95-99% Performance must be established in each laboratory.

Can be more sensitive than other rapid tests. Cytospin preparation of slides improves results. Use of pooled antibodies can be used to screen a single cell spot for multiple respiratory viruses. IF allows assessment of sample quality.

95-99% Only rapid method available. Cytospin preparation of slides improves results. Antibodies to types 1, 2, 3, but not type 4, are included in commercial antibody pool.

99% IF for adenovirus not as sensitive as for other respiratory viruses. Cytospin preparation of slides improves results.


Adenovirus, enteric types 40,41 Rotavirus

Astrovius Noroviras

Herpes simplex

EIA, agglutination EIA, agglutination, immunogold


Varicella zoster

Stool Stool

Stool Stool

Skin lesions, genital lesions, oral lesions, BAL, brain tissue Skin or genital lesions

Skin lesions, BAL


Blood leukocytes 90-97%

99% Test available for detection of all adenovirus types in culture fluids or stools; does not differentiate among types.

99% Test available to detect only enteric types 40 and 41.

Ad40 and Ad41 do not grow in routine cell cultures.

90-98% Rotavirus does not grow in routine cell cultures, so rapid tests are compared with EM. Rotavirus shed in high titers in stools of infants and young children. Titers decline after day 8.

99% Rapid tests compared to EM.

94—99% Limited by antigenic variation; rapid onset and resolution of illness.

>99% Sensitivity enhanced by cytospin preparation of slides.

Sensitivity is higher for skin lesions than for mucosal lesions. HSV and VZV antibodies labeled with different fluorochromes can be used to test for both viruses in a single cell spot.

99% EIA available in reference laboratories. Most sensitive for fresh vesicular skin lesions. Amenable to automation.

>99% IF for VZV in skin lesions is more sensitive than culture. VZV and HSV antibodies can be pooled for dual detection using two fluorochromes.

>99% Quantitative detection of CMV pp65 antigenemia is very useful in rapid diagnosis and in monitoring therapy. More sensitive than culture and equivalent to PCR in plasma.


Table 3.2 (Continued)







Human immunodeficiency vims

Human immunodeficiency virus

Hepatitis B surface antigen (HBsAg) and e antigen (HBeAg)

Real-time immuno-PCR




<50 to 6000 viral copies/mL plasma

Once antibody appears in blood, sensitivity of antigen detection decreases. Immune complex dissociation and signal amplification boost sensitivity. Rheumatoid factor can cause false-positive results. Neutralization test needed to confirm specificity of result.

Detects ultralow level of protein. Combines traditional ELISA with PCR. Much less expensive than current molecular tests.

Free HBsAg is produced in 100- to 1000-fold excess over complete virus particles. Thus HBsAg is generally more sensitive than DNA techniques. HBeAg has been the standard marker for high levels of viral replication.

POC, point of care; ELISA, enzyme linked immunosorbent assay; EIA, enzyme immunoassay; IF, immunofluorescence; PCR; polymerase chain reaction.

Other rapid formats: IA, immunoassay; IC, immunochromatography; OIA, optical immunoassay; EVEA, endogenous viral encoded enzyme assay. LA, latex agglutination;

NP, nasopharyngeal; BAL, bronchoalveolar lavage; EM, electron microscopy.

Antigen testing for genital Chlamydia infections has been almost entirely replaced by nucleic acid testing, which is substantially more sensitive and specific. Rapid tests have the potential for POC use, but none is yet FDA approved (Mahony et al., 2003).

Bacterial antigen testing for meningitis is rapid but has fallen out of use in recent years due to inadequate sensitivity and specificity and the use of empirical antibiotic therapy. The presence of neutrophils in cerebrospinal fluid (CSF) generally leads to therapy in patients with compatible syndromes, whereas the positive predictive value of antigen testing performed on patients with acellular CSF is dismal. Empirical antibiotic choices cover the organisms detected by the antigen tests (Kiska et al., 1995; Thompson et al., 2003).

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