Rapid diagnostic testing in microbiology. (Clinical Issues).
Generally, the first step in examining clinical material for bacteria is a direct microscopic examination, and the most commonly used stain is the Gram stain. Most bacteria and fungi can be quickly visualized with this stain. The Gram stain not only provides staining characteristics of bacteria--gram-positive or gram-negative and cocci or bacilli--it can also detect inflammatory cells. Although the Gram stain can be insensitive, it still is useful in many types of specimens, most notably male urethral smears. While the presence of gram-negative diplococci is no longer confirmatory for Neisseria gonorrboeae, their presence is highly indicative of infection.
A number of other stains are part of the microbiologist's arsenal, such as the acid-fast stain to visualize the Mycobacterium. This stain is useful because the pathogenic mycobacteria are slow growing and can take several weeks to be isolated in culture. Additional stains include the Giemsa and Wright stains used to visualize blood parasites, Pneumocystis (carinii) jiroveci in lower respiratory tract specimens, and inclusion bodies of Chlamydia trachomatis.
Because of ease of use and increased sensitivity, fluorescent stains have become very common to diagnostic microbiology laboratories. These procedures utilize fluorochromes--fluorescent dyes--that stain various molecules in bacterial cells. These stains are not fluorescent-labeled antibodies. Three commonly used fluorescent stains are auramine-rhodamine, acridine orange and calcofluor white.
Auramine-rhodamine binds to mycolic acids found in the mycobacteria and Cryptosporidium. Acridine orange is intercalated into the nucleic acid of cells. This stain is particularly useful in specimens that are likely to contain few bacteria, such as blood culture broths, buffy coat preparations and cerebrospinal fluid (CSF). Because host cells will also bind acridine orange, excessive fluorescence can make the stain difficult to interpret. Calcofluor white binds cellulose and chitin in the cell walls of fungi. Calcofluor white can be mixed with 10 percent potassium hydroxide to clear the specimen for easier detection of fungal elements. This stain has also been used to detect parasites such as Acanthamoeba, Microsporidium, Pneumocystis jiroveci, and Naegleria. (1) Assays using fluorescent stains are generally rapid, but they do require a fluorescent microscope.
For a number of years, the India ink (nigrosin) wet mount was commonly performed on CSF to detect the encapsulated yeast Cryptococcus. A drawback is the low sensitivity, especially in patients without acquired immunodeficiency syndrome. (2,3) Not all C. neoformans isolates will produce a capsule, making their detection more difficult. In addition, artifacts such as white blood cells, air bubbles and talc powder from gloves can be misidentified as encapsulated yeast. Due to these difficulties, latex agglutination tests (Meridian, Cincinnati, OH and Wampole, Princeton, NJ) have largely replaced the India-ink method. Latex agglutination tests for cryptococcal antigens are sensitive, specific and easy to perform. (4) The polysaccharide capsular antigen is produced in large quantities and can be detected in both CSF and blood.
Antigen-detection assays are a type of immunoassay. By definition, immunoassays are procedures measuring antigens or antibodies to determine if patients are infected or immunologically responding to infection or immunization. Many antigen-detection tests are rapid and can be performed directly on a clinical specimen. Antigen-detection tests are often performed on a solid phase, such as plastic, nitrocellulose, polyvinyl chloride or polyacrylamide. The three most common types of antigen-detection methods are competitive, direct (double-antibody sandwich) and indirect. The antigen or antibody is labeled with an enzyme or other easily detected molecule. Many easy-to-perform kits are available.
In the competitive assay, antigen in the patient sample competes with labeled antigen for a limited number of antibodies attached to a solid phase. The amount of labeled antigen is detected and is inversely proportional to the amount of antigen in the original specimen. With the direct-detection assay, a capture antibody attached to a solid phase binds antigen in the patient sample. After a washing step, a labeled antibody also specific to the antigen is added. The amount of labeled antibody is measured and is directly proportional to the amount of antigen in the original specimen. The indirect antigen-detection assay is similar to the direct in that a capture antibody is used; however, the second antibody is not labeled. After the second antibody binds to the antigen, a third labeled anti-immunoglobulin antibody is added. The third antibody will bind to the second antibody, and again the amount of labeled antibody measured is directly proportional to antigen in the clinical specimen.
A number of antigen-detection kits are available; generally they are easy to perform, do not require specialized equipment and are easy to interpret. The ColorPAC Giardia/Cryptosporidium Rapid Assay (Becton Dickinson, Sparks, MD) and ProspecT Giardia/Cryptosporidium are two examples. These assays can detect either parasite in a stool sample. The disadvantage is that other parasites would not be detected, as they could be with a standard, albeit time-consuming, microscopic examination of the stool. In a recent study, the ColorPAC Rapid Assay had a reported sensitivity of 100 percent for both Giardia and Cryptosporidium and a specificity of 100 percent and 95 percent, respectively. (5) In the same study, the ProspecT assay had a sensitivity of 100 percent for both Giardia and Cryptosporidium and a specificity of 98.4 percent and 98.6 percent, respectively.
Other examples of rapid antigendetection methods are direct fluorescent antibody (DFA) and agglutination. With the DFA assay, clinical material is fixed onto a microscope slide. A fluorescent-labeled antibody is added, and after appropriate incubation and wash steps, the slide is examined for fluorescence. DFA tests are generally rapid and easy to perform, but they do require some expertise in interpreting results. DFA tests are available for a number of microorganisms, including Bordetella, Legionella, Pneurnocystis, and Treponema pallidum.
In agglutination procedures, antibodies are attached to carrier particles, such as red blood cells, but more often latex beads. Material from the patient is mixed with the coated particles on a slide and observed for agglutination. In some cases, such as tests for group A streptococci, an extraction step is performed on the clinical specimen to make the antigen more accessible for reacting with the antibody. In other cases, such as assays for group B streptococci in CSF, the patient sample can be mixed directly with the carrier particles. These tests tend to be rapid, sensitive and specific, and it is easy to interpret a positive reaction.
Molecular detection and identification of microorganisms is a relatively new but important aspect of microbiology. Molecular detection, or nucleic acid testing (NAT), is a broad area encompassing nucleic acid probes (hybridization), nucleic acid amplification, plasmid analysis, fingerprinting, ribotyping and DNA sequencing. Hybridization tests can be performed on microarrays, or biochips, which can have thousands of single-stranded nucleic acid samples arranged in a grid on a solid surface. This allows screening a clinical sample for many different nucleic acid sequences at one time in an automated system.
NAT is used to detect and identify microorganisms, to help determine relatedness for taxonomy, and in epidemiological studies. Nucleic amplification tests generally fall into one of three categories: target amplification systems, such as polymerase chain reaction (PCR); probe amplification, such as DNA ligase chain reaction (LCR); and signal amplification--for example, branched DNA (bDNA) technology. Most of these assays can provide results in a couple of hours.
NAT can enhance the speed, sensitivity and specificity of laboratory diagnosis. While some methods can be completed in a few hours, others can take several days. NAT lends itself best to microorganisms that are difficult to grow or are slow growing. With NAT, in vitro growth is not always necessary for microbial identification.
The disadvantages are the price of reagents and instruments, and the technical expertise required. In addition, the nucleic amplification tests may be too sensitive; they are prone to contamination and cannot distinguish between living and dead bacteria. Because of the advantages, however, clinical microbiology laboratories have been gradually adopting these tests.
Rapid biochemical tests
There are few rapid biochemical tests that can be performed on clinical specimens, and generally, they are screening tests. Two of the most commonly used are leukocyte esterase (LE) and nitrite. These two assays are available on urine dipsticks. LE is present in granules found in white blood cells. The LE assay detects the presence of granulocytes and monocytes in urine (pyuria). According to the package insert, the sensitivity is five to 15 cells/high power field (Chemstrip 2LN, Roche Diagnostics, Indianapolis, IN). A number of bacteria causing urinary tract infections reduce nitrate to nitrite; therefore, the presence of nitrite in urine is suggestive of infection. The reagents for the two tests are impregnated onto pads attached to a plastic strip. The strip is inserted into a freshly collected urine sample and observed for color change; the nitrite test is read at 60 seconds and the LE is read at two minutes.
Because as many as 60 percent to 80 percent of urine cultures processed by clinical laboratories contain no etiological agent, (6) a screening test to eliminate the negative cultures would be cost effective. Even when used in combination, however, the LE and nitrite tests are not very sensitive. In a study of 479 women with symptoms suggestive of urinary tract infection, LE and nitrite results were correlated to urine culture results. (7) It was reported that a positive LE and nitrite had a sensitivity of 84 percent and specificity of 98 percent when the colony count was greater than [10.sup.5] colony-forming units/mL. However, when colony counts were low--for example, [10.sup.4] colony forming units/mL, the sensitivity dropped to 25 percent, even though the specificity remained 98.3 percent. In addition at the lower colony count, the positive predictive value was 42.9 percent. LE and nitrite could possibly be used to screen clean-catch, voided urine, but they would not be appropriate for catheter or suprap ubic aspirate specimens when lower bacterial colony counts are significant.
Diagnostic tests for Chlamydia trachomatis
Chlamydia trachomatis is one of the most prevalent sexually transmitted bacterial pathogens. This organism can cause mild chronic infections, such as cervicitis and urethritis, that progress to more serious diseases, such as ectopic pregnancy; pelvic inflammatory disease, salpingitis and infertility Because infections can be asymptomatic, infected individuals can transmit the organism to their sexual contacts. Due to the fact that the organism is clinically unrecognized, screening with laboratory tests followed by treatment is the most important means of control.
C. trachomatis is an obligate intracellular parasite, and as such, cannot grow on artificial media. In the past, because it was the most sensitive method available, cell cultures were considered the gold standard, or reference method, for the detection of this bacterium. Cell cultures are technically difficult, not standardized, and time consuming. Performance can vary widely, 50 percent to 80 percent sensitivity depending on the skill and experience of the laboratory scientist. (8)
One difficulty in determining the sensitivity and specificity of a diagnostic test for C. trachomatis is what test to use as the reference method. A number of studies have used an expanded gold standard, based on culture results adjudicated with nucleic amplification assays. In these studies, discrepant results are resolved using a third assay. A sample positive by two of the three assay methods is considered a true positive.
A number of nucleic acid probes and amplification assays have been developed to overcome some of the shortcomings of cell cultures. The COBAS AMPLICOR CT assay (Roche Diagnostic Systems Inc., Branchburg, NJ) uses PCR to amplify target DNA, along with an internal control. The PACE 2 assay (Gen-Probe, San Diego, CA) uses a chemiluminescent labeled, single-stranded DNA probe, complementary to the rRNA of C. trachomatis. The Hybrid Capture 2 (HC2, Digene, Gaithersburg, MD) is a chemiluminescent signal amplification assay, using an RNA probe to target DNA. The resulting RNA:DNA hybrid is detected with antibodies. The AMPLIFIED Chlamydia trachomatis (AMP-CT) assay (Gen-Probe, San Diego, CA) uses transcription-mediated amplification (TMA) technology to amplify target rRNA. Nucleic acid tests can provide results in a few hours and have been shown to be more sensitive than cultures. (8-11) Some of these assays, such as the PACE 2 and the HC2, have the advantage of detecting both C. trachomatis and Neisseria gonorrhoea e. With most assays, the specimen tested affects the performance; urine tends to be a less sensitive sample (Table 1).
Recently, a number of rapid tests for the detection of C. trachomatis have reached the market. Many of these tests can be performed on urine samples in about 30 minutes and do not require sophisticated equipment. Most of the rapid tests are EIAS using monoclonal or polycolonal antibodies against the major outer membrane protein or lipopolysaccharide of C. trachomatis.
The performance of three rapid tests, Testpack Chlamydia (Abbott Laboratories, North Chicago, IL), Surecell Chlamydia (Kodak, Rochester, NY) and Clearview Chlamydia (Unipath, Bedford, UK), was evaluated by comparison to the highly sensitive ligase chain reaction (LCx Chlamydia, Abbott Laboratories, S. Pasadena, CA). (12) A total of 128 first-voided urine samples from selected asymptomatic men was tested. Also in this study, an LE urine dipstick test (Chemstrip 2LN, Roche Diagnostics, Indianapolis, IN) was performed. The rapid tests ranged in sensitivities from 62.9 percent to 70.9 percent (Table 2). It was interesting to note that the LB dipstick was the most sensitive test; however, as expected, it was the least specific. The study designed required using frozen urine, which is not recommended by the manufacturers; this could have affected the sensitivity of the assays. The sensitivity of these assays increases when cervical specimens are submitted. For example, the sensitivity of the Clearview assay with ce rvical specimens is 79 percent to 95 percent, the Surecell is 75 percent to 90 percent, and the Testpack is 48.5 percent to 90 percent. (13-18)
Two additional rapid assays have been evaluated, the Quick Vue-Chlamydia (Quidel Corp., San Diego, CA) and the Biostar optical immunoassay (Biostar Inc., Boulder, CO). The Quick Vue, a lateral-flow immunoassay technique, had a sensitivity of 92.0 percent and a specificity of 99.1 percent using cervical swabs. (19) The Biostar immunoassay was 73.8 percent sensitive also on cervical specimens. (20) These results may not be truly indicative of the assay methods; while the assays are designed for physician offices, in these studies the tests were performed by experienced laboratory personnel.
The rapid assays for diagnosing C. trachomatis are appealing, because they can be quick point-of-care tests used on specimens collected by noninvasive procedures, such as urine. Rapid tests are important m clinics where patients may not return several days later for test results. Also, the rapid assays often have very few false-positives. (12) The disadvantage is the lower sensitivity compared to cervical and urethral specimens and NAT. Because the sensitivity is also affected by the number of bacterial cells present, the sensitivity is often increased in symptomatic patients.
In the age of managed healthcare, laboratorians must consider the cost effectiveness of rapid tests. In an evaluation of an EIA test for influenza A, it was reported that the rapid test not only reduced duration of influenza A outbreaks, but also lowered laboratory costs, compared to a control group. (21) This study, however, involved a nursing home, which, to a degree, is a closed population, making infection control easier. In another study, the financial benefits of rapid tests for the diagnosis of viral respiratory pathogens were evaluated. (22) The virology laboratory instituted a cytospin fluorescent antibody assay with pooled fluorescent antisera to influenza A and B viruses, respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3 and adenovirus (Imogen respiratory screen, DAKO Diagnostics Ltd., U If the sample was positive with the pooled antisera, follow-up testing with monoclonal specific antisera was performed. For inpatients testing positive, the mortality rate was lower by 10 percent, alt hough not statistically significant (P = 0.326), compared to the previous year when the rapid test was not available. The mean length of stay was shorter (10.6 days compared to 5.3 days, P 0.065) and the mean total cost per patient was $11,817 less (P = 0.104). The authors reported that the cytospin FA was 100 percent specific for all viruses. The sensitivities for influenza A and RSV were 90 percent and 98 percent, respectively, but the sensitivities for influenza B virus and adenovirus were 14.3 percent and 0 percent, respectively. However, influenza A and RSV accounted for over 85 percent of the respiratory viruses isolated in cell cultures.
When a laboratory decides to offer a rapid test, this can result in increased demand for services. The Hawaii Department of Health (HDH) began offering a rapid diagnostic test for influenza in addition to viral cell cultures in the 19992000 influenza season. (23) The HDH reported an increase in requests for influenza diagnosis from 396 to 2,169 between the 1998-1999 and 2000-2001 influenza seasons. The number of influenza isolates also increased from 64 to 491.
A wide variety of rapid tests exists today, and each laboratory must decide on an individual basis which tests to offer primary care providers. In addition to cost effectiveness, another consideration is to identify infectious diseases for which there are rapid tests available, and from which patients would benefit the most from rapid treatment. Also, the present or conventional diagnostic methods need to be evaluated to determine if they are providing accurate results in a timely manner. The incidence of a disease in the population the laboratory serves needs to be considered, because incidence affects the performance of diagnostic tests (e.g. positive predictive value).
Clinical laboratory scientists need to assess the impact on patients of false-positive and false-negative results. False-negative results will prolong initiation of treatment, which could obviously have severe consequences to the patient. In addition, delayed treatment can promote transmission of contagious diseases, such as those that are sexually transmitted. False-positive results can result in unnecessary expensive treatment and produce undue stress in a patient, particularly with sexually-transmitted diseases. Cross contamination of M. tuberculosis cultures resulted in misdiagnosis of three patients that had total costs of $32,618. (24) Although cultures are not a rapid test, this example points out the costs of misdiagnoses.
Each new test needs to be evaluated. Laboratorians cannot always rely on the sensitivities and specificities reported in package inserts. Performance evaluations are affected by the choice of the reference method. In the case of C. trachomatis, if cell cultures alone are used as the reference method, the sensitivities of comparison methods will be falsely increased. Before adapting a particular rapid test, the clinical laboratory scientist must decide which microorganisms need a better diagnostic test, which clinical specimens should be tested (this can affect the sensitivity and specificity), and if the rapid test fulfills the laboratory's criteria of sensitivity, specificity, speed, simplicity and cost.
Table 1 Comparison of nucleic acid tests in the diagnosis of Chlamydia trachomatis infection Percent Sensitivity Specificity PPV NPV Endocervical (a) AMP-CT 100 99.2 87.5 100 COBAS 100 98.5 80.0 100 PACE 2 85.7 98.9 82.8 99.2 Urethral, male (a) HC2 CT 97.0 99.8 98.0 99.7 PACE 2 69.3 98.3 78.7 97.2 Urine (b) AMP-CT 80.8 100 100 99.0 COBAS 88.5 99.4 88.5 99.4 (a)Data is from Semeniuk, et al. (25) (b)Data is from Templeton, et al. (26) Note: Table made from bar graph Table 2 Diagnostic performance compared to LCx for asymptomatic male urine specimens Percent Sensitivity Specificity Testpack Chlamydia 70.9 95.5 Surecell Chlamydia 62.9 100 Clearview Chlamydia 57.7 95.5 LE dipstick Chlamydia 87.5 92.4 Data is from Chernesky, et al. (12) Note: Table made from bargraph
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(20.) Pate MS, Dixon PB, Hardy K, Crosby M, Hook EW III. Evaluation of the Biostar Chlamydia OIA Assay with specimens from women attending a sexually transmitted disease clinic. J Clin Microbiol. 1998:36:2183-2186.
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(25.) Semeniuk H, Zentner A, Read R, Church D. Evaluation of sequential testing strategies using non-amplified and amplified methods for detection of Chlamydia trachomatis in endocervical and urine specimens from women. Diagnostic Microbial In fact Dis. 2002;42:43-51.
(26.) Templeton K, Roberts J, Jeffries D, Forster G, Aitken C. The detection of Chlamydia trachomatis by DNA amplification methods in urine samples from men with urethritis. International Journal of STD & AIDS. 2001;12:793-796.
Don Lehman teaches medical bacteriology, parasitology, virology and serology/immunology in the department of Medical Technology, University of Delaware, in Newark.
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|Publication:||Medical Laboratory Observer|
|Date:||May 1, 2003|
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