Laboratory diagnosis of Mycoplasma pneumoniae infection

D2 LABORATORY DIAGNOSIS OF Mycoplasma pneumoniae INFECTION R. J. Harris^ J. Williamson^ C. Hahn^ and B. P. Marmion

Introduction and Overview The early expectations for laboratory techniques allowing the rapid diagnosis and immediate clinical management of atypical pneumonia caused by Eaton agent (Mycoplasma pneumoniae), which were raised by the isolation of the causative organism (Eaton et aL, 1944), its identification as a mycoplasma, and its growth in cell-free media (Marmion and Goodbum, 1961; Chanock et aL, 1962), and the subsequent development of various antibody assays, are only now nearing reality. However, there is no single test or array of tests which will provide a rapid, economical diagnosis which can direct clinical chemotherapeutic decisions in a reasonable time frame (:^24 hours) and also detect a realistic percentage of infections (>:90%), although some tests are approaching that ideal. One established test is the antigen capture or antigen-enzyme immunoassay (Ag-EIA) of Kok et al. (1988). Simplified, quantitative polymerase chain reaction (PCR) DNA amplification, now under development in a number of centers, will no doubt provide another rapid test. The development of effective tests has been hampered by two major constraints. First, and principally, confirmation by culture of the organism is lengthy and difficult. The difficulty in culturing the organism presumably arises from its minimal absorptive and synthetic capability. Few other major human pathogens offer similar difficulties. Even with improved diphasic medium (Kenny et al., 211 Molecular and Diagnostic Procedures in Mycoplasmology, Vol. II

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1990), the growth, subculture onto solid medium, and identification of colonies may take 7 to 42 days. Moreover, even when completed evidence shows that culture only detects a proportion (68%) of those infected as judged by serodiagnosis. Second, and conversely, serodiagnosis by the commonly used complement fixation test is also slow. The conventional criterium for identification of a current infection is a fourfold rise in specific antibody (total or IgM generally) and requires collection of paired sera over an interval of 5-8 days. Additionally, serodiagnosis falls short of detecting all culture-positive patients. Thus both culture and serodiagnosis are too slow to direct early clinical intervention. Despite the limitations, the routine laboratory diagnosis of M. pneumoniae infection often relies on serodiagnosis alone, principally by complement fixation or, more recently, on EI A techniques or agglutination of antigen-coated particles. In many clinical settings a tentative diagnosis of M. pneumoniae infection is based on a number of clinical and epidemiological factors (Clyde, 1993). These selective factors are at present the mainstay of diagnosis because they allow rapid chemotherapeutic intervention. Strategies for laboratory diagnosis which have been developed (but not necessarily clinically assessed or widely available commercially) are summarized in Fig. 1 and Table I. Three categories of tests have been devised. These comprise first, direct detection of the organism by culture; second, detection of cellular components (DNA, rRNA, antigens); and third, quantification of the nonspecific and specific immunological responses of infected individuals. Direct methods comprise culture of the organism via liquid culture, agar, diphasic, and cell sheet. Methods for detection of cellular components involve detection of the antigens of the organism (total proteins, PI adhesin, or glycolipids), detection of specific DNA sequences, and detection of specific ribosomal RNA sequences. Assays of the immunological response of infected individuals include those for nonspecific cold hemagglutinins; M. pneumoniae-specific IgM, IgG, and IgA; and a general (nonspecific) T-cell activation marker, i.e., raised serum adenine deaminase activity. The three categories of tests are presented in detail in the following discussion.

Detection of Cellular Components of M. pneumoniae Detection of M. pneumoniae Antigens by El A Kok et al. (1988) have developed a simple and effective antigen capture method (Ag-EIA) for detecting M. pneumoniae antigens (proteins) in respiratory exudates. The antigen capture assay protocol involves first coating of microtiter wells

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D2 Laboratory Diagnosis of M. pneumoniae

rRNA

M. pneumoniae

t

Culture 1 Liquid Culture 2 Agar 3 Diphasic 4 Cell Sheet

In-Solution Hybridisation Assay (Gen-Probe)

P1 Adhesin

Immune Response

IgM igG igA IgE

Antik)ody Assays 1 CF 2 IHA(t) 3 IHA (M) 4 IgM-EiA 5 IgA-EIA 6 IgE-EIA

DNA

Antigen Assays 1 Immunofluorescence ^ 2 Antigen-EIA (Ag-capture) 3 P1 specific EIA 4 T cell response (raised adenine deaminase) DNA Assays 1 Dot blot hybridisation 2 PCR - with DNA detected by: (a) Agarose gel electrophoresis (b) Dot blot hybridisation (c) Solid phase capture (d) Solid phase PCR

Fig. 1. Listings of diagnostic targets of M. pneumoniae and corresponding assays for detection of infections mediated by the organism.

with "capture" rabbit antiserum to M. pneumoniae. A point of central importance is the adsorption of the rabbit antiserum with human fetal lung. Specimens are diluted in skim milk (or casein)-phosphate-buffered saline (PBS)-Tween, sonicated briefly, and then added to the coated wells. The detector layer consists of guinea pig antisera to M. pneumoniae followed by the rabbit anti-guinea pig sera coupled to horseradish peroxidase (indirect kg capture). Substitution of a PI adhesin-specific, monoclonal antibody either for "capture," or in the detection system, was less effective. Similarly, the use of a high-titer polyclonal, Plspecific, rabbit antiserum did not improve sensitivity. The latter was raised by transfection of RK13 rabbit cells with a "universal" coding equivalent of the PI adhesin gene (i.e., UGA terminator codons removed by in vitro mutagenesis;

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Professor P.-C. Hu, University of North Carolina) under the control of a zincinducible metallothionein promoter. Zinc induction of the PI gene led to the production of PI protein which was then used (unpurified) to raise high titer Plspecific antibodies in rabbits (Williamson et al, 1994). Attempts to express the PI gene in Escherichia coli have been unsuccessful. Of a wide range of mycoplasmas and bacteria (many of which can be found in the respiratory tract), only M. genitalium reacted to a minor extent in the AgEIA as devised by Kok et al. (1988). The assay detected 10"^ colony-forming units (CFU)/ml in specimens artificially "laced" with known numbers of M. pneumoniae. The Ag-EIA detects 59% of serologically proven cases; about the same rate as improved culture reported by Kenny et al. (1990). The 100% or reference "gold" standard was provided by serodiagnosis, either a high, unchanging CF antibody titer with IgM detected by hemagglutination-IgM capture (see later) or a fourfold or greater rise in specific antibody titer. Antigen capture-EIA is much quicker (18 hours) than culture and provides an initial diagnosis for clinical management, with later consolidation of the diagnosis by serological examination. A similar Ag-EIA has been developed by Kleemola et al. (1993) and is marketed as Enzygost by Behring. Since 1988, in the virus diagnostic laboratory of the Institute of Medical and Veterinary Science, Adelaide, the Ag-EIA for M. pneumoniae has been included with other Ag-EIA for direct diagnosis of infection due to respiratory viruses. This has been highly effective in detecting M. pneumoniae infections in the late winter/spring (southern hemisphere) of 1987, 1988, 1989, and 1990, but with only sporadic cases in 1991. Detection of M. pneumoniae Antigens by Immunofluorescence Understandably, modem microbiological laboratories are avoiding the more labor-intensive (and therefore expensive), subjective visual immunofluorescencebased diagnostic methods for antigen detection and prefer semiautomated test systems which are principally EIA in nature. Nevertheless, an indirect IF test, based on previous observations by Hers and Masurel (1967), has been successfully reexplored by Hirai etai (1991). Smears made from throat swabs were examined with a carefully absorbed polyclonal rabbit antiserum against M. pneumoniae and samples from 42/49 (86%) patients with serological evidence of current infection were positive. Detection of M. pneumoniae by Probing for Specific Nucleotide Sequences Cloned genomic fragments of M. pneumoniae have been used by us and others (Hyman et al., 1987) in dot-blot hybridization-based assays for detection of

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M. pneumoniae. In our laboratory, two M. pneumoniae-spQcific probes, labeled with 32P, comprising ~1000-bp double-stranded (denatured) molecule and a single-stranded 500 nucleotide Ml3 probe were prepared and tested (R. Harris et al., unpublished). However, their limit of detection, 10^ CFU, of M. pneumoniae was considered too insensitive for further development (see Chapters Al and A3, this volume; and review Razin, 1994). Attention was therefore turned to the M. pneumoniae rapid diagnostic system (Gen-Probe, San Diego, CA). This assay involves an "in solution" hybridization of a i25i.iabeled probe against a specific ribosomal RNA (rRNA) sequence of the mycoplasma. The assay was considerably more sensitive with simulated samples than was Ag-EIA or hybridization with the DNA probes, presumably reflecting the larger number of target rRNA molecules versus genomes per cell; —10^ CFU of M. pneumoniae was detected. However, rather surprisingly with clinical specimens, the rapid diagnostic system detected only about one-fourth of the specimens detected by Ag-EIA. This insensitivity was speculatively attributed to selective degradation (or rapid selective clearance) of the target rRNA in respiratory secretions during natural infections, once the membrane of the organism had been breached by immune reactions or chemotherapy. Proteins and glycolipids detectable by Ag-EIA were speculated to be cleared from the respiratory tract more slowly (Harris et al., 1988). In view of these findings, attention was turned to PCR amplification of sequences within two genes of M. pneumoniae as a method of direct detection of the organism. Primers and probes (described in detail in Williamson et al., 1992) were designed to detect a section of the PI or cytadhesin gene with the product identified by dot-blot hybridization (DBH) following PCR amplification (PlPCR-DBH assay). Similarly, sequences within the 16S rRNA gene were assayed (16S rDNA-PCR-DBH assay). The latter assay was established to exclude certain antigen-positive, PCR-negative results that may arise from a possible strainto-strain variation in the PI gene; it is presumed that the rRNA gene sequence would be highly conserved between strains of M. pneumoniae. The PCR product from each assay was detected and quantified by DBH with a synthetic ^^p. labeled hairpin probe. The bound counts in the blots were quantified by scintillation counting, and a sample ratio was derived: sample ratio = (sample counts per minute - background counts per minute)/background counts per minute (Williamson et al., 1992). The sensitivity was excellent; 50 CFU of M. pneumoniaelmX were detected: 200-fold less than could be detected by Ag-EIA. Tests with a panel of organisms including M. genitalium showed no crossreactions. A quantification curve was constructed from the Pl-PCR-DBH sample ratios obtained from dilutions of a laboratory culture of M. pneumoniae, which, in turn, allowed an approximation of CFU of M. pneumoniaelml in the range of 50-»10^ CFU/ml for any clinical sample. Certain samples were also tested with a PCR-DBH assay for M. genitalium, constructed along the same lines to ex-

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elude the possibility that Ag-EIA-positive, PCR-DBH-negative samples were the result of the presence of M. genitalium, which cross-reacts antigenically with M. pneumoniae. The antigen-positive/PCR negative samples proved negative for M. genitalium. However, the latter organism was detected in samples from two other subjects negative for M. pneumoniae. While this study was in progress, other investigators developed PCR-based assays for M. pneumoniae mostly using simulated positive samples. Those working with material from naturally infected subjects include de Barbeyrac et at. (1993) and Luneberg et al. (1993). The latter detected, via PCR, 83% of serologically and/or culture proven infections (see Chapter A6, this volume; and Razin, 1994).

Isolation of M. pneumoniae by Culture Considerable advances have been made in improving both the percentage of isolations (i.e., relative to the "gold" standard of serologically proven infections) and in reducing the time in culture to obtain a positive result. Improvements have included development or application of special liquid media (SP4; Tully et al., 1979; Chapter A2, Vol. I) and diphasic (liquid-solid) media; the latter improving isolation by 26% to an impressive 68% of those with serological evidence of infection (Kenny et al., 1990). Also, culture on live but cycloheximide-inhibited feeder cell sheets (Marmion et al., 1993) reduced culture time to as little as 5 days. Detection of M. pneumoniae in the cell sheet system is by immunofluorescence, or preferably Ag-EIA. Direct comparisons of culture in the cell sheet system and in diphasic media with SP4 as the liquid phase were carried out with homogenized lung samples from artifically infected guinea pigs. These comparisons consistently demonstrated that the cell sheet culture was slightly more sensitive and certainly more rapid. However, the same study showed that Pl-PCR-DBH assay was considerably more sensitive and exposed the poor plating efficiency of both culture systems (Marmion et al., 1993).

Detection of M. pneumoniae Infections by Measurement of the Immunological Response of Individuals with Respiratory Infection A number of assays have been used for detection and quantification of M. pneumoniae antibodies, including the older ones based on complement fixation (CF), metabolic inhibition (e.g., tetrazolium reduction-inhibition technique), mycoplasmacidal test, radioimmunoprecipitation, radioimmunoassay, and hemagglutination (Taylor-Robinson et al., 1966; Busolo and Meloni, 1983; Hu et al., 1993) and the newer techniques based on EI A (Jacobs, 1993). Those relying on

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culture are too cumbersome and slow for modem diagnostic services. However, CF (which predominantly detects IgM) and EIA are widely used and particle agglutination assays have become available. Measurement of IgM and Other Immunoglobulin Class Responses by Modified Indirect Hemagglutination Assay (IHA) (Kok et aL, 1989) Sheep erythrocytes are glutaraldehyde fixed, tanned, and coated with sonicated M. pneumoniae. Sensitized erythrocytes can be stored up to 2 months at 4°C without loss of activity. The assay is based on the antibody capture principle. Microtiter plates are coated with antisera to human |JL, a, or 7 chains, dilutions of the patients' sera are added and incubated, and the wells are subsequently washed. When antigen-coated erythrocytes are added, the settling pattern in those wells in which immunoglobulins to M. pneumoniae have been captured leads to the cells forming a shield rather than a central button in the cup or well. The |x chain capture version of the assay [IHA(M)] has been shown to be consistently more sensitive than the CF test. For example, Kok et al. (1989) found that with the CF assay as the standard, IHA(M) detected 89% of the patients who had CF antibody, whereas in reverse, with IHA(M) as the standard, the sensitivity of the CF test was 64%. Although the results of the IHA(M) assay correlated well with those of a locally developed solid-phase indirect IgM-EIA in formal comparisons, investigations of respiratory infection in the Newcastle survey (see later) showed that IgM-EIA tests were positive for only 2 of 24 subjects who were seropositive by IHA(t) (for total specific antibody) and also by IHA(M). Finally, the combined use of IHA(t)—in essence the test as devised by Dowdle and Robinson (1964) which does not utilize antibody capture—for measurement of the total antibody along with the 7 and a chain capture variants would allow additionally the detection of IgG or IgA responses in the absence of IgM responses in persons who are experiencing reinfection with M. pneumoniae (see Hu et al., 1983; Jacobs, 1993). However, it is possible that the use of antibody capture for the measurement of responses of these immunoglobulin classes might be more sensitive than IHA(t); this requires investigation. Other workers have developed EIA for M. pneumoniae-spQcific IgM (Samra and Gadba, 1993; Wreghitt and Sillis, 1985). These also offer greater sensitivity than the CF test. Care is required to eliminate false IgM positives arising from the presence of rheumatoid factor complexes with M. pneumoniae-specific IgG. Suspect sera can be adsorbed with an IgG-specific latex bead adsorbent. Bead-Based Agglutination Tests for Detection of M. pneumoniae-Specific IgG A high density particle agglutination test for M. pneumoniae IgG has been developed. The test is very rapid (30 minutes) and is more sensitive than the CF

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test (Shitara et al., 1990). However, a latex agglutination test was assessed by Karppelin et al. (1993) and found to be insensitive.

Cross-Reaction of M. genitalium in M. pneumoniae Tests M. genitalium is occasionally found in the respiratory system and shares antigens with M. pneumoniae (Baseman et al., 1988; Lind et al., 1984). M. pneumoniae antibodies have been shown in laboratory tests to cross-react with M. genitalium. Consequently, strictly speaking, a definitive diagnosis cannot be made with most available tests based on the detection of M. pneumoniae antigens or antibodies. However, PCR-based tests are specific. This is expected in view of the small degree of total genomic sequence homology between the two organisms.

Concluding Remarks One of the problems with the laboratory diagnosis of M. pneumoniae infection lies with the well-known insidious nature of disease onset. The patient may not seek medical attention for days or weeks until increasing malaise and respiratory distress force a visit. By that time the initial phase of the respiratory infection may have passed when organisms might have been more easily cultivated or demonstrated. Also, the serological response may have reached a plateau, excluding the possibility of meeting the conventional criterion for a current infection that a fourfold or greater increase in antibody titer must be demonstrated. Several rapid tests are available to assist clinical management: first, antigen detection in respiratory secretions by enzyme immunoassay (Kok et al, 1988; Kleemola et al., 1993) and second, a number of alternative techniques for detection of M. pneumoniae-spQcific total or IgM or other immunoglobulins, early in infection from a single serum sample. Presumptive evidence of infection is indicated by a significant titer, for example, a titer >:32 via CF (Jacobs, 1993) or via EI A or |x chain-specific indirect hemagglutination (Kok et al., 1989). Similarly, detection of early M. pneumoniae-spQcific IgA levels, particularly in cases of reinfection, may be useful. In our experience one of the more convenient and rapid diagnostic procedures is the Ag-EIA of Kok et al. (1988): this detects the "complete" antigenic mosaic of the organism. In its present usage in Adelaide it provides routine detection of the M. pneumoniae antigen in secretions of patients with severe respiratory infections which might be due to M. pneumoniae. Results are timely (18 hours) for directing appropriate chemotherapy. Specificity is excellent, and approximately 59% of serologically proven cases are detected.

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Surprisingly, PCR-based tests, although more rapid, do not always detect a greater percentage of cases (Williamson et ai, 1992). However, Luneberg et aL (1993) detected 83% of culture and/or serologically proven infections using PCR coupled with anchored liquid-phase hybridization detection of the amplified DNA product. In view of the problems associated with PCR—principally the ease of occurrence in false positives due to contamination of laboratories with amplicons arising from previous amplifications—it may be premature to embark on routine screening with this method. Instead, PCR may be used for the more serious systemic infections (e.g., of the central nervous system) or for infections in immunocompromised individuals with respiratory infections. As stated, serological examination of patients is the predominant method for diagnosis of M. pneumoniae infection. Detection of diagnostically significant levels of total antibody to M. pneumoniae and specific IgM and IgG (and perhaps IgA) from a single blood sample taken from patients shortly after presentations also provides timely presumptive information for clinical decisions on the form of appropriate chemotherapy. According to Granstrom et al. (1994), M. pneumoniae-^^tciiic IgA (via EIA) develops more often and more rapidly than IgM. Additionally, elevated IgM is less often seen in infections in older age groups. For IgM, approximately 40% of infected individuals were positive in the first serum sample (Samra and Gadba, 1993). This percentage is higher if all three antibody classes are determined (Granstrom et al., 1994). However, sensitive M. pneumoniae-specific IgM capture methods are required (e.g., EIA); the conventional complement fixation test is less sensitive. There are therefore a range of diagnostic methods offering improved speed and sensitivity which potentially allow timely chemotherapeutic intervention in M. pneumoniae infections.

References Baseman, J. B., Dallo, S. F., Tully, J. G., and Rose, D. L. (1988). Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract. J. Clin. Microbiol. 26, 22662269. Busolo, F., and Meloni, G. A. (1983). Serodiagnosis of M. pneumoniae infections by enzyme-linked immunosorbent assay (ELISA). Yale J. Biol. Med. 56, 517-521. Chanock, R. M., Hayflick, L., and Barile, M. F. (1962). Growth on artificial medium of an agent associated with atypical pneumonia and its identification as a PPLO. Proc. Natl. Acad. Sci. U.S.A. 48, 41-49. Clyde, W. A., Jr. (1993). Clinical overview of typical Mycoplasma pneumoniae infections. Clin. Infect. Dis. 17(Suppl. 1), S32-S36. de Barbeyrac, B., Bemet-Poggi, C , Febrer, F., Renaudin, H., Dupon, M., and Bebear, C. (1993). Detection of Mycoplasma pneumoniae and Mycoplasma genitalium in clinical samples by polymerase chain reaction. Clin. Infect. Dis. 17(Suppl. 1), S83-S89. Dowdle, W. R., and Robinson, R. Q. (1964). An indirect haemagglutination test for diagnosis of Mycoplasma pneumoniae infections. Proc. Soc. Exp. Biol. Med. 116, 947-950.

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Eaton, M. D., Meiklejohn, G., and van Herick, W. (1944). Studies on the etiology of primary atypical pneumonia: A filterable agent transmissible to cotton rats, hamsters and chick embryos. J. Exp. Med. 79, 649-668. Femald, G. W., and Clyde, W. A., Jr. (1989). Epidemic pneumonia in university students. J. Adolesc. Health Care 10, 520-526. Granstrom, M., Holme, T., Sjogren, A. M., Ortqvist, A., and Kalin, M. (1994). The role of IgA determination by ELISA in the early serodiagnosis of Mycoplasma pneumoniae infection, in relation to IgG and |x-capture IgM methods. J. Med. Microbiol 40, 288-292. Harris, R., Marmion, B. P., Varkanis, G., Kok, T., Lunn, B., and Martin, J. (1988). Laboratory diagnosis of Mycoplasma pneumoniae infection. 2. Comparison of methods for the direct detection of specific antigen or nucleic acid sequences in respiratory exudates. Epidemiol. Infect. 101, 685-694. Hers, J. F. P., and Masurel, N. (1967). Infection with Mycoplasma pneumoniae in civilians in the Netheriands. Ann. N. Y. Acad. Sci. 143, 447-460. Hirai, Y., Shiode, J., Masayoshi, T., and Kanemasa, Y. (1991). Application of an indirect immunofluorescence test for detection of Mycoplasma pneumoniae in respiratory exudates. J. Clin. Microbiol. 29, 2007-2012. Hu, P . - C , Powell, D. A., Albright, F., Gardner, D. E., Collier, A. M., and Clyde, W. A., Jr. (1983). A solid-phase radioimmunoassay for detection of antibodies against Mycoplasma pneumoniae. J. Clin. Lab. Immunol. 11, 209-213. Hyman, H. C , Yogev, D., and Razin, S. (1987). DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium. J. Clin. Microbiol. 25, 726-728. Jacobs, E. (1993). Serological diagnosis of Mycoplasma pneumoniae infections: A critical review of current procedures. Clin. Infect. Dis. 17(Suppl. 1), S79-S82. Karppelin, M., Hakkarainen, K., Kleemola, M., and Miettinen, A. (1993). Comparison of three serological methods of diagnosing Mycoplasma pneumoniae infection. J. Clin. Pathol. 46, 1120-1123. Kenny, G. E., Kaiser, G. G., Cooney, M. K., and Foy, H. M. (1990). Diagnosis of Mycoplasma pneumoniae pneumonia: Sensitivities and specificities of serology with lipid antigen and isolation of the organism on soy peptone medium for identification of infections. J. Clin. Microbiol. 28, 2087-2093. Kleemola, M. Raty, R. Karjalainen, J., Schuy, W., Gerstenecker, B., and Jacobs, E. (1993). Evaluation of an antigen-capture enzyme immunoassay for rapid diagnosis of Mycoplasma pneumoniae infection. Eur. J. Clin. Microbiol. Infect. Dis. 12, 872-875. Kok, T., Varkanis, G., and Marmion, B. P. (1988). Laboratory diagnosis of Mycoplasma pneumoniae infection. 1. Direct detection of antigen in respiratory exudates by enzyme immunoassay. Epidemiol. Infect. 101, 669-684. Kok, T., Marmion, B. P., Varkanis, G., Worswick, D. A., and Martin, J. (1989). Laboratory diagnosis of Mycoplasma pneumoniae infection. 3. Detection of IgM antibodies to M. pneumoniae by a modified indirect haemagglutination test. Epidemiol. Infect. 103, 613-623. Lind, K., Lindhart, B. O., Schiitten, H. J., Blom, J., and Christiansen, C. (1984). Serological crossreactions between Mycoplasma genitalium and Mycoplasma pneumoniae. J. Clin. Microbiol. 20, 1036-1043. Luneburg, E., Jensen, J. S., and Frosch, M. (1993). Detection of Mycoplasma pneumoniae by polymerase chain reaction and nonradioactive hybridization in microtitre plates. J. Clin. Microbiol. 31, 1088-1094. Marmion, B. P., and Goodbum, G. M. (1961). Effect of an organic gold salt on Eaton's primary atypical pneumonia agent and other observations. Nature (London) 189, 247-248. Marmion, B. P., Williamson, J., Worswick, D. A., Kok, T., and Harris, R. J. (1993). Experience with newer techniques for the laboratory detection of Mycoplasma pneumoniae infection: Adelaide, 1978-1992. Clin. Infect. Dis. 17(Suppl. 1), S90-S99.

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