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ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 13, No. 2Copyright © 1983, Institute for Clinical Science, Inc.
Acute-Phase, Indirect Fluorescent Antibody Procedure for Diagnosis of M ycoplasma pneumoniae InfectionJ. B. CARTER, M.D.* andS. L. CARTER, MT(ASCP)SM, SIf
*Immunology Laboratory, Henrotin Hospital, and D epartm ent o f Pathology,
Northwestern University Medical School, Chicago, IL 60610
and\Immunology Laboratory, Henrotin Hospital,
Chicago, IL 60610
ABSTRACTAn indirect fluorescent antibody (IFA) technique was developed to de
tect IgG and IgM-specific antibodies to Mycoplasma pneumoniae. The presence of IgM-specific mycoplasma antibody was in terpreted as reflecting active infection in patients w ith atypical pneum onia or other clinically compatible illness. The procedure is suitable for use in routine clinical laboratories, correlated well with com plem ent fixation test results and did not show cross reaction with Legionella pneum ophila antibody. The ready availability of an acute-phase procedure for diagnosis of M ycoplasm a pneumoniae infection permits therapeutic judgm ents based on testing of the acute serum sample.
IntroductionLaboratory diagnosis of M ycoplasma
pneumoniae infection has been lim ited to non-specific procedures such as detection of cold agglutinins and streptococcus MG antibody, or to com plem ent fixation (CF) techniques and cultural isolation procedures.7,8 The latter procedures are av ailab le only in la rg e r or re fe ren ce laboratories and often involve a delay of 10 days to two weeks prior to receipt of te s t resu lts . R ecen t rev iew s of m ycoplasma infection refer to the need for a rapid and readily available diagnostic procedure to assist in clinical evaluation of m ycoplasm al d iseases.4,5 This com
m unication p resen ts a rap id , in d irec t f luo rescen t antibody (IFA) p rocedure specific for M ycoplasm a pneum oniae that is su itable for use in the routine m edical laboratory.
M aterials and M ethodsE q u i p m e n t
The procedure requires only routinely available immunology laboratory equipment. A good quality fluorescent microscope is important, particularly fluorescein isothiocyanate (FITC)-specificity of th e excitor and b a rrie r filte rs . W hile transm ission flu o rescen t illu m in a tio n
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IFA MYCOPLASMA PROCEDURE 151
may be acceptable, the IFA Mycoplasma procedure is significantly easier in performance and interpretation using epi- illum inated fluorescent microscopy.
R e a g e n t s
A ntigen: A com m ercially available,freeze-dried, whole-organism M ycoplasma pneum oniae preparation (#VA-10)* serves as the antigen substrate.
Fluorescein Labeled Antiserum : F luorescein isothiocyanate (FITC) anti-human IgM (sheep) (#MF04)* and anti-hum an IgG (sheep) (#MF03).* These are reconstituted according to m anufacturer’s instructions. Purity of the conjugate is confirm ed according to th e p ro ced u re of Nakamura and associates.9
FA Rhodam ine Counterstain: f This is reconstitu ted according to m anufacturer’s instructions. It is d iluted 1:20 in PBS w ith w ork ing c o n c e n tra tio n of F IT C -co n ju g a te (e.g ., 1.0 ml F IT C - conjugate of stock concentration, 8.5 ml phosphate buffered saline (PBS) 0.5 ml reconstituted rhodamine).
Three Percent Normal Yolk Sac Suspension: T his is av a ilab le from th eCenter for Disease Control. |
Phosphate B uffered Saline: The pHis 7.2 to 7.4.
Glycerol Mounting Medium: Glycerol/PBS—9/1.
M ultiw ell Slides: These are commercially available. §
Cover-Slips: #1 thickness, 24 x 50mm.
C o n t r o l s
Commercially available positive control sera for M ycoplasm a pneum oniae are positive for th e IgG -specific an tibody. The IgM-specific control serum is
* Wellcome Reagents, Research Triangle Park, NCf Difco, Detroit, MI| CDC, Atlanta, GA§ J. Melvin Freed Inc., Perkasie, Pa. 18944.
obtained from confirm ed active cases of M ycoplasm a pneum oniae infection, pooled and stored at — 20°C in 0.5 ml aliquots. Negative control serum may be obtained commercially or prepared as a pool of known negative patients. Known positive and negative control sera should be run w ith each test batch.S a m p l e
The patient sample consists of a single, routinely collected and separated serum specim en. Sam ples are usually fasting ow ing to rou tine laboratory collection procedures. However, non-fasting samples have not posed a problem. Separated serum is stable for testing purposes at am bient tem peratures for a b rie f period of storage or transport and is stored at 4°C for periods greater than 24 hours. M inor hemolysis has not affected test results.S u b s t r a t e S l i d e P r e p a r a t i o n
1. C om m ercia l M ycop lasm a p n e u m oniae an tigen is reh y d ra ted according to m anufacturer’s instructions.
2. Using a calib rated p ipet, 3 fil of antigen suspension are placed on each well of a m ulti-well fluorescent microscopic slide. The slides are dried for 30 m inutes at room tem perature.
3. Slides are fixed in acetone at room tem perature for 15 minutes.
4. Slides are dried at room tem peratu re and sto red at —20°C u n til used . Slides are stable at this tem perature for at least six months.T e s t i n g P r o c e d u r e
1. A single routinely drawn and separated patient serum sample constitutes the testing specim en in m ost circum stances. All serum samples are retained at 4°C for a minimum of two weeks in the event of need for follow-up testing, at which tim e initial and follow-up sera are tested in parallel.
152 CARTER AND CARTER
2. Previously prepared substrate slides are rem oved from the freezer and equilibrated at room tem perature for five to 10 minutes.
3. Using a calib rated p ipet, the patient’s serum is serially diluted. The initial dilution (1:8 or 1:10) is made in three percent normal yolk sac suspension; subsequent dilutions are made in PBS.
4. Using a 25 /u.1 calibrated pipet, 25 /¿I of appropriately titered serum are added to each well or reaction site.
5. S lides are in cu b a ted in a m oist cham ber at 37°C for 60 minutes.
6. S lides are rin sed in at least two changes of PBS for at least 10 minutes.
7. S lides are b lo tted carefully w ith smooth absorbent paper to avoid disruption of the antigen substrate.
8. Twenty-five ju.1 of appropriate IgG- or IgM -specific anti-hum an FIT C are added w ith rhodam ine counterstain to each reaction site.
9. S lid es are in c u b a te d in a m oist cham ber at 37°C for 30 minutes.
10. Slides are rinsed and dried, as in steps 6 and 7.
11. Using PBS buffered glycerol as the mounting m edium , coverslips are placed on slides.
12. Slides are read using incident light fluorescence at 200 x magnification with an FITC-specific filter combination.
13. A positive resu lt appears as an apple-green, fluorescent slurry of particulate m atter consistent w ith the almost submicroscopic morphology of the Mycoplasma pneumoniae organism (figure 1). Incorporation of the rhodamine countersta in su p p re sse s n o n -sp ec ific b ack ground fluorescence. A negative result appears as a muted, reddish-orange granular field. Strict comparison with known positive and negative sera of both antibody classes and blind reading betw een observers to establish reproducible visual th resholds w ill provide consistent and reproducible results.
14. T ite rs or d ilu tio n s c o n s id e re d
“positive” should be individualized for each laboratory em phasizing clinical correlation and reproducibility betw een obse rv e rs . B o rd e rlin e or w eak re su lts should be interpreted w ith caution and repeated with follow-up samples if clinical suspicion persists.
ResultsDevelopm ent of the IFA mycoplasma
procedure included correlation of test re sults w ith the patient’s clinical status and parallel testing with reference laboratory CF results in an initial series of 128 cases presenting as febrile pneum onitis w ith “normal flora” bacterial cultures (table I). Results of this comparison show the IFA test to be an adequate screening test as it produced no “ false-negatives” relative to CF results. All CF-positive cases w ere also positive w ith the IFA procedure. Four cases which w ere borderline positive, using IFA criteria, involved patients w ith febrile illness of unknown etiology; two of these responded to erythromycin therapy, and two recovered spon taneously.
An additional 165 healthy, asym ptomatic persons were tested using the IFA mycoplasma procedure to determ ine the incidence of com m unity antibody frequency for Mycoplasma pneum oniae. All 165 sera were negative for mycoplasma- IgM; 48 percent were positive for low
TABLE I
Comparison of Mycoplasma Complement Fixation and Indirect Fluorescent Antibody* Results
CF R e s u l t IFA R e s u l tNumber o f
c a s e s
Negative Negative 120Negative Borderline 4Positive Positive 4Positive Negative 0
♦Consecutive samples submitted for mycoplasma testing.
IFA MYCOPLASMA PROCEDURE 1 53
Figure 1. Positive IFA M ycoplasma pneum onia preparation. (400 x)
levels of mycoplasma-IgG (titers of 8 to 32) suggesting previous infection or antigen exposure in these patients. To date, a total in excess of 300 patients has been tested and clinical correlation has rem ained excellent.2
Clinical in terpretation of the significance of class-specific antibody titers for Mycoplasma pneumoniae is outlined in table II, according to criteria established in our own laboratory. Initial dilution titers of mycoplasma-IgM (8 or 10) are interpreted with caution and with correlatio n w ith th e to ta l c lin ica l p ic tu re . Follow-up studies are suggested if the clinical situation warrants.
C o n cu rren t te s tin g of th e o rig ina l series of 128 p a tien ts for L egionella pneum ophila d isclosed n ine serologically positive cases of which five were confirmed using direct fluorescent antibody (DFA) studies on pulmonary tissue or culture isolation of the organism.1 No patient had a duel positive result for both legionella and mycoplasma antibodies.
DiscussionThe I FA mycoplasma procedure is pre
sen ted as a rap id d iagnostic te s t for
M ycoplasma pneumoniae infection and is recom m ended for testing febrile patien ts w ith pneum onitis w ith norm al sputum bacterial studies (“atypical pneumonia”). Interpretation of a positive my- coplasma-IgM titer as indicative of active infection is analogous to current testing in terpretation for hepatitis A virus in fection.6 An initially negative or borderline result in a clinically suspect patient should be followed with serial studies w hich may disclose seroconversion to positive titers.
In our experience, approxim ately 90 percent of test results are clearly positive or negative on testing of the initial, acute serum sample. Only two initially nega-
T A B L E II
Clinical Correlation of Mycoplasma Indirect Fluorescent Antibody Test Results
I n t e r p r e t a t i o n IgM t i t e r Ig G t i t e r
Negative result neg and negPrevious infection neg and > 8Active or recent infection* > 16 and > 16Equivocal resulti 8 - 1 6 and neg - 8
♦Correlate with clinical picture.tSuggest follow-up study if clinically indicated.
154 CARTER AND CARTER
tive patients tested thus far have shown conversion to mycoplasma-IgM antibody positivity, illustrating the usefulness of te s tin g for IgM -specific m ycop lasm a antibody in the acute serum sample.
All patients having positive titers of mycoplasma-IgM antibody w ere tested for rheum atoid factor (RF) with no evidence of dual positivity or cross reaction encountered thus far. A mycoplasma-IgM positive serum also positive for rheum atoid factor w ould be interpreted cautiously w ith close correlation w ith the patie n t’s clinical p resentation and other laboratory data. The low incidence of rheum atoid factor positivity in the genera l p o p u la tio n shou ld n o t p re c lu d e ro u tin e use of an Ig M -sp ec ific IFA m ycoplasm a procedure, particularly if the IFA procedure is restricted to testing febrile patients w ith “bacterial negative” pneum onitis.
M ost p a tie n ts p re su m p tiv e ly d iag nosed as having mycoplasma pneum onitis using IFA test results p resen ted w ith the expected clinical findings of low-grade fever and m ild pneum onitis and responded rapidly to erythromycin th erap y . O ne p a tie n t p re se n te d w ith acute pericard itis , dem onstra ted seroconversion from negative to positive mycop lasm a IFA tite rs , and re sp o n d e d promptly to antibiotic therapy. Another patient presented with acute polyarthritis, positive mycoplasma IFA titers, and responded promptly to appropriate therapy.
Subsequent to presentation of the procedure abstract,3 communication with the director of the M ycoplasma R eference Laboratory in Norwich, E ngland ind icated that a sim ilar, IgM -specific IFA procedure for Mycoplasma pneumoniae had b een found, in th e ir experience, equally sensitive to cultural isolation and identification of the organism.10
The use of class-specific antibody detection in the immunologic diagnosis of M ycoplasma pneum oniae infection re flects a personal bias and experience with
fluorescent microscopy. The basic concept should readily translate to enzyme labeled im m unosorbant assay (ELISA) and /or rad io im m unoassay p ro ced u res and thus prove susceptible to automation.
ConclusionIn response to a need for a rapid and
specific testing procedure for M ycoplasma pneum oniae infection , an IgM and IgG -specific in d irec t f lu o rescen t a n tibody technique was estab lished using w hole-organism M ycoplasm a pneum oniae as the antigen substrate for detection of class-specific antibodies in the acute- phase serum sample. The technique has proven to be a successful on-site screening test in the analysis of more than 300 patients presenting w ith clinical “atypical pneum onitis” and offers rapidly availab le resu lts of com parable sensitiv ity and specificity to traditional com plem ent fixation procedures. Increased case finding resulting from use of the IFA mycoplasma procedure has led to an increased appreciation of the spectrum of the clinical disease.
AcknowledgmentsThanks are extended to Dr. Bruce Rabin for re
view of the manuscript and Ms. Tandy Morris and Ms. Jay Mahoney for preparation of the manuscript.
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