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

1500091-7370/83/0300-0150 $00.90 © Institute for Clinical Science, Inc.

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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