Cumitech 34: Laboratory Diagnosis of Mycoplasmal Infections

Laboratory Diagnosis of Mycoplasmal Infections KEN B. WAITES, CiClLE M. BiBiAR, JANET A. ROBERTSON, DEBORAH F. TALKINGTON, AND GEORGE E. KENNY COORDINATING

EDITOR

FREDERICK

S. NOLTE

Cumitech CUMULATIVE

TECHNIQUES

AND PROCEDURES

IN CLINICAL

MICROBIOLOGY

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IB 2B 3A 4A

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Blood Cultures III Laboratory Diagnosis of Urinary Tract Infections Quality Control and Quality Assurance Practices in Clinical Microbiology Laboratory Diagnosis of Gonorrhea Practical Anaerobic Bacteriology New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide Laboratory Laboratory

Diagnosis Diagnosis

of Lower Respiratory of Bacterial Diarrhea

Tract Infections

Laboratory Laboratory Laboratory Laboratory Laboratory

Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis

of of of of of

Laboratory Laboratory Therapeutic

Diagnosis of Hepatitis Viruses Diagnosis of Chlumydia trachomatis Infections Drug Monitoring: Antimicrobial Agents

Ocular Infections Central Nervous System Infections Viral Infections the Mycobacterioses Female Genital Tract Infections

Laboratory Diagnosis of Viral Respiratory Disease Immunoserology of Staphylococcal Disease Infections of the Skin and Subcutaneous Tissues Rapid Detection of Viruses by Immunofluorescence Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing Laboratory Diagnosis of Viral Infections Producing Enteritis Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion oratory Animals Laboratory Diagnosis of Zoonotic Infections: Chlamydial, tained from Companion and Laboratory Animals Laboratory Safety in Clinical Microbiology

and Lab-

Fungal, Viral, and Parasitic Infections

Ob-

Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract Verification and Validation of Procedures in the Clinical Microbiology Laboratory Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Infections Obtained from Food Animals and Wildlife Laboratory Laboratory

Safety, Management, and Diagnosis of Biological Diagnosis of Mycoplasmal Infections

Cum/techs should be cited as follows, e g Waites, Cumttech 34, Laboratory diagnosis of mycoplasmal Washington, DC Board for ASM &mite&s: Linda Cook, Lynne Garcia, Richard James W Snyder, Allan Truant Editorial

Agents Associated with Rioterrorism

K B , C M BBbear, J A Robertson, D F TalkIngton, and G E Kenny 2001 Society for MIcrobiology, InfectIons Coordlnat\ng ed , F S Nolte American

Alice S Weissfeld, Chair, Vlckle Baselskl, M Jamlson, Karen Knsher, Susan L Mottice,

B Kay Buchanan, Mltchell Michael Saubolle, David

I Burken, L Sewell,

Roberta Carey, Daniel Shapiro.

Effective as of January 2000, the purpose of the Cumitech series IS to provide consensus recommendations regarding the judlclous use of clinical microbiology and Immunology laboratorles and their role In patient care Each Cum/tech IS written by a team of cl~rxclans, laboratonans, and other Interested stakeholders to provide a broad overview of various aspects of infectious collectlon, transport, processing, and disease testing These aspects include a dlscusslon of relevant clinical considerations, Interpretive guIdelInes, the cl~nlcal utility of culture-based and non-culture-based methods and emerging technologtes, and issues surrounding coding, medical necessity, frequency limits, and reimbursement The recommendations in CumZechs do not represent the offlclal views or policies of any third-party payer CopyrIght 0 2001 ASM Press American Society for Microbiology 1752 N Street NW Washington, DC 20036-2904

Laboratory Mycoplasmal

Diagnosis of Infections Ken B. Waites

Department of Pathology, University of Alabama at Birmingham, 618 South 18th Street, WP P230, Birmingham, AL 35233-7331

C&zile M. Bib6ar Laboratoire de Bacte’riologie, Universite’ Victor Segalen Bordeaux 2, 33 076 Bordeaux, France

Janet A. Robertson Department of Medical Microbiology

and Immunology, University of Alberta, Edmonton, Alberta TGG 2H7, Canada

Deborah F. Talkington Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333

George E. Kenny Department of Patbobiology, University of Washington School of Public Health, Seattle, WA 98195 COORDINATING EDITOR: Frederick S. Nolte Emory University Hospital, Emory University, Atlanta, GA 30322 Introduction Taxonomy Significance

......................................................................................... ................................................ and Biologica! Characteristics in Human Diseases .............................................................

2 2 3

............................................................. Mycoplasma pneumoniae Respiratory Disease .............................................................................................. Genitourinary Infections .................................................................................................... Neonatal Infections .................................................................................................... Systemic Infections

3 3 4 4

Laboratory

4

Diagnosis

Culture-Based Tests Nonculture Methods

Serologic

Diagnosis

............................................................................

................................................................................................... .................................................................................................

13

...........................................................................

................................................................................... Cold Agglutinins Complement Fixation .................................................................................. Indirect Immunofluorescence Assays ......................................................................... ......................................................................... Particulate Antigen-Antibody Assays ..................................................................... Enzyme-Linked Immunosorbent Assays Genital Mycoplasma Assays ......................................................................................

Antimicrobial

4 12

Susceptibility

Testing

.................... .............

.....................................................

13 13 14 14 14 15

16

............................................................................................. General Considerations ................................................................................................ Antimicrobial Agents ................................................................................................ lnoculum Preparation ................................................................................................... Broth Microdilution Agar Dilution ............................................................................................................ Agar Gradient Diffusion ............................................................................................ Reporting and Interpretation of Results ...................................................................... ...................................................... Minimum Mycoplasmacidal Concentration Testing

16 17 17 18 19 19 19 20

Appendix 1: Medium Formulations for Cultivation of Mycoplasmas from Humans ..................................................................................... Appendix 2: Suppliers of Kits, Media, and Reagents ............................... .............................................................. Appendix 3: Coding Guidance

20 22 23

References

26

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2

Waites

et al.

CUMITECH

INTRODUCTION

M

ycoplasmas and ureaplasmas represent a complex and unique group of microorganisms that has previously been ignored by many diagnostic laboratories because of their fastidious growth requirements, lack of commercially prepared media for cultivation, and absence of rapid diagnostic procedures and a long-standing and widespread clinical perception that these organisms are of minor importance and that diagnostic tests are not readily available. This situation is now changing because of greater appreciation for the clinical importance of these organisms, improved methods for detection, and the need for documented eradication from infected persons. While considerable progress has been made in the areas of traditional methods for culture-based and serologic diagnosis of mycoplasmal infections, the most significant and exciting advances have occurred in the domain of nucleic acid amplification technology such as PCR and other molecular genetic methods. These techniques have greatly expanded our understanding of the cell biology of these organisms, allowed the sequencing of entire genomes for some species, and provided a basis for the identification and characterization of many new species, and they may eventually be the methods of choice for laboratory detection for slow-growing and/or extremely fastidious species. This Cumitech provides a comprehensive compilation of the biology, clinical significance, methods for laboratory detection, and characterization of mycoplasmal and ureaplasmal species of human origin. It is intended to be a resource for clinicians, researchers, clinical microbiologists, technologists, and students who are interested in learning more about this fascinating group of microorganisms.

TAXONOMY AND CHARACTERISTICS

Table 1. Primary sites of colonization, pathogenicity of mollicutes of human

metabolism, origin

Primary site of colonization

Species

Oropharynx

+ + + + + + + ?+b + ? + + ?

M salivanum M. orale M. buccale M faucium M. lipophilum M. pneumoniae M hominis M. genitalium M. fermen tans M. prima turn M. sperma tophilum M. pirum M. penetrans Ureaplasma spp.” A. laidla wii A. oculi a Metabolize urea ’ -)+ I organism has common or primary

and

Metabokm of.

Genltounnary tract

been found In oropharynx, locatlon IS not known

Patho-

G lucase

Argonine

+ + + + + + +

+ + + + + + + + + + + -

+ + + + + ? + + ?

but

34

whether

genlcltY

+ + + + ? + that

IS a

genome size and cellular dimensions, and thus are considered the smallest life-forms capable of an independent existence (Fig. 1). Mycoplasmas associated with humans range from coccoid cells 0.5 to 0.4 pm in diameter, as in Ureaplasma spp. and Mycoplasma hominis (80), to spindle-shaped cells 1 to 2 km in length and 0.1 to 0.2 pm in width, as in M. pneumoniae (122). The practical consequencesof such a small cell size and massinclude the inability to detect the organisms by the usual method of light microscopy, the lack of turbidity in broth cultures, and the need for micros-

BIOLOGICAL

Bacteria commonly referred to as mycoplasmas (“fungus-form”) are included within the class Mollicutes (“soft skin”), which is composed of 4 orders, 5 families, 8 genera, and over 150 known species. Table 1 lists 16 species isolated from humans, excluding occasional animal mycoplasmas that have been detected in humans from time to time but are generally considered transient colonizers. Mollicutes evolved from clostridium-like cells through the process of many gene deletions (123) and as a consequence have alterations of cellular structures and fastidious nutritional requirements. Mycoplasmas are smaller than most other bacteria, in both

t FIGURE 1 pared to other

Relative sizes mlcroorganlsms

of M

horn/n/s

and

I um

Ureaplasma

spp

com-

CUMITECH

Mycoplasmal

34

copy to visualize colonies on agar. Mycoplasmas permanently lack cell walls and peptidoglycan precursors. They differ from L-forms of bacteria, for which the lack of the cell wall is but a temporary reflection of environmental conditions. Unlike other bacterial cells, mycoplasma cell membranes have a significant sterol content. This accounts in part for the need for serum supplementation in their growth media. Mycoplasmas and ureaplasmas require preformed amino acid precursors and nucleotides as well as other nutrients supplied by the base, the serum, and the yeast extract supplements. Ureaplasmas uniquely require urea, whereas mycoplasmas utilize glucose and/or arginine (Table 1). Growth rates in culture are species dependent and modulated by culture conditions. Ureaplasmas, M. hominis, M. pneumoniae, and M. genitalium have generation times of approximately 1, 1.5, 6, and 16 h, respectively (57). Mollicutes colonize mucosal surfaces of the respiratory and urogenital tracts but may cause invasive disease in immunocompromised hosts or under other . special circumstances. Many species exist as commensals in the human oropharynx and can cause diagnostic confusion with M. pneumoniae when they occasionally spread to the lower respiratory tract. Most mycoplasmal species reside extracellularly; however, some may localize within cells. Even though M. hominis and Ureaplasma spp. are frequently detected in the lower genitourinary tract of normal adults, they can cause disease in some instances, as does M. genitalium. Recent studies, particularly those using the PCR assay, have expanded our understanding of where mycoplasmas may localize in the human body, demonstrating the presence of M. fermentans in synovial fluid of persons with rheumatoid arthritis (85, 86), M. genitalium in the urogenital tract (25,47,52), and M. penetrans in the urine of homosexual males and children with human immunodeficiency virus (48, 118, 119). Mycoplasmas and ureaplasmas may be transmitted by direct contact between hosts, i.e., venereally through genital-genital or oral-genital contact, vertically from mother to offspring either at birth or in utero, by respiratory aerosols or fomites in the case of M. pneumoniae, and even by nosocomial acquisition through transplanted tissues.

SIGNIFICANCE

IN HUMAN

DISEASES

An exhaustive review of the disease associations and pathogenic mechanisms of human mollicutes is not the focus of this publication. The reader is encouraged to consult other references and reviews for further details of diseases associated with human mycoplasmas and their management (7, 19, 95, 97, 117). Among mollicutes isolated from humans, three organisms are of major concern. M. pneumoniae is a well-established pathogen, whereas M. hominis and

Infections

3

Ureaplasma spp. are generally considered opportunists. The two biovars of Ureaplasma urealyticum have been proposed for division into separate species: U. urealyticum and U. parvum (83). Separation of these species is not possible except by PCR (50, 83, 101). Therefore, they will be considered together as Ureaplasma spp. Serologic studies and PCR have enhanced our knowledge of M. genitalium, 111.fermentans, M. pirum, and M. penetrans and their possible roles in certain pathologic condition in humans. Due to their extremely fastidious nature and the lack of reliable means for cultivation on artificial media, detection of these mycoplasmas rests primarily on the use of molecular techniques currently outside the realm of most diagnostic laboratories. Mycoplasma

pneumoniae

Respiratory

Disease

M. pneumoniae occurs endemically and epidemically in persons of all age groups. The most frequent clinical syndrome is tracheobronchitis, often accompanied by upper respiratory tract symptoms. Typical complaints can persist for weeks to months and include hoarseness, fever, cough, sore throat, headache, chills, coryza, and general malaise (34,95). M. pneumoniae accounts for approximately 20% of communityacquired pneumonias requiring hospitalization in the United States (66) and probably an even greater proportion of those not requiring hospitalization. The incubation period is 1 to 3 weeks, and spread throughout households often occurs. M. pneumoniae can persist in the respiratory tract for several months after initial infection and sometimes for years in hypogammaglobulinemic patients (34, 100). Attachment of M. pneumoniae to host cells in the respiratory tract is mediated by the I?1 adhesin protein, followed by induction of ciliostasis, local inflammation, and tissue destruction that may be further mediated by liberation of peroxides. Some people may experience extrapulmonary complications including skin rashes, pericarditis, hemolytic anemia, arthritis, meningoencephalitis, peripheral neuropathy, and pericarditis. M. pneumoniae has been isolated from extrapulmonary sites such as synovial fluid and cerebrospinal fluid, pericardial fluid, and skin lesions. Acute mycoplasma1 infection may also be associated with exacerbations of chronic bronchitis and asthma. The clinical manifestations are not sufficiently distinctive to allow differentiation from infections caused by other common microorganisms. Genitourinary

Infections

Ureaplasma spp. and M. hominis can be isolated from the lower genital tract in many sexually active adults, leading to difficulty in accepting these organisms as causes of disease. Nevertheless, there is evidence that these organisms play etiologic roles in some condi-

Waites

et al.

tions. Ureaplasma spp. and 211.genitalium are causes of nonchlamydial, nongonococcal urethritis in men (27,47,95). Th ere is no evidence that M. hominis is a cause of the female urethral syndrome, but ureaplasmas may be involved (95). There is considerable controversy about whether genital mycoplasmas play an etiologic role in prostatitis. Some investigators have detected these organisms by culture or PCR, whereas others were unsuccessful in doing so (29). Overall, it seems likely that a role for mycoplasmas in prostatitis is minimal. Jalil and coworkers suggest that ureaplasmas can cause acute epididymo-orchitis (5 1). Ureaplasmas induce crystallization of struvite and calcium phosphates in urine in vitro and calculi in animal models (43, 102). M. hominis has been isolated from the upper urinary tract only in patients with symptoms of acute pyelonephritis, often with an antibody response (lOS), and probably causes a small portion of cases of this disease. However, Ureaplasma spp. have not been associated in the same way. Mollicutes do not cause vaginitis but may proliferate in patients with bacterial vaginosis and may contribute to the condition (95). M. hominis has been isolated from the endo metrium an.d fallopian tu bes of 10% of women with salpingitis, accompanied by a specific antibody response. M. genital&m but not ureaplasmas may also play a role in salpingitis (70, 95). Whether ureaplasmas cause involuntary infertility remains speculative (94). Ureaplasmas have been isolated from internal organs of spontaneously aborted fetuses and from stillborn and premature infants more often than from the products of induced abortions or normal full-term infants (19). Ureaplasmas can cause placental inflammation and may invade the amniotic sac early in pregnancy in the presence of intact fetal membranes, causing persistent infection and adverse pregnancy outcome, including premature birth (16, 19). M. hominis has been isolated from the blood of about 10% of women with postpartum or postabortal fever, often in association with seroconversion, but not from afebrile women who have had abortions or from healthy pregnant women (19, 95). Similar observations have been made for Ureaplasma spp. (19,95). Neonatal

Infections

Colonization of infants by genital mycoplasmas may occur by organisms ascending from the lower genital tract of the mother at the time of delivery or in utero ( 19). Congenital pneumonia, bacteremia, progression to chronic lung disease of prematurity, and even death have occurred in very low birth weight infants and have been attributed to ureaplasmal infection of the lower respiratory tract ( 18,19). Both 211.hominis and Ureaplasma spp. have been isolated from maternal and umbilical cord blood, as well as the blood of neonates. Both can also invade the cerebrospinal fluid

CUMITECH

34

(112). Colonization of healthy full-term infants declines after 3 months of age, and fewer than 10% of older children and sexually inexperienced adults are colonized with genital mycoplasmas. While M. fermentans has been detected in pure culture from the placenta and amniotic fluid in the presence of inflammation, no studies have been performed to determine its occurrence and significance in neonates. Systemic

Infections

Mycoplasmas and ureaplasmas can cause invasive disease of the joints and respiratory tract with bacteremit dissemination, especially in individuals with hypogammaglobulinemia (19, 38, 69, 95, 98-100). M. hominis bacteremia has been demonstrated after renal transplantation, trauma, and genitourinary manipulations and has also been found in wound infections (69). M. ff ermentans, Ureaplasma spp., and M. hominis can be detected by culture and/or PCR in synovial fluid of persons with rheumatoid arthritis, although the precise contribution of these organisms to this disease is uncertain ( 85,8 6). The significance of M. fermentans, 111. penetrans, M. pirum, and other mycoplasmas in persons infected with human immunodeficiency virus has received a great deal of attention and is currently a matter of debate (5,12,48,56, 118,119). M. f ermentans has been recovered from the throats of children with pneumonia, but the frequency of its occurrence in healthy children is not known (95). Respiratory infection with M. fermentans is not necessarily linked to immunodeficiency but may also be an opportunistic respiratory disease (2, 64).

LABORATORY

DIAGNOSIS

There are no proficiency tests or any specific regulations concerning mycoplasma testing as distinct from other laboratory requirements in the United States. Clinical laboratories must develop internal procedures for quality control and proficiency and meet the licensing requirements for local or other agencies to which they are responsible. A few specialized reference laboratories provide diagnostic testing for mycoplasmas, as do a small number of laboratories that specialize in mycoplasma research. Resources for mycoplasmal strains, immunological reagents, and suppliers of diagnostic media and kits are provided in the appendixes. Culture-Based

Tests

Cultures should focus on the species known to cause human disease and for which cultivation techniques are best defined. Unusual organisms and extremely fastidious species for which cultivation conditions are not well established may be best detected by PCR, which is offered through a few specialized laborato-

CUMITECH

34

ries. Such organisms should be sought only after consultation with clinicians and with the personnel from the reference laboratory. Infections due to M. hominis are sometimes discovered accidentally, either by observing tiny colonies growing on bacteriologic media such as Columbia agar or because treatment failure with antimicrobials ineffective against mycoplasmas aroused suspicion of a mycoplasmal infection. Growth of any mycoplasma on bacteriologic media cannot be presumed, due to their complex nutritional requirements. Therefore, it is essential to perform specific procedures to detect mycoplasmas in clinical material. It is appropriate to perform diagnostic evaluations for mycoplasmas in patients who are suspected of having a condition for which these organisms have been shown to be of etiologic significance but not in circumstances where there has been no such association. Specimen Collection and Transport Mycoplasmas are extremely sensitive to adverse environmental conditions, particularly to drying and heat. Specimens should be inoculated at bedside whenever possible, using appropriate transport or mycoplasma culture medium, such as SP-4 broth (106) or 10B broth (87), 2 SP (10% [vol/vol] heat-inactivated fetal calf serum with 0.2 M sucrose in 0.02 M phosphate buffer [pH 7.2]), or Trypticase soy broth with 0.5% (vol/vol) bovine serum albumin. 2 SP can also be used for sample preparation for PCR assays. Other media available commercially for transport and storage of specimens include Mycotrans (Irvine Scientific), A3B (Remel, Inc.), and arginine broth (Remel, Inc.). Specialized transport media for mycoplasmas are also available in Europe from various suppliers. Liquid specimens or tissues do not require special transport media if cultures can be inoculated within 1 h, provided that the specimens are protected from drying. Tissues can be placed in a sterile container and delivered to the laboratory immediately. Otherwise, tissue specimens should be placed in transport medium if a delay in culture inoculation is anticipated. When swabs are obtained, care must be taken to sample the desired site vigorously to obtain as many cells as possible, since mycoplasmas are cell associated. Calcium alginate, Dacron, or polyester swabs with aluminum or plastic shafts are preferred. Wooden-shaft cotton swabs should be avoided because of potential inhibitory effects. Specimens should be refrigerated if immediate transportation to the laboratory is not possible. If specimens must be shipped and/or if the storage time prior to processing is likely to exceed 24 h, the specimens in transport medium should be frozen to prevent loss of viability. Mollicutes can be stored for long periods in appropriate growth or transport media at -70°C or in liquid nitrogen (37). Storage at -2OOC for even short peri-

Mycoplasmal

Infections

5

ods will result in loss of viability. Frozen specimens may be shipped in dry ice to a reference laboratory if necessary. When specimens are to be examined, they should be thawed rapidly in a 37OC water bath. Specimens Appropriate for Culture Culture is considered relatively insensitive for detection of M. pneumoniae, and alternative techniques such as PCR and serologic testing should be considered even if culture is attempted. Respi ratory tract specimens, including nasopharyngeal and throat swabs, sputum, pleural fluid, broncheoalv *eolar lavage fluid, endotracheal asp irates, and lung ti ssue are acceptable for culture-based detection of M. pneumoniae. Urethral swabs in men are preferred over urine for detection of genital mycoplasmas. Prostatic secretions, semen, and urinary calculi can be cultured. For females, urine and cervical or vaginal swabs are acceptable. Specimens contaminated by lubricants or antiseptics should be avoided. Urine samples from females are most meaningful when obtained by catheter or suprapubic aspiration and if numbers of organisms are quantitated. Endometrial tissue, tubal samples, or pouch of Douglas fluid can be obtained to confirm mycoplasmal etiology of pelvic inflammatory disease or postpartum fever. For women with clinical amnionitis, amniotic fluid, blood, and placenta should be cultured. Culture of nasopharyngeal, throat, and endotracheal secretions of neonates is appropriate, especially if the birth weight is less than 1,500 g and there is clinical, radiographic, laboratory, or other evidence of pneumonia. Extragenital or extrapulmonary specimens submitted for culture should reflect the site of infection and the disease process. Ureaplasmas and mycoplasmas should always be sought from synovial fluid in the setting of acute arthritis in hypogammaglobulinemia. Other sterile fluids, including peritoneal fluid, pericardial fluid, cerebrospinal fluid, and blood, are suitable for culture. Bone chips from patients with chronic osteomyelitis without a proven bacterial etiology are also appropriate for culture, as are wound aspirates and tissue collected at biopsy or autopsy. Mollicutes are inhibited by sodium polyanetholsulfonate, which is present in most commercial blood culture media, but the effect can be overcome by adding 1% gelatin (74). Commercial blood culture media designed for use in automated instruments may support the growth of M. hominis, but the instruments usually do not flag the bottles containing this organism as positive. M. hominis and Ureaplasma spp. can be successfully isolated from blood by inoculating 210 ml directly into liquid mycoplasmal growth media in at least a 1: 10 ratio. Smaller volumes can be used for neonates or children. There is sometimes difficulty in detecting a color change in liquid media in the presence of large amounts of blood due to hemolysis, and there may be

6

Wattes

et al.

a slight color change immediately after introducing blood into liquid media. Serial dilution of the original specimen and subcultures to agar will help distinguish such nonspecific color changes. Growth Media No single formulation is ideal for all pertinent species due to their different properties, optimal pH values, and substrate requirements. SP-4 broth and agar (106) and Hayflick’s modified broth and agar (35) can be used for both M. pneumoniae and M. hominis. These media can also be used to cultivate other fastidious species and slow growing species. Shepard’s 10B broth (87) can be used for cultivation of M. hominis and Ureaplasma spp., with A8 agar (88) as the corresponding solid medium. Use of manganese as the urease indicator in agar should be avoided because of its toxicity to some ureaplasmal strains (8 1). Bromothymol blue (B broth) has also been recommended for detection of Ureaplasma spp. and will also support the growth of M. hominis (79). Positive and negative controls are recommended to ensure adequate detection of growth in all media. The compositions of these media and general comments about their use are included in the Appendixes. Biphasic media have been used successfully for many years for cultivation of M. pneumoniae. An agar slant is prepared in a small screw-cap bottle, to which broth is added to fill half to two-thirds the height of the agar. The rationale for this approach is that it supplies a wide range of atmospheric conditions and that inhibitor metabolites present in the specimen may be absorbed by the agar, further promoting the possibility of growth. This idea has been adapted in some commercial medium formulations. 211. pneumoniae can also be successfully isolated using the broth-toagar technique. For self-prepared media, quality control is crucial for each of the main components. New lots or batches of broth are considered satisfactory if the numbers of organisms that grow are within lo- to loo-fold of the number in the reference batch. Agar plates should support growth of at least 90% of the colonies supported by the reference media. Type strains of 211. pneumoniae, M. hominis, and U. urealyticum are available for quality control testing Lo w-passage clinical isolates and a minim urn of ;wo strains of Ureaplasma spp. should be included in quality control testing. Recommended choices include serotypes 3 and either 5, 7, or 8. Almost any formulation of mycoplasma media that is prepared correctly can be expected to grow prototype strains. The real challenge is the ability to isolate mycoplasmas and ureaplasmas from clinical specimens, hence the need for inclusion of known positive specimens or recent clinical isolates. Testing of the inhibitory properties of media against the growth of various other organisms likely

CUMITECH

34

to be present in specimens from nonsterile sites may also be worthwhile to prevent the loss of mycoplasmas due to overgrowth of contaminating organisms. If commercially prepared media or kits are to be utilized, it is advisable that laboratories perform internal quality control tests, and users should be aware of the potential limitations of existing products. Several companies sell transport and growth media patterned after the original formulations developed by researchers in mycoplasmology, and some have developed kits for detection and preliminary identification of the most common clinically significant species. The products available vary from one country to another. Mycoscreen GU (Irvine Scientific) is available as a broth culture for screening for M. hominis and Ureaplasma spp. Vials showing a color change must be subcultured to the Mycotrim GU triphasic flask system or other solid medium for isolation and identification. The triphasic flask system incorporates an agar growth surface and enrichment broth separated by a humid air phase in a single flask. Using this product, growth in the broth medium, as indicated by a color change, can be verified by observation of colonies. A comparable system, adapted to 211.pneumoniae detection, Mycotrim RS, is also available. Remel, Inc., markets several formulations of growth media, including 10B broth, arginine broth, A7 and A8 agars, and P-4 broth and agar. Some of these media can be purchased in freeze-dried form to prolong their shelf life. Mycoplasma Experience, a British company, and three French companies produce several different liquid and solid media and market them primarily in Europe. There have been a few studies examining the capabilities of commercial media sold in the United States to detect M. hominis and ureaplasmas in clinical specimens. The results indicate that certain products may be comparable to self-prepared media but that problems continue to exist, especially with recovery of ureaplasmas. Wood et al. (124) reported that the Mycotrim GU system worked as well as nonproprietary argine and urea broths, glucose agar, and A7B agar. However, Phillips et al. (73) found that Mycotrim detected fewer Ureaplasma and 211.hominis isolates than did A7 agar. Broitman et al. (13) compared a combination of Remel’s A8 agar and Mycotrim GU broth with the Mycotrim triphasic flask system and found that the triphasic flask system detected Ureaplasma spp. in only 25% of 64 positive specimens versus 100% by GU broth-A8 agar. The Mycotrim triphasic flask system detected 94% of 18 positive cultures for M. hominis, all 18 of which were detected using GU broth-A8 agar. Welborn et al. (W. Welborn, L. Skodack-Jones, and K. Carroll, Abstr. 97th Gen. Meet. Am. Sot. Microbial. 1997, abstr. G-8, p. 281,

CUMITECH

34

1997) compared the Mycotrim triphasic flask system with Remel 10B broth and A7 agar in 195 clinical specimens. Of 79 positive cultures, 32 were detected by the Remel media versus 28 by Mycotrim. Mycotrim had a 13 % contamination rate versus < 1% for the Remel media. Several types of broths, agars, and diagnostic kits are sold in European countries for the detection and preliminary identification of Ureaplasma spp. and 211. hominis. Sillis (90) reported that the MycoplasmaLyo, an arginine-urea broth and agar system (bioMerieux), provided qualitative and quantitative results suitable for diagnostic use. Abele-Horn et al. (1) also reported that the performance of nonproprietary and various commercial media was similar for identification of Ureaplasma spp. and 211.hominis but that standard methods were superior for quantitative identification. Diagnostic kits consist of strips with wells containing specific dried or lyophilized substrates and inhibitors. Specimens are placed in a suspension medium that is used to inoculate the wells. The presence and identification of organisms is based on the color change of specific wells containing biochemical substrates and inhibitors. Some products also contain antimicrobial agents so that in vitro susceptibilities can be determined simultaneously. Poulin and Kundsin (S. A. Poulin and R. B. Kundsin, Abstr. 97th Gen. Meet. Am. Sot. Microbial. 1997, abstr. G-9, p. 281, 1997) reported that the Mycofast Evolution 2 and the Mycofast US kits (International Microbio) correctly identified all cultures positive for Ureaplasma spp. and/or 211.hominis determined by standard methods using Boston broth and A7 agar, with no false-positive results. Abele-Horn et al. (1) evaluated the Mycoplasma IST (bioMerieux), the Mycoplasma DUO (Sanofi Diagnostics Pasteur), and the quantitative Mycoplasma “All-In” (International Microbio) products in 298 clinical specimens and found results roughly comparable to those obtained with standard media and procedures. Clegg et al. (22) compared the Mycoplasma IST kit with arginine broth, 1OC broth, and A7 agar to determine the prevalence of M. hominis and Ureaplasma spp. in vaginal specimens and found that the kit gave a sensitivity and specificity of 92.9 and 86.7% for M. hominis versus 97.4 and 72.7% for Ureaplasma, respectively. They noted that false-positive reactions may occur in the Mycoplasma IST kit if contaminating bacteria are present. Therefore, organism identification and purity should be verified by determination of colony morphology on agar plates. Lower organism numbers detected by commercial kits in comparison to standard methods have been attributed to the fact that standard methods include recommenda tions for serial di 1ution of the original specimen to remove I.nhibitors that mav be present. Some of the kits and media such as those

Mycoplasmal

Infections

7

described above have a shelf life of several months, making them attractive for use in laboratories that have only occasional needs for performing mycoplasma cultures, but they are considerably more expensive to purchase than media and reagents prepared in-house. Diagnostic Approach Detection of mycoplasmas and ureaplasmas in clinical specimens involves careful consideration of the type of specimen to be cultured, the type of patient suspected of having infection, and the organisms sought. For respiratory specimens, if M. pneumoniae is the only organism of interest, it is sufficient to inoculate a single medium such as W-4 broth and agar and follow the protocol outlined in Fig. 2. Likewise, if only 211. hominis or Ureaplasma spp. are of interest, it is sufficient to set up cultures using a single broth and agar formulation such as 10B or B broth and A8 agar and follow the protocol shown in Fig. 3. However, if the specimen to be cultured is from an extragenital or extrapulmonary site and is a normally sterile body fluid or tissue and/or the patient is immunosuppressed, any mycoplasma of human origin or “accidental infection” with a mycoplasma of animal origin should be considered. Even though many of these species are not reliably detected in culture by the culture protocols described, and alternative non-culture-based tests should also be performed, culturebased isolation may occasionally be successful, usually after prolonged incubation. To maximize the potential yield, SP-4 broth and agar, containing glucose and arginine, should also be inoculated and incubated anaerobically and cultures should be processed as outlined in Fig. 2 and 3. Specimen Inoculation Specimens should be mixed, and fluids should be centrifuged (600 X g for 15 min) and the pellet inoculated. Tissues are to be minced in broth prior to diluting. Serial dilution of liquid specimens and tissues in broth to at least 10e3 with subculture of each dilution onto agar is an extremely important step in the cultivation process since it will help overcome possible interference by antibiotics, antibodies, and other inhibitors, including bacteria, that may be present in clinical specimens. Omission of this critical dilution step is one reason why some laboratories have difficulty in recovering the organisms, since the undiluted specimen is often negative when subsequent dilutions are positive. Dilution also helps to overcome the problem of rapid decline in culture viability, which is particularly common with ureaplasmas, and it provides information about the number of organisms present in the specimen.

8

Wattes

CUMITECH

et al

1 Specimen

34

I

Inoculate serial dilutions of SP-4 broth and agar. Incubate broths aerobically and agar plates in a CO;! incubator or candle jar at 37 ’ C.

Subculture any positive broths to agar if I---there is no growth on primary agar culture

1

to agar I Performat leastblindonce,subculture days 1O-21

I

Examine plates from primary culture and any 4 subcultures at 2-3 day intervals after 4 days of incubation

Spherical colonies 10 - 100 pm in diameter develop, usually after 4-20 days.

Perform tetrazolium

PCR, hemadsorption or reduction tests on colonies

Positive I

FIGURE 2 lsolatlon of M pneumonlae from cllnlcal specrmens Rate of growth, glucose metabolism, confirmatory tests can usually differentiate M pneumonlae from other mycoplasmas and acholeplasmas resprratory tract Noting the presence of a mycoplasma other than M pneumonlae will allow cllnrcrans charactenzatlon IS needed

Incubation

and Subcultures

Broths should be incubated at 3.5 to 37OC under atmospheric conditions. The use of a humidified incubator is helpful to delay dehydration of the medium when performing cultures in dry climates or when incubation will be prolonged. Agitation of broths on a mechanical rotator during incubation may hasten the detection of M. pneumoniae by about 1 day. Colonies of genital mycoplasmas develop best when agar plates are incubated in an atmosphere of 95% N,-5% CO, (78), but successful isolation is also possible using an atmosphere of room air plus 5% CO2 or in a candle jar if specialized incubators are not available. M. pneumoniae grows best in 5% CO2 in air. All broths that have changed color should be subcultured onto agar, unless colonies are already evident from the primary agar culture. Subcultures must be performed soon after the color change occurs, particularly if the

colony morphology, and simple that may be present In the to determine whether further

organism is Ureaplasma, because the culture can lose viability within hours. Subculture also increases the diagnostic yield since some strains may not grow sufficiently from the original specimen inoculated initially onto the solid medium. Blind subculture may improve the yield of 211.pneumoniae and other slowgrowing mycoplasmas since a color change may not always be evident. Turbidity in broth cultures indicates bacterial contamination. For fastidious, slowgrowing organisms such as 111.fermentans, M. genitalium, and other mycoplasmas of human origin, cultivation conditions are not well established, but 6 weeks or more may elapse before growth becomes evident. Due to the advent of PCR assays for use in research and reference laboratories, the need to refine culture techniques for these slow-growing and fastidious mycoplasmal species is less critical than previously.

CUMITECH

34

Mycoplasmal

Infections

9

Specimen broculate serial dilutions of 1OB broth and A8 agar, incubate broths aerobically and agar plates in 5% CO2 + 95% N5 5% CO2 in air, or candle jar at 37 ’ C. Observe broths twice daily for color change.

Alkaline shiR (yellow to pink) after l-4 days of incubation Subculture any positive broths to agar if there is no growth on primary agar culture Examine plates from primary culture and any subcultures daily.

Brown granular colonies 15-60 pm in diameter develop in l-3 days

Fried egg colonies 200-300 pm in diameter develop in 3-4 days

I

I

I I No colonies develop by 7 days

Report as presumptive M hominis

FIGURE 3. lsolatlon of M homuxs and Ureaplasma spp. from cllnlcal specimens M. homuxs IS the most common mycoplasmal species Isolated from the genttounnary tract and from extragenital sites Prellmlnary dlfferentlatlon of M horn/n/s from other potentially pathogenic organisms can usually be made based on a comblnatlon of colony morphology, growth rate, and differential utlllzatlon of glucose and/or arglnlne. Rare tsolatrons of other human mycoplasmas or species not normally associated with humans can cause confusron, necessttatlng species determlnatlon procedures outside the purview of most clInIcal laboratories

Interpretation Identification

of Cultures and Species

Mycoplasma cells are too small to be visualized in Gram-stained preparations. However, the Gram stain may prove useful to exclude contaminating bacteria that may be present. Giemsa stains may be used, but the results can be difficult to interpret because of debris and artifacts in clinical specimensthat can be confused with mycoplasmas due to their small size. DNA fluorochrome stains such as Hoechst 33258 may be useful to determine whether microorganisms are present in a clinical specimen or culture, but they do not distinguish mycoplasmas from other bacteria (67) Agar cultures can be examined for mycoplasma colonies with a hand lens, but microscopic examination using a low-power (4 X to 10 X ) objective of a stereomicroscope or a standard microscope is more reliable. To seemycoplasmas, it is necessary to focus on the surface of the agar. A heavy inoculum can result in contact inhibition, and none of the colonies will be sufficiently large to be identified. Therefore,

when examining agar cultures for mycoplasmas, it is important to pay attention not only to the initial point of inoculation but also to the area where the inoculum is most sparse and thus allows the best colony development. Colonies must be distinguished from artifacts such as air bubbles, water or lipid droplets, or other debris. Pseudocoloniesmay form as a result of precipitants in the medium (Fig. 4a). Pseudocolonies share some of the characteristics of true mycoplasma colonies, including the ability to be transferred. However, most mycoplasmas have a spherical, mulberry, or fried-egg appearance that differs from the spiral pattern in the center of the pseudocolony. A vital stain can rapidly distinguish living from inanimate structures. Dienes methylene blue stain or neutral red can be usedfor this purpose. If necessary, it is possible to filter-sterilize neutral red stain, verify that the colonies contain living cells, and then transfer the red colonies to a fresh culture by removing a plug of agar. Colonies of Ureaplasma spp. (Fig. 4b) can be identified on A8 agar by ureaseproduction in the presence

Waltes

et al.

CUMITECH

34

FIGURE 4. (a) Colonies of M. hominisand pseudocolonies. Pseudocolonies (P) formed by medium components are sometimes mistaken for mycoplasmal colonies (M). Magnification, ~53. Photomicrograph courtesy of F. L. Jackson. (b) Colonies of ii/l. hominis and Ureaplasma spp. on A8 agar. Fried-egg colonies of M. hominis are approximately 200 to 300 pm in diameter and are urease negative. Ureaplasma colonies are 15 to 60 pm in diameter and have a brownish granular appearance in the presence of the CaCI, urease indicator. Magnification, ca. x40. Photomicrograph courtesy of J. A. Robertson. (cl Colonies of M. pneumoniae. M. pneumoniae produces spherical colonies 10 to 100 pm in diameter that are smaller than M. hominis and usually do not demonstrate the fried-egg appearance. Magnification, x212. Photomicrograph courtesy of W. L. Thacker. (d) M. pneumoniae hemadsorption reaction. M. pneumoniae and M. genital&m are differentiated from other human mycoplasmal species by their abilities to hemadsorb guinea pig and human type 0 erythrocytes. Magnification, x53. Photomicrograph courtesy of F. L. Jackson

of CaCl, indicator. M. hominis colonies (Fig. 4b) are larger and m-ease negative and often have the typical fried-egg appearance. The occurrence on media such as Columbia agar of pinpoint colonies that do not produce a recognizable Gram reaction warrants subculture to appropriate mycoplasma media because of the possibility that they are M. hominis. M. pneumoniae will produce much smaller spherical colonies when grown on mycoplasma agar (Fig. 4~). To perform subcultures of mycoplasmas from agar to broth, a sterile scalpel or Pasteur pipette is used to cut out a block of agar that contains colonies and the block is placed into 1 to 2 ml of broth, mixed well, and incubated until a color change is evident. To transfer colonies from one agar plate to another, a plug of agar is cut out, placed upside down on a fresh agar plate, and pushed over the surface with a scalpel blade or other flat tool. When transferring mycoplasmas from broth to agar, a loop or a spreader should be used. It is helpful to damage the surface of the agar at the point of inoculation so that new growth can be easily sought at the point of inoculation. Mycoplasmas of human origin can be classified

according to whether they ferment glucose, hydrolyze arginine, or hydrolyze urea (Table 1). Except for hydrolysis of urea, which is unique for ureaplasmas, these biochemical features are insufficient for definitive species distinction. However, colony morphology in conjunction with the biochemical profile, body site of origin, rate of growth, and other confirmatory tests shown in the flowcharts will usually allow presumptive identification of the most common clinically significant species. A glycolytic organism from the respiratory tract that produces spherical colonies on P-4 agar after 4 to 20 days of incubation is likely to be M. pneumoniae. Further confirmation can be achieved by relatively simple tests that are readily adaptable for use in the clinical laboratory. The hemadsorption test (Fig. 4d) involves flooding colonies on agar with a 5 % suspension of washed guinea pig erythrocytes, incubating at room temperature for 30 min, and washing gently two or three times with sterile saline. Colonies are then examined microscopically to determine whether the erythrocytes have adhered. Initially, the surface of freshly isolated M. pneumoniae colonies

CUMITECH

34

will be covered with erythrocytes, but over time these will be lysed by H,O, produced by the organisms. Commensal mycoplasmas that may be isolated in culture from the respiratory tract during this time frame do not hemadsorb in this manner. Acholeplasma laidlawii is glycolytic but usually grows more rapidly and produces large fried-egg colonies. Unlike 211.pneumoniae, A. laidlawii can be subcultured to fresh medium and grown at room temperature in 24 to 48 h, and it will also grow on serum-free medium. M. genitalium, an organism closely related to M. pneumoniae that may occur in the respiratory tract, may also hemadsorb, but it is extremely fastidious and grows even more slowly. Unless more specific techniques such as colony epi-immunofluorescence or PCR are used, the identification of 211.pneumoniae should be considered presumptive. A second technique to presumptively identify M. pneumoniae is based on its unique ability to reduce the colorless compound triphenyl tetrazolium to the red compound formazan. This test is performed by flooding colonies on agar with 2 ml of sterile 0.21% (wt/vol) 2-(piodophenyl)-3-nitrophenyl-5-phenyl tetrazolium chloride (INT) and incubating at 37OC for 1 h. Colonies of M. pneumoniae will appear pink during incubation and become purple to black after several hours, whereas colonies of other human mycoplasm .as (except M. genitalium) will be unaffected. M. saliuarium can be used as a negative control for both of these confirmatory tests. A8 agar contains CaCl,, the substrate for urease detection in Ureaplasma spp., thereby eliminating the need for a separate test to detect the presence of this enzyme. However, urease activity can also be detected on agar without the substrate by simply adding approximately 0.2 ml of sterile 1% CaCl, solution to isolated colonies and observing whether they change from colorless to yellow to dark brown within 15 min. 211.hominis can be used as a negative control for the spot urease test. To identify an unknown mycoplasma definitively to species level, a number of different techniques are available, but none is practical for a diagnostic laboratory that may encounter such organisms very rarely. Agar growth inhibition is one method, but several antisera used at end point dilutions may be required to encompass multiple strains within a given species. Because of possible cross-reactions between closely related species, such as M. pneumoniae and 211.genitalium, rigorous proof of the specificity of the method must be documented by testing multiple isolates of the same species as controls. To perform the growth inhibition test, a logarithmic-phase broth culture of the unknown isolate is diluted l/50 and l/500 in fresh broth. Three drops of each dilution are plated onto the surface of the appropriate agar medium and

Mycoplasmal

Infections

11

spread uniformly. Sterile paper disks are saturated with 25 ~1 of serum. Normal animal serum is used as a control, and hyperimmune serum prepared in the same animal against the suspected species is used for the test. The serum disks are placed onto the agar, making sure the disk touches the plate only where it is deposited. The plates are then incubated and inspected for growth around the disks. Colonies should grow right up to the disks with normal serum or heterologous antisera but should form a distinct zone of inhibition around the disk with homologous antisera. The appearance of a few colonies within a zone of inhibition suggests either a mixture of species or the presence of variants within the identified species. Epi-immunofluorescence or immunoperoxidase techniques enable colonies on agar to be identified directly so that mixtures of different species can be readily discerned. Immunoblotting with monoclonal antibodies, metabolism inhibition tests, mycoplasmacidal tests, and PCR assays have also been employed in specialized laboratories to identify mycoplasmas to the species level (19, 93, 95, 117). Reporting Results 111.pneumoniae isolation from respiratory tract specimens is clinically significant i n most instances and should be correlated with the presence of clinical respiratory disease since a small proportion of asymptomatic carriers may exist. Detection by PCR is becoming more widely available through reference laboratories, but a positive result must still be correlated with clinical events. Reliable serologic tests, including both acute- and convalescent-phase sera, are critical for accurate diagnosis of M. pneumoniae respiratory disease. CcUreaplasma spp. ” is sufficient for reporting purposes since the two biovars of U. urealyticum will be given separate species status. There is no phenotypic means or urgent clinical necessity to differentiate between them or among their various serovars in laboratory reports. Isolation of ureaplasmas in any quantity from normally sterile body fluids or tissues is significantly associated with disease. The presence of fewer than lo4 organisms in the male urethra is unlikely to be significant. For genital specimens yielding an arginine-hydrolyzing, urease-negative organism that produces friedegg colonies after 3 to 4 d ays of incubation, a report of pres umptive M. hominis is appropriate, and in most instances no further work-up is required. 211.hominis isolation in any quantity from normally sterile body fluids or tissues is significantly associated with disease, but reports quantitating the numbers of organisms present may be of value in other circumstances. Mycoplasmas other than 111.pneumoniae that require more definitive identification can be submitted to a reference laboratory in the event that this is

12

Waites

et al.

CUMITECH

Isolation by culture

Species

Requirement for PCR

M. pneumoniae

Occasional

Very

U. M. M. M.

Frequent Frequent Very rare Very rare

Sometimes Sometimes Essential Essential

Very

Essential

urealyticum hominis genital&m fermen tans

M. penetrans

rare

Examples

useful useful useful

clinically indicated. This may be relevant when the organism is present in a clinically significant infection of a normally sterile site, particularly if the patient is immunosuppressed. Nonculture

Methods

Culture is well adapted to species which can be isolated easily and rapidly from clinical specimens, such as M. hominis and Ureaplasma spp., and has the advantages of being able to provide quantitative results and an isolate for susceptibility testing. However, it is not satisfactory for detection of fastidious and/or extremely slow-growing organisms such as M. genitalium and, to some degree, M. pneumoniae. Therefore, alternative rapid methods have been devised based on knowledge concerning the characterization of epitopes and DNA sequences. These methods have been recently reviewed (7). Antigen Detection Techniques Several types of rapid methods have been developed for antigenic detection of 111. pneumoniae (39, 61). Their utility and general acceptance have been limited by low sensitivity and specificity, due to the antigenic similarity between M. pneumoniae and 211. genitalium, and by nonspecific reactions with other bacteria. Antigen detection tests have been replaced by more powerful methods such as PCR. DNA Probes DNA hybridization techniques for the diagnosis of M. pneumoniae infection were developed in the early 1980s (75). 16s rRNA genes have been widely used as targets, as have probes consisting of rDNA. Kits were previously commercialized in the United States but are no longer available. Because probes are relatively insensitive ( lo3 to lo6 CFU), amplification-based techniques such as PCR have supplanted them. PCR The major advantage of PCR is that it provides a very large number of copies of a chosen DNA sequence. PCR systems have been reported for almost all mycoplasmas pathogenic for humans. For species detection, the amplified target must be selective, whereas for the simultaneous detection of several species it

25, 44, 99th 24, 82, 42 25,52 20, 28, Meet. 42

34

of PCR references

54, 65, 91, 108, 109, Gen. Meet. Am. Sot. 83

121; Zhang et al., Abstr. Microbial. 1999

85, 86, 120; Zhang et al., Abstr. Am. Sot. Microbial. 1999

99th

Gen.

must be highly conserved. Published PCR techniques for human mycoplasmas are listed in Table 2 and in references 7 and 40. Table 2 gives examples of the utility of PCR for detection of the most important species. PCRs based on 16s rRNA genes have been widely used (7.5, 82, 83). New fluorogenic-probe based PCRs (TaqMan; Perkin-Elmer) have been designed to detect the ATPase gene of M. pneumoniae and the ftx gene of M. fermentans (M. Zhang, B. P. Holloway, W. L. Thacker, S. B. Schwartz, and D. F. Talkington, Abstr. 99th Gen. Meet. Am. Sot. Microbiol. 1999, abstr. G-18, p. 325, 1999). These assays are very sensitive and specific and can be designed to detect at a specific threshold or to quantitate gene copies in clinical samples. Other sequences which have been used are the repetitive insertion-like elements of M. fermentans (120), the specific genes of Pl adhesin of M. pneumoniae (25), and the MgPa adhesin gene of M. genitalium (52). Urease genes have been used for U. urealyticum (11). Multiplex PCR tests, detecting several mycoplasmas or even combinations of mycoplasmas and other agents such as Chlamydia pneumoniae and Legionella pneumophila, may eventually prove useful for screening purposes (20, 44), although multiplexing can result in a decrease of analytic sensitivity. The detection limit of the PCR is very high, corresponding to a single organism or a single copy of the gene when purified DNA is used. Culture requires having at least 1 to 10 CFU; larger numbers are probably needed when clinical specimens are used. PCR is also a very good tool for identification of an unknown mycoplasma previously obtained by culture. It can be used for characterization of strains within a species and for detection of a specific feature, such as the presence of an antibiotic resistance determinant (26). Another advantage of PCR is that it can be useful for detection of mycoplasmas in tissue samples when the tissue has already been processed for histologic examination or is contaminated, making culture impossible (91, 121). Practically, PCR technology appears to be less valuable for routine diagnostic purposes in the case of the more rapidly growing and easily cultivable organ-

CUMITECH

34

Mycoplasmal

isms, such as M. hominis and U. urealyticum, except in some specific cases where isolation by culture could be difficult. For instance, PCR may more easily detect urogenital mycoplasmas in various specimens such as synovial fluids and biopsy specimens. For slow-growing organisms such as M. pneumoniae, and especially for extremely fastidious species for which optimum cultivation techniques are not established, such as M. genitalium and M. fermentans, the use of PCR assays may be the only practical means of detecting their presence in clinical material, despite attempts at improving culture-based detection (53). Comparison of the PCR technique with culture and/or serologic testing for M. pneumoniae has yielded varied results, and large-scale experience with this procedure is still limited. Positive PCR results for M. pneumoniae in culture-negative persons without evidence of respiratory disease suggests inadequate specificity, persistence of the organism after infection, or its existence in asymptomatic carriers, making interpretation of such results difficult. Presently, PCR detection for mycoplasmas is still too labor-intensive, expensive, and complex to be carried out routinely in most clinical microbiology laboratories. Some drawbacks must still be corrected, such as the presence of inhibitors in the specimens and laboratory contamination. The possible development of commercial PCR kits in the future should bring about better standardization of the technique, and if it becomes available at a reasonable cost, PCR could become a major method for the diagnosis of mycoplasma1 infections. Detailed information concerning PCR methodology and controls has been described elsewhere (40, 75).

SEROLOGIC

DIAGNOSIS

Serologic testing has played a historically prominent role in diagnosing M. pneumoniae infections, despite its limitations in immunocompromised patients, and it is particularly valuable when cultures are not possible. M. pneumoniae possesses both protein and glycolipid antigens that elicit antibody responses in infected individuals. Following an initial infection, the normal immune system responds by rapidly producing antibodies which peak at 3 to 6 weeks and then gradually decline over months to years. M. pneumoniae-specific immunoglobulin M (IgM) alone is often interpreted as evidence of acute infection, and this approach has the practical advantage in that only one specimen, taken approximately 7 to 10 days after infection, is required. The presence of IgM is considered most significant in pediatric populations, in whom there have been fewer opportunities for repeated exposures. Adults who have been infected repeatedly over a period of years do not respond to mycoplasma antigens with a brisk IgM response. The IgM response may

Infections

13

persist for months or years following infection (l25), and in these cases a positive IgM test result may not reflect a current or recent infection. In addition, IgM antibodies to protein antigens targeted in most commercial serologic tests may be absent in adults after repeated infections. In these cases, reinfection leads directly to an IgG response; therefore, the absence of a positive IgM result does not rule out an acute infection. In view of these considerations, it is advisable to test for both IgM and IgG for the most accurate diagnosis of recent M. pneumoniae infection, especially in adults (104). IgA, while often overlooked as a diagnostic antibody class, may actually be a better indicator of recent infections in all age groups (89). IgA antibodies are produced early in the course of disease, and their levels peak quickly and decrease earlier than those of IgM or IgG (41). In theory, a single early specimen could detect an acute infection even after multiple reinfections. Research into IgA responses in adult and pediatric populations is warranted. Cold Agglutinins Cold agglutinins are IgM antibodies to the I antigen of erythrocytes and are detected by agglutination of type 0, Rh-negative erythrocytes at 4*C. They are produced 1 to 2 weeks after the initial infection and persist for several weeks. Cold agglutinins are found in only 30 to 50% of M. pneumoniae infections (49). Therefore, a negative result does not exclude mycoplasma infection. Because other bacteria, viruses, or even collagen vascular diseases can induce cold agglutinins, a positive test result is not specific for mycoplasma1 infection. Better tests are available that have eliminated the need for this assay, and cold-agglutinin testing is no longer recommended for diagnosis of M. pneumoniae infections. Complement

Fixation

The complement fixation (CF) test using a chloroform-methanol lipid extract of M. pneumoniae has long been used for serodiagnosis of M. pneumoniae pneumonia (59). CF tests with whole-organism antigen give similar results to those with the lipid antigen. The CF test is a two-step procedure based on the ability of an antigen-antibody complex to bind added complement. If added complement is bound by specific antibody-mycoplasma complexes, it cannot later initiate lysis of the sheep erythrocyte indicator system. Therefore, absence of erythrocyte lysis correlates with the presence of antimycoplasma antibodies in serum. The titer is usually defined as the greatest dilution of serum that shows 0 to 30% hemolysis in a test using a specific amount of antigen and complement and serial twofold dilutions of serum. CF tests measure both IgM and IgG and hence do not differentiate between

14

Waites

et al.

CUMITECH

antibody classes. Therefore, detection of fourfold or greater increases in antibody titers between an acutephase serum specimen and a convalescent-phase specimen is commonly used as a diagnostic measure. A fourfold or greater decrease has been considered evidence of a resolving infection. Paired sera, run simultaneously, are necessary for proper interpretation of test results. Historically, the CF test gained early popularity among laboratories that routinely ran CF tests for viral agents. However, mycoplasmas are much more antigenically complex than are viruses, leading to nonspecific reactions. The glycolipids of M. genitalium are highly cross-reactive with M. pneumoniae due to shared lipid antigens (63), causing problems for CF tests. Sera from patients with bacterial meningitis also tend to have high CF titers. Kenny et al. (60) reported that among M. pneumoniae culture-positive, X-ray-proven pneumonia patients, 53% showed a fourfold titer increase and 36% showed antibody titers of 232. Using both high titers and high stationary titers as criteria, the sensitivity of the CF test was 90% and the specificity was 88%. Single titers of >32 are sometimes considered to be indicative of recent infection. However, this end point varies greatly among laboratories, and, as stated above, antibodies to glycolipid antigens may persist for long periods. Confirmation of CF test results by Western immunoblotting can aid in interpretation but greatly adds to the time and expense of testing (57). In view of the many limitations of CF tests, they have largely been replaced by improved methods using alternative technologies. Some of these formats are available in various commercial kits and are discussed below. Specific examples and comments about them are shown in Ta ble 3. Indirect

Immunofluorescence

Assays

M. pneumoniae antigen is affixed to glass slides, and specific antibody is detected after staining with antihuman IgM or IgG fluorochrome conjugate. The indirect immunofluorescence assay (IFA) is relatively simple to perform and requires only about 90 min, but it is subjective to interpret and requires a fluorescence microscope. Results can be affected by rheumatoid factor and/or high IgG levels. This technique has been compared to other methods, including enzyme immunoassays (EIAs) and CF (3, 33). Particulate

Antigen-Antibody

Assays

Passive-agglutination tests make use of particles such as latex, gelatin, or erythrocytes coated with M. pneumoniae-specific antigen. When erythrocytes are used, the test is referred to as the indirect hemagglutination assay (IHA) (3 1). Erythrocytes are treated with tannic acid or chromium chloride to facilitate adherence of

34

the antigen. The antigen-coated particles are incubated with test serum, and if the serum contains specific antibodies, the particles agglutinate, resulting in a visible reaction. A modification of the IHA and an eryth .rocyte IgM anti body capture assay (23) detects I&f antibodies that are a.bsorbed to immobilized IJ, heavy chains. All of these tests use a mixture of M. pneumoniae antigens, and they require paired sera to demonstrate rises in titer (4, 32, 55, 62). Commercial kits utilizing particle agglutination technology to detect IgM and/or IgG separately or simultaneously are available, but they do not offer any advantages over other techniques such as EIAs or IFAs. Enzyme-Linked

lmmunosorbent

Assays

EIAs, first developed in the 197Os, have become widely adapted in laboratories, achieving the largest market share of commercial mycoplasma serologic tests in the United States. They are amenable to a variety of assay conditions, detect very small amounts of antibody, and can be made isotype specific. Crude multiantigen preparations, purified proteins, p-capture approaches, and synthetic peptides have all been used (4, 17, 32, 45, 46, 49, 71, 84, 107, 125). The basic format is to immobilize an antigen to a solid phase. Patient sera are incubated with the solid phase, and bound antibodies are visualized using substrate and enzyme-labeled conjugates directed against the primary Ig. Most EIAs are sold as 96-well microtiter plate formats, although some can be obtained as breakaway microwell strips, which allow smaller numbers of sera to be tested economically. Comparative evaluations of some of these commercial assays have been published (3, 4, 21, 33, 103, 104; W. L. Thacker and D. F. Talkington, Abstr. 97th Gen. Meet. Am. Sot. Microbial. 1997, abstr. G-5, p. 280, 1997). Two EIAs are packaged as qualitative membranebased procedures for the detection of single test specimens. These are truly rapid EIAs (10 min or less) and are simple to perform. The Meridian ImmunoCard is an IgM-only assay that is simple to read and is especially useful for testing pediatric samples. Typical of IgM-only assays, the specificity may be somewhat compromised in patients with autoimmune disease and there are limitations in interpreting the results of IgM-only assays when testing sera from adults, as discussed above. In evaluations using sera from patients with confirmed M. pneumoniae cases, the Immunocard performed better than the CF test (3, 68, 103, 104; Thacker and Talkington, Abstract). The Remel EIA is a membrane-based assay that detects IgM and IgG simultaneously and has shown good sensitivity and specificity compared to other tests (33, 103). One disadvantage is that frozen sera must be filtered, but fresh or refrigerated sera may be simply diluted and tested. Neither the Meridian Immuno-

CUMITECH Table

3.

34 Examples

Mycoplasmal of commercial

Screntifrc

tests

used

for

Antibody class(es) detected

Test format

Company Zeus

serologic

diagnosis Test

of AA pneumonhe name

IFA

IgM and IgG separately

Fqirebro & Fulrrebro America

Particle agglutinatron

IgM and IgG multaneously

SI-

SERODIA-MYCO tin agglutination

lnternatronal

Particle agglutination

IgM and IgG multaneously

SI-

SEROFAST

Particle agglutination

IgM and IgG multaneously

SI-

MERISTAR-MP agglutination

Alexon-Trend

EIA

IgM and IgG separately

ImmunoWELL-IgM or IgG

Drasonn

EIA

IgM

and IgG

EPI-MP-IgM

EIA

IgM

only

ImmunoCaro’Mycoplasma Test

EIA

IgM and IgG multaneously

EIA

IgG and IgM separately

EIA

IgG and IgM separately

Mendran

Mendran

Mrcrobro

Dragnostrcs

Diagnostics

Remel

Zeus

Scientific

Bio-Rad

II gelatest

latex test

and -IgG

M. pneumonlae lgG/lgM antibody test system Mycoplasma IgG and IgM ELISA test system Platelra IgG and IgM

‘There are many other serologic tests sold In Europe and other countrres that are not avatlable sentative examples of some of the best-studied assays, several of which have been validated peer-reviewed publlcatlons.

Card nor the Remel EIA membrane test can be reliably run with lipemic, contaminated, or hemolyzed samples or on samples containing debris. Genital

Mycoplasma

Assays

The ubiquity of most genital mycoplasmas in humans makes interpretation of antibody titers difficult, and the mere existence of antibodies alone cannot be considered significant. However, if invasive extragenital

Reference(s)

Drstnbutes for Wampole Labs, available as a mycoplasma slurry or “CrownTrtre” affixed to mrcroscope slides Japanese parent company; drstnbuted In Europe by Bayer Dragnostrcs; sold In Europe, Asia, Australia, New Zealand, and Canada French parent company; drstnbutes rnternatronally In Europe Drstnbutes In United States for another company Drstnbutes for GenBro, now markets former Seradyn products; 96well plate format coated with “punfred” glycolrprd mycoplasma antigen Drstnbutes for Savyon Dragnostrcs; multrwell breakaway strips of 8, coated with a membrane preparation containing the PI protein; 192 tests per krt Membrane-based, rapid, single-sample test, 30 tests per krt

Membrane-based, rapid, single-sample test, 25 tests per krt MultIwell breakaway strips of 8, 96 tests per kit Multiwell breakaway strips of 8, 96 tests per krt; drstnbutes Internatronally In Europe In the Unlted States externally and the

15

infections

Notes

M. pneumonlae Antrbody (MP) Test System

SI-

respiratory

Infections

3, 33

4, 62

55

21

3, 68, 104; Thacker and Talkrngton, Abstr. 97th Gen. Meet Am Sot Mlcroblol. 1997 104

The products results have

shown are reprebeen published rn

disease occurs, elevation of antibody titers is often apparent. Serologic methods that have been described for 111. hominis, M. genitalium, and/or Ureaplasma spp. include IFAs and EIAs, microimmunofluorescence, and metabolic inhibition (14, 15, 36, 92). No single type of serologic test has proven satisfactory in identification of genital mycoplasmal infections, and none are currently produced and sold commercially in the United States. Noncommercial EIA-based tests for

16

Wartes

Table 4. MIC and Ureaplasma

et al. ranges spp.

CUMITECH of various

antimicrobials

for /IA pneumoniae,

MIC

Anttmrcrobral

M pneumonlae

Tetracycline Doxycycline Erythromycin Roxrthromycrn Clarithromycrn Azrthromycrn Josamycrn Telrthromycrn Clrndamycrn Lincomycin Pristinamycin QurnupnstrtVdaIfopnstrn Chloramphenrcol Gentamrcrn Ciprofloxacrn Ofloxacin Levofloxacin Sparfloxacin Moxrfloxacrn Gemrfloxacrn Rrfamprn Nrtrofurantorn

0.63-0.25 0 02-0.5 ~0004-0.06 SO.01 ~0004-0125 ~0.004-0.01 ~0.01-0.02 0.008-0.06 50.008-Z 4-8 0.02-0.05 0.008-0.06 2 4 0.5-Z 0.05-Z 0.5-I ~0.008-0.5 0.06-0.12 0.05-O 1 ND ND

M. horn/n/s 0.2-Z 0.1-Z 32->I,000 >I6 16->256 4-64 0.05-Z 2-32 ~0.008-2 0 2-l 0.1-0.5 0.25-8 4-25 2-16 01-4 01-64 0.1-0.5 I ,000 6-500

genital mycoplasmas may have some value in cases of intrauterine infection and predicting pregnancy complications, but they are not widely used because of very limited availability and therefore cannot be recommended for routine diagnostic purposes in the United States. Most commercial tests have not been evaluated for cross-reactions that could potentially occur with sera containing antibodies to M. genitalium. PCR-based studies done at the Centers for Disease Control and Prevention showed that respiratory specimens rarely contain M. genitalium. However, this does not preclude the potential for crossreacting antibodies arising from a genital infection with this organism.

ANTIMICROBIAL TESTING General

IM. hominis,

SUSCEPTIBILITY

Considerations

The Mycoplasmal Chemotherapy Working Team of the International Research Program on Comparative Mycoplasmology has addressed a number of issues as they relate to antimicrobial susceptibility testing of mycoplasmas of human origin. The recommendations by this group for performance of antimicrobial susceptibility testing of mycoplasmas of humans are reflected in this document. Recent publications have provided step-by-step procedures for performing antimicrobial susceptibility tests on human mycoplasmas and should be consulted for the development of specific laboratory protocols (6, 96). Information on in vitro susceptibilities is most

(r-g/ml)

/LX genifalium,

34

M. fermentans,

for:

M. genl tallurn

M. fermen

ND ~0.01-0.3 SO.01 0.01 SO.01 50 01 0.01-0.02 co.015 0.2-I ND ND ND ND ND 2 l-2 0.5-l 0.05-0.1 0.03 0.05 ND ND

0.1-I 0.05-l 0.5-64 32-64 l-64 ~0.003-0.05 0.1-0.5 0.06-0.25 0.01-0.25 ND ND ND 05-10 0.25->500 0.02-~64 0.02-25 0.05 ~0.01-005 ~0.015-0.06 0‘001-0.01 25->50 0.1-2.5

tans

Ureaplasma

spp.

0.05-Z 0.02-I 0.02-4 01-Z ~0.004-2 0.5-4 0.5-4 ~0.015-0.25 0.2-64 8-256 0.1-l 0.12-0.5 04-8 0.1-I 3 0.1-16 0.2-25 0.2-I 0.003-I 0.12-O 5 0.1-0.25 >I ,000 13->I,000

abundant for 211.pneumoniae, M. hominis, and Ureaplasma spp. 111.genitalium has susceptibilities generally similar to those of M. pneumoniae, while M. fermentans has susceptibilities generally similar to those of M. hominis, with some exceptions. A comparison of the MICs of several antimicrobial agents is shown in Table 4. Mollicutes are innately resistant to all p-lactams and glycopeptides, sulfonamides, trimethoprim, polymyxins, nalidixic acid, and rifampin. Potentially active antimicrobials include tetracyclines, macrolides, lincosamides, streptogramins, fluoroquinolones, aminoglycosides, and chloramphenicol. Susceptibility to macrolides and lincosamides is variable according to species, as shown in Table 4. M. pneumoniae is predictably susceptible to tetracyclines and macrolides, so that susceptibility testing is not indicated except for the in vitro evaluation of new and previously untested agents. Tetracycline resistance has been well documented in recent years in both M. hominis and Ureaplasma spp., mediated by the tetM transposon (77). The extent to which tetracycline resistance occurs in genital mycoplasmas varies geographically and according to prior antimicrobial exposure in different populations. Extragenital infections, often in immunocompromised hosts, may be caused by multidrug-resistant mycoplasmas, making guidance of chemotherapy by in vitro susceptibility tests important in this clinical setting. Eradication of infection under these circumstances can be extremely difficult, requiring prolonged therapy, even when the organisms are susceptible to the expected agents. New fluoroquinolones (Table 4)

CUMITECH

34

tend to have greater in vitro activities than older agents such as ciprofloxacin and can be mycoplasmacidal. However, Bebear et al. recently described in vitro mutants and clinical isolates of M. hominis that demonstrated resistance to fluoroquinolones (8- 10) This resistance is associated with alterations rn both DNA gyrase and topoisomerase IV. Recommendations for antimicrobial treatment of specific diseases caused by mollicutes are based largely on in vitro susceptibilities since so few studies of clinical outcomes have been paired with microbiologic efficacy data of individual antimicrobials. Agar disk diffusion has not been recommended in testing mycoplasmas for antimicrobial susceptibilities because the relatively slow growth of many mycoplasmal species on agar will allow widespread diffusion of the antimicrobial from the disk throughout the agar plate before visible growth can be detected and because there are no data to correlate the zone diameters with MICs. Broth and agar dilution techniques and, more recently, the agar gradient (Etest) technique have been adapted for use with these organisms. When properly performed with appropriate controls, these techniques can provide reliable susceptibility data to aid in treatment of infections caused by or associated with mycoplasmas and ureaplasmas. The culture medium used should be appropriate for the organism being tested, providing for the most rapid and sustained growth, and must contain an indicator. It is not possible to standardize on a single medium or pH since growth requirements differ among species. Shepard’s 10B broth and A8 agar work well for ureaplasmas, whereas P-4 and Hayflick modified broth and agar work equally well for M. hominis, M. pneumoniae, and other species. Bromothymol blue broth has also been used successfully for ureaplasmas. Its theoretical advantages are less serum binding of antibiotics due to reduced serum component and lower concentration of urea, which protects against the death of ureaplasmas and makes mycoplasmacidal testing easier. Addition of high concentrations of cations to medium for ureaplasmas can cause serious problems for certain antimicrobials. Cations, such as calcium, used for visualization of ureaplasmal colonies are just as effective when added to agar at the end of incubation for agar-based tests. The pH can affect activities of macrolides, tetracyclines, and quinolones in vitro and is therefore a critical factor in the interpretation of results (58). Ureaplasmas require media with acidic pH (6.0 to 6.5) for good growth, so that testing at neutral pH is inappropriate. In contrast, M. hominis and M. pneumoniae can be tested at pH 7.3 to 7.4. The temperature range for incubation should be 35 to 37OC. Even though incubation under CO, can affect the pH and

Mycoplasmal

Infections

17

ultimately the MICs obtained, ureaplasmas will grow poorly on agar in the absence of supplemental CO2 unless the medium is buffered at pH 6.1 to 6.4. Brothbased tests can be incubated under atmospheric conditions. The length of the incubation will depend on the growth rates of the species being tested and the technique; e.g., growth in broth will typically be evident before colonies develop on agar, so that the MIC may be determined after shorter periods in brothbased tests. Antimicrobial

Agents

Antimicrobials should be obtained in powder form from the manufacturer and should be accompanied by a statement of potency in relation to base with details of optimum storage conditions, expiration date, and solubility. Publications by the National Committee for Clinical Laboratory Standards (NCCLS) that are updated yearly include specific instructions on the preparation of antimicrobial dilutions and general guidelines for testing that are widely accepted in many countries (72). The number and types of antimicrobials that should be tested depend somewhat on the mollicute species, the type of illness, whether the patient is a child, an adult, or a pregnant woman, and which drugs are being considered for treatment purposes. Not all drugs that have activity against mycoplasmas are suitable in every clinical setting, and not all are available in every country. A laboratory performing susceptibility tests for patient management purposes may choose to individualize drugs according to patient-specific needs determined by consultation with the physician ordering the test and by the organism identity. lnoculum

Preparation

At maximal growth, some mycoplasmal species achieve cell counts in the range of lo* to lo9 cells per ml, so that dilution of actively growing cultures or use of frozen stock cultures of known titer is necessary to obtain the proper inoculum of lo4 to 10’ colorchanging units (CCU) per ml. Mollicutes do not produce significant turbidity in broth, making determination of the organism density more complex. Guidelines published by the NCCLS (72) for performing susceptibility tests are based on the use of an actively growing culture with a known organism density in logarithmic growth phase. However, experience with various mycoplasmal species suggests that MICs of common drugs are not greatly affected when tested soon after thawing of a frozen stock culture, and some laboratories prefer to use frozen stocks of known organism titer because of the complexity in determining the number of mycoplasmas in a test system. When testing ureaplasmas by using bromo-

18

Waltes

et al.

thymol blue broth, there will be approximately lo7 CCU/ml when the broth just turns to chartreuse green. Therefore, a 1:lOO dilution will generally yield the desired inoculum. Other species such as M. hominis can also be diluted as soon as a color indicator in broth demonstrates adequate growth. It is important to verify the inoculum by serially diluting an aliquot, typically 0.1 ml, in 0.9 ml of broth and incubating the mixture until a color change is evident. The greatest dilution to show a color change denotes the reciprocal of the number of CCU present in the original inoculum. Setting up MIC tests with multiple dilutions, e.g., 1:lOO and l:l,OOO, of an actively growing culture is one method to ensure that tests do not have to be repeated if the wrong inoculum is used. The obvious drawback to this exercise is the added time and expense incurred. Results are reported only for the dilution that corresponds to lo4 to 10’ CCU/ml. If frozen stocks are to be used, organisms are thawed and grown in broth. As soon as a color change is apparent, an aliquot is serially diluted to determine the number of CCU per milliliter, and the undiluted tube is frozen. Once the number of CCU per milliliter is determined, the original undiluted sample can be diluted to yield the desired inoculum and volume needed according to the type of test and number of drugs being evaluated. If dilutions from frozen stocks are used, they should be incubated for 2 h prior to adding to antimicrobials, to lessen the lag phase of growth.

Broth

Microdilution

The broth microdilution test is based on the principle that a constant number of microorganisms are added to serial doubling concentrations of antimicrobial agents diluted in broth in a 96-well microtiter plate. Broth microdilution is the most practical and widely used method for determining antimicrobial susceptibilities of human mycoplasmas and ureaplasmas. It is economical and allows several antimicrobials to be tested in the same plate, and mycoplasmacidal testing can be performed in the same system. The test provides a quantitative MIC for each antimicrobial agent tested, with susceptibility or resistance determined based on the ability of the organism to metabolize substrates when grown in the presence of the antimicrobial agent and indicator of growth. Preparation of antimicrobial dilutions is labor-intensive and the endpoint tends to shift over time for some drugs, so that careful attention must be paid to the time when the end point is read. Comparisons of agar dilution with broth microdilution indicate that categorically similar results may be obtained for erythromycin, tetracycline, and fluoroquinolones, but the actual MICs de-

CUMITECH

34

termined by microbroth dilution may be slightly lower (8, 58, 113). The broth microdilution procedure involves adding 100 ~1 of broth to wells 2 to 12 in a horizontal row of a 96-well microtiter plate. Then 100 l..~lof broth containing the antibiotic in the highest concentration to be tested is added to wells 1 and 2, and doubling dilutions are performed in wells 2 to 12. Organisms, prepared in the appropriate dilution, are then added to the wells. Mandatory controls include the antimicrobial in highest concentration, sterile medium growth medium, and inoculated growth without antimicrobial. If organic solvents are used to dissolve the antimicrobial, an inoculated solvent control should be included. The effect of medium on the activity of the antimicrobial is evaluated by comparing parallel MICs for a type strain of a reference bacterium with known MICs obtained in a standard medium such as Mueller-Hinton in addition to mycoplasma medium. A control strain of the mollicute species being tested, for which the MICs of the drugs being evaluated are reproducible, should also be included in each assay for validation purposes. Tests should be carried out with the plates sealed with an acetate or other adhesive cover to prevent drying out and color changes because of gases that may be released during the metabolism of biochemical substrates. However, the wells should be vented by puncturing the acetate cover prior to incubation. The MIC should be read as the lowest concentration of antimicrobial agent that prevents a color change at the time when the growth controls first show a color change. Presumptive MICs for ureaplasmas will be available at 16 to 24 h, and those for M. hominis will be available at 36 to 48 h. MICs for M. pneumoniae may require 5 days or more until evidence of growth in the control wells is evident. Turbidity or color change in broth control indicates bacterial contamination. Susceptibility testing kits such as Mycoplasma IST (bioMerieux), Mycoplasma SIR (Bio-Rad), Mycofast “All-In” (International Microbio), and MYCOKITATB (PBS Orgenics) are available in Europe. They consists of microwells containing dried antimicrobials, generally in two or more concentrations corresponding to the threshold proposed for conventional bacteria to classify a strain as susceptible, intermediate, or resistant. Some of these kits combine organism growth and identification with susceptibility tests in the same product. Abele-Horn et al. (1) evaluated the above kits in comparison to standard methods; they found that the results for tetracycline correlated better than those for other agents, which showed more widespread differences, and did not endorse the use of these products for clinical purposes. Inoculum size can influence MICs, and direct inoculation with the clinical specimen without a defined inoculum, as is commonly done with some of these kits, can contrib-

CUMITECH

34

Mycoplasmal

ute to error. Renaudin and Bebear (76) also evaluated the Mycoplasma SIR; they reported that, using a defined inoculum, this product gave results comparable to those obtained by established MIC determination and considered it a reasonable choice for diagnostic laboratories in countries where the product is available. This product is adapted for use with Ureaplasma spp. and M. hominis after a primary culture. Agar Dilution Dilution of antimicrobials in agar has been adapted for use with several mycoplasmal species and ureaplasmas (6, 58, 77). It has the advantages of a relatively stable end point over time, allows the detection of mixed cultures readily, and is suitable for testing larger numbers of organisms simultaneously. However, this technique is not practical for testing small numbers of strains or occasional isolates which may be encountered in diagnostic laboratories, and mycoplasmacidal testing cannot be done in the same system. If an inoculum contains too many cells or the agar is inadequate, colonies may not form and the test will be uninterpretable. Media must be used when fresh. Agar plates containing the appropriate antimicrobial concentrations prepared in advance are inoculated with multiple strains of the organism to be tested by using a multipoint replicator, micropipettor, or calibrated loop to deliver 10 ~1. The goal is to obtain 30 to 300 colonies per spot of inoculum on the growth control plate. A lo-p1 volume of a dilution of an actively growing culture prepared as described above can be used as the initial inoculum, verified by serial dilution and plate counts. Controls required include an inoculum on antimicrobial-free agar, an inoculum of a reference strain for which the MIC of the agent being tested is known, and plates incorporating the highest concentration of solvent used to dissolve the antimicrobial being tested. The effect of the mycoplasma medium on MIC can be assessed as described above for broth microdilution. Inoculated plates are incubated in a sealed container containing moistened paper towel to prevent desiccation and are examined microscopically. The MIC is the lowest concentration of the agent that prevents colony formation when read at the same time the antimicrobial-free control plate demonstrates growth. This usually occurs in 1 to 2 days for U. urealyticum, 2 to 4 days for M. hominis, and 5 or more days for M. pneumoniae and other human mycoplasmas. Agar Gradient

Diffusion

Preliminary studies using the agar gradient diffusion or E-test (AB BIODISK) technique for detection of tetracycline and fluoroquinolone susceptibilities in M. hominis yielded results comparable to

Infections

19

those of broth microdilution and agar dilution (114-l 16). The E test has also been successfully used for testing the in vitro susceptibilities of Ureaplasma spp. (30). Susceptibility testing with the E test can be done by adapting the procedure from methods used for other techniques. A 0.5-ml suspension of at least 10’ CCU of organisms per ml, prepared as described above, is inoculated onto the appropriate agar plate. The plate is rotated to allow the liquid to spread uniformly across the surface and then allowed to dry for 15 min. Two E tests can be placed on each standard-size plate. The agar plates are incubated until colonies are apparent in the periphery of the plate and an ellipse becomes apparent. The MIC is read under the microscope as the number on the E-test strip corresponding to the intersection of mycoplasmal growth. Staining plates with Dienes stain helps visualize the ellipse. The E test has the advantages of the simplicity of agar-based testing, has an end point that does not shift over time, does not have a large inoculum effect, and can easily be adapted for testing single isolates. This technique is readily adaptable to laboratories not specializing in mycoplasma diagnosis that may encounter isolates needing s uscepti bility tests only on an occasional basis. E tests are commercially available, can be maintained frozen for 3 to 5 years, and work well with commercial mycoplasma media (115, 116). The strips can be expensive if a large number of different drugs are evaluated, but many of the drugs used for testing mycoplasmas may also be appropriate for use with other bacteria if this technique is also being employed in other settings. Reporting

and I nterpretation

of Results

Although some laboratories have adopted MIC breakpoints for Ither bacteria for use in interpretation of MICs wl en testing mycoplasmas, this practice should be used with caution, and it may be preferable to merely report MICs since no breakpoints specific for these organisms are endorsed by any regulatory agency. However, in general terms, MICs of 51 pg/ml should be considered predictive of potentially effective treatment. For most antimicrobial agents of potential use against mollicutes, there will be a clear distinction between susceptible and resistant strains. Tetra cycline-resistant M. hominis and Ureaplasma spp. can readily be distinguished by broth- or agar-bas ed methods sin .ce the resistan t strains generally hav .e MICs of 28 f-%/ml whereas susceptible strains consistently have MICs of 52 pg/ml, with no overlap between the two distinct populations (58, 113, 114). Ureaplasmas will often demonstra te erythromyci .n MICs of 0.5 to 2 kg/ml, technically not indicative of full suscep ti-

20

Waites

CUMITECH

et al

bility if one considers breakpoints published for other bacteria. However, in view of the testing conditions at acidic pH, these MICs are probably indicative of clinical susceptibility, as borne out by limited clinical studies (110, 111). Minimum Testing

Mycoplasmacidal

34

color change and no colonies formed on subculture to agar. APPENDIX 1 MEDIUM FORMULATIONS FOR CULTIVATION OF MYCOPLASMAS FROM HUMANS

Concentration

In classic bacteriological time-kill experiments, antimicrobial agents are added to actively growing cultures. These mixtures are incubated along with a control antimicrobial-free culture. At various time points, samplesare taken and diluted in control broth to stop the killing reaction. Dilutions are plated on agar, and the counts of the experimental cultures are compared with those at time zero and with those of the control cultures at the chosen times. The usual end point is a 99.9% decreasein counts from the control at time zero. For mycoplasmas, broth containing the antimicrobial agent at several concentrations of antimicrobial agent (0, 1 X, 2X, 4X and sometimes 8 X the amount needed for the MIC) is mixed with actively growing organisms to a concentration of lo6 to 10’ organisms per ml (total volume, 25 ml). At 0,2, 4, 8, and 24 h, samples are removed and diluted in control broth to lob4 and 0.1 ml of each dilution is plated on agar. Depending on the amount of agent used, dilutions of 1: 10 or 1:lOO are needed to stop the reaction. The undiluted mixture cannot be plated becausethe killing reaction will continue on the surface of the plate. Plate counts are obtained, and the end point is the amount of agent needed to produce a 99% reduction in the initial plate count. The 99% killing, rather than 99.9%, is recommended for mycoplasmas because of their limited growth compared to bacteria. Times for killing vary markedly: aminoglycosides kill within minutes, and quinolones show a 6-h latent period. Time-kill studies for M. hominis and organismsthat show steepdeath phasesin control media are difficult to interpret because of the loss of viability in the control cultures after peak growth. Tetracyclines can be used as a control because they are mycoplasmastatic and viability is preserved for 248 h. The mycoplasmacidal concentration of an antibiotic can also be determined by direct subculturing of 0.2 ml of fluid from wells of a microbroth dilution MIC systemthat do not show a color change and from the growth control at the time this control first shows a color change. Sufficient volumes (20 ml) to dilute the antibiotic beyond the MIC must be used to dilute the fluid from the MIC system. The broths are incubated and subcultured to agar if a color change occurs. The mycoplasmacidal concentration is the lowest concentration of antibiotic at which there is no evidence of a

GENERAL CONSIDERATIONS Although a number of other formulations are available, a choice from the media described below should serve the requirements of the diagnostic laboratory. The small petri plates (60 mm) used for agar require 7.5 ml for cultures that will receive prolonged incubation, e.g., M. pneumoniae, but 5.0 ml is sufficient for specimens cultured for M. hominis and Ureaplasma spp. The volume of broth is also dictated by the length of incubation required, e.g., 1.8 to 4.5 ml for M. pneumoniae and 0.9 ml for the genital mycoplasmas. Selective media to separate M. hominis from ureaplasmas can be prepared by incorporation of clindamycin or lincomycin (10 pg/ml). Ureaplasmas can be excluded by addition of erythromycin, but this is usually not necessary because they die more rapidly than M. hominis.

A8 AGAR Purpose A8 is a differential agar medium useful for isolating genital mycoplasmas. Urea is included in this medium to enhance the differentiation of U. urealyticum from non-urea-hydrolyzing mycoplasmas. Ingredients

and Preparation

To prepare 1 liter, mix the following ingredients in a flask in the order specified. Adjust the pH to 5.5 with 2 N HCl. Autoclave for 15 min at 121OC. Cool in a 56°C water bath. Base Ultrapurified water . . . . . . . . . . . . . . . . . . . . . . . . . . . . CaCl, dihydride (dissolve before adding other ingredients) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trypticase soy broth (Becton-Dickinson [BD]) . . Yeast extract (Difco) . . . . . . . . . . . . . . . . . . . . . . . . . . . Putrescine dihydrochloride .. ... ... ... .... ... ... DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Select Agar (BD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

82.5 ml 0.15 g 24 g 2g 1.7 g 0.2 g 10.5 g

Supplements Prepare and filter sterilize (0.2~pm-pore-size filter) each supplement separately (except serum). Mix, add mixture to base agar, and adjust the pH to 6.0. Pour plates, invert after 2 h, and keep at room temperature overnight. Place in plastic bags and refrigerate at 4°C for up to 3 months. Horse serum (HyClone) . . . . . . . . . . . . . . . . . . . 200 ml IsoVitaleX (BD) . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ml 10 ml 10% urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GHL tripeptide solution (CalbiochemNovabiochem) . . . . . . . . . . . . ..*........... 1 ml 2% L-cysteine (prepared fresh on day of use) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ml Penicillin (to prevent bacterial overgrowth) . 1,000 IU/ml

CUMITECH

34

Mycoplasmal

IOB BROTH Purpose 1OB is an enriched broth medium Ureaplasma spp. and M. hominis. Ingredients

useful for cultivation

of

and Preparation

To prepare 1 liter, add the following ingredients to a flask. Adjust the pH to 5.5 with 2 N HCl. Autoclave for 15 min at 121°C and cool before adding supplements. Ultrapurified water .............................. 825 ml Mycoplasma broth base without crystal violet W) ......................................... 14 g Arginine ............................... ......... 2g DNA ........................................... 0.2 g 1% phenol red (prepared fresh monthly). . . . . . . . . 1 ml Supplements Prepare and filter sterilize (0.2~pm-pore-size filter) each supplement separately (except serum), and add the supplements to the base. Adjust the pH to 5.9 to 6.1. Store at 4°C for up to 3 months. Horse serum (HyClone) . . . . . . . . . . . . . . . . . . . . . . . . 200 ml 25%1 yeast extract (Difco) . . . . . . . . . . . . . . . . . . . . . . 100 ml IsoVitaleX (BD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ml 4 ml 10% urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4% L-cysteine (prepared fresh on day of use) . . . . 2.5 ml Heat-inactivated fetal bovine serum can be used instead of horse serum.

SP-4 BROTH AND AGAR Purpose SP-4 broth is an enriched growth medium used for the cultivation of many MycopZasma species, including M. pneumoniae. Agar may be added to SP-4 broth for preparation of solid medium, and glucose and/or arginine and urea may be added as metabolic substrates, depending on which mycoplasmas are being sought. Ingredients and Preparation To prepare 1 liter, add the following ingredients to a flask. Autoclave for 15 min at 121°C and cool before adding supplements. For agar, cool in a 56°C water bath. Base Ultrapurified water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643 ml Mycoplasma broth base without crystal violet PW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 g Tryptone (Difco) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . log Peptone (Difco) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 g Arginine (only if medium is to be used to isolate M. hominis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . % 1% phenol red (prepared fresh monthly). . . . . . . . . 2 ml DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g Noble agar (only if preparing SP-4 agar) (Difco). . 15 g Supplements Prepare and filter sterilize (0.2~pm-pore-size filter) each supplement separately (except serum), and add the supplements to the base. Adjust the pH to 7.4 to 7.6 if the medium

Infections

21

will be used primarily for M. pneumoniae. If agar is added, pour plates, invert after 2 h, and keep at room temperature overnight. Place in plastic bags, and refrigerate at 4°C for up to 3 months. 10X CMRL 1066 (Gibco) . . . . . . . . . . . . . . . . . 50 ml 25 % yeast extract (Difco). . . . . . . . . . . . . . . . . . 35 ml 2% yeastolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ml Heat-inactivated (56OC for 30 min) fetal bovine serum (HyClone). . . . . . . . . . . . . . . . . 170 ml 50 % glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 ml Penicillin (to prevent bacterial overgrowth) . 1,000 IU/ml

HAYFLICK MODIFIED BROTH AND AGAR Purpose Hayflick modified broth is an enriched growth medium for cultivation of many mycoplasmal species, including pneumoniae and M. hominis. Agar may be added to flick modified broth for preparation of solid medium, glucose and/or arginine may be added as metabolic strates. Ingredients

used M. Hayand sub-

and Preparation

To prepare 1 liter, add the following ingredients to a flask. Autoclave for 15 min at 121 “C, and cool before adding supplements. For agar, cool in a 56°C water bath. Base for Broth Ultrapurified water ............................ Heart infusion broth (BD). ..................... 25% yeast extract (freshly prepared) ........... Phenol red (1 mg/ml) ..........................

800 17.5 100 20

Base for Agar Ultrapurified water ............................. Heart infusion broth (BD) ...................... 25% yeast extract (freshly prepared) ............

300 ml 17.5 g 100 ml

ml g ml ml

Supplements Prepare and filter sterilize (0.2~pm-pore-size filter) each supplement separately (except serum), and add the supplements to the base. Adjust the pH to 7.4 to 7.6 for M. pneumoniae or to 7.0 if arginine is added for M. hominis. If agar is added, pour plates, invert after 2 h, and keep at room temperature overnight. Place in plastic bags, and refrigerate at 4°C for up to 3 months. For Broth

Heat-inactivated (56OC for 30 min) fetal bovine or foal serum . . . . . . . . . . . . . . . . . . . . 200 ml Arginine or glucose. . . . . . . . . . . . . . . . . . . . . . . . 5g Penicillin (to prevent bacterial contamination) . . . . . ..*.................. 1,000 IU/ml Ampicillin (1 mg/ml) can be used instead of penicillin. For Agar

Heat-inactivated (56OC for 30 min) fetal bovine or foal serum . . . . . . . . . . . . . . . . . . . . Ultrapurified sterile water.. . . . . . . . . . . . . . . . . Noble agar or purified agar . . . . . . . . . . . . . . . . Arginine or glucose. . . . . . . . . . . . . . . . . . . . . . . . Penicillin (to prevent bacterial contamination) . . . . . . . . . . . . . . . . . . . . ...*.. Ampicillin

200 ml 500 ml 10 g sg 1,000 IU/ml

(1 mg/ml) can be used instead of penicillin.

22

Wattes

BROMOTHYMOL

et al.

CUMITECH

BLUE BROTH

Purpose Bromothymol blue broth (B broth) is useful for cultivation, detection, and preliminary identification and differentiation of Ureaplasma and M. hominis. Unlike phenol red-containing media, the indicator changes color before the end of logarithmic growth. As a consequence, at the color change the organism is in good physiologic condition. Furthermore, because of the relatively low urea concentration, the death rate of the culture is reduced and cultures can maintain viability for up to 1 day thereafter. B broth can be stored indefinitely at -2OOC. It should not be stored, even for short periods, in a thawed state. Uninoculated broth is yellow. If the color does not progress past chartreuse, only M. hominis is present. If a full green color develops, only ureaplasmas are present. A blue-green color indicates the presence of ureaplasmas and M. hominis. Ingredients

and Preparation

To prepare 1 liter, add the following ingredients to a flask. Autoclave for 1.5 min at 121°C and cool to at least 50°C before adding supplements. Base PPLO broth base without crystal violet (BD). . . . . . 21 g Yeast extract (BD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lg Bromothymol blue solution (0.4%, wt/vol) . . . . . . . 10 ml Distilled-deionized water. . . . . . . . . . . . . . . . . . . . . . . . . 900 ml Bromothymol blue powder is available from several chemical companies. Dissolve in a minimal volume of 1 N NaOH, and make up to volume with water. Sterilize. Supplements Prepare and filter sterilize (0.2-pm-pore-size filter) each supplement separately (except serum), and add the supplements to the base. Sterile, pooled normal horse serum, preadjusted to pH 6.0. . . . . . . . . . . . . . . . . . . . 100 ml 10% (wt/vol) urea solution, stored at -20°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 ml Glycyl-L-histidyl-L-lysine acetate (20 pg/ml; Calbiochem-Novabiochem), stored at -20°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 ml Ampicillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 mg/ml Nystatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 U/ml Readjust the pH of supplemented medium to 6.0 to 6.1 with sterile 1 N HCl. Greater acidity will reduce the ability of the medium to support growth. Store at -20°C until use.

APPENDIX 2 SUPPLIERS OF KITS, MEDIA, AND REAGENTS Companies that supply kits, media, reagents, and other products tend to change rapidly, and some products are available only in Europe and not North America. Therefore, the products listed from these suppliers may not always be available as they are described. The list of suppliers is provided as an initial source for the reader to use as a basis for seeking information. Contact information for products mentioned in this Cumitech is provided below.

KITS AND MEDIA AB BIODISK North America Inc. 200 Centennial Ave. Piscataway, NJ 08854-3910 Phone: (800) 874-8814 (United States) Fax: (732) 457-8980 Website: http://[email protected] Alexon-Trend 14000 Unity St. NW Ramsey, MN 55303 Phone: (800) 366-0096 (United States) Website: http://www.alexon-trend.com BD Micro biology Systems 7 Loveton Cir. Sparks, MD 21152 Phone: (410) 316-4000 Website: http://www. bdms.com bioMerieux SA 692 80 Marcy L’Etoile France Phone: 33 4 78 87 20 00 Fax: 33 4 78 87 20 90 French website: http://www. biomerieux.fr U.S. website: http:llwww. biomerieux-vitek.com Bio-Rad 3 blvd Raymond Poincare 92430 Marnes-la-Coquette prance Phone: 33 147 95 60 00 FAX: 33 147 4191 33 Website: http:llwww. bio-rad.com Calbiochem-Novabiochem International, P.O. Box 12087 La Jolla, CA 92039-2087 Phone: (800) 854-3417 (United States) Fax: (800) 776-0999 (United States) Website: http://www.calbiochem.com DiaSorin 1990 Industrial Blvd. Stillwater, MN 55082 Phone: (800) 328-1482 (United States) Fax: (612) 439-9710 Website: http:llwww.diasorin.com Difco Laboratories P.O. Box 331058 Detroit, MI 48232-7058 Phone: (313) 462-8500 Fax: (313) 462-8517 Fujirebio (U.S. office) 30 Two Bridges Rd., Suite 250 Fairfield, NJ 07004-1550 Phone: (973) 227-8888 Fax: (973) 227-8585 HyClone 1725 South Hyclone Rd. Logan, UT 84321 Phone: (800) 492-5663 (United States) Fax: (800) 533-9450 (United States) Website: http://www.hyclone.com

Inc.

34

CUMITECH

Mycoplasmal

34

ICN Biomedicals, Inc. 3300 Hyland Ave. Cosa Mesa, CA USA 92626 Phone: (800) 854-0530 (United States) Fax: (800) 834-6999 (United States) Website: http://www.icnbiomed.com International Microbio BP 705 83030 Toulon Cedex 9 France Phone: 33 4 94 88 5 00 Fax: 33 4 94 88 55 22 Website: http://www.int-microbio.com Irvine Scientific 2.511Daimler St. Santa Ana, CA 92705 Phone: (800) 437-5706 (United States) Fax: (949) 261-6533 Website: http:llwww.irvinesci.com Meridian Diagnostics, Inc. 3471 River Hills Dr. Cincinnati, OH 45244 Phone: (800) 543-1980 (United States) Fax: (513) 271-3762 Mycoplasma Experience 1 Norbury Rd. Reigate, Surrey RH2 9BY England Phone: 44 1737 226662 Fax: 44 1737 224751 Website: http://www.mycoplasma-exp.com PBS Orgenics 19 rue de Lambrechts 92404 Courbevoie Cedex France Phone: 33 141 99 92 92 Fax: 33 14199 92 Remel Laboratories 12076 Santa Fe Dr. Lenexa, KS 66215 Phone: (800) 255-6730 (United States) Fax: (800) 447-5750 Website: http://www.remelinc.com Wampole Laboratories Division of Carter-Wallace Half Acre Rd. Cranbury, NJ 08512 Phone: (800) 257-9525 (United States) Website: http:llwww.wampolelabs.com Zeus Scientific, Inc. P.O. Box 38 Raritan, NJ 08869 Phone: (800) 286-2111 (United States) Website: http:llwww.zeusscientific.com

Infections

23

STRAINS University of Florida Mollicutes Collection Maureen Davidson, Director Division of Comparative Medicine 1600 SW Archer Rd., P.O. Box 100006, Room CB 159 Gainesville, FL 326 10 Phone: (352) 846-2788 Fax: (352) 846-278 1 E-mail: [email protected] American Type Culture Collection 10801 University Blvd. Manassas, VA 20110-2109 Phone: (703) 365-2700 Website: http:llwww.atcc.org

REAGENTS Plain and fluorescein isothiocyanate-conjugated antisera for the identification of mollicutes can be obtained from the University of Florida Mollicutes Collection (address given under “Strains” above).

EXPERT ASSISTANCE LABORATORIES Centers for Disease Control and Prevention Deborah Talkington, Laboratory Chief Mycoplasma Laboratory Division of Bacterial and Mycotic Diseases National Center for Infectious Diseases 1600 Clifton Rd. Atlanta, GA 30333 Phone: (404) 639-3563, -3918, or -2215 (Respiratory Diseases Branch) Fax: (404) 639-4215 E-mail: [email protected] Cecile Bebear Department of Bacteriology University of Victor Segalen Bordeaux 146 rue Leo Saignat 33076 Bordeaux Cedex France Phone: 33 5 57 57 16 25 Fax: 33 5 56 93 29 40 E-mail: [email protected]

2

Additional information and resources concerning mycoplasmas can be obtained through the International Organization for Mycoplasmology website (mycoplasmas.vm .iastate.edu).

APPENDIX 3 CODING GUIDANCE Correct coding (CPT-4 and ICD-9-CM) (Tables A3.1 and A3.2) facilitates accurate workload determination and utilization review, allows benchmark comparisons, and ensures appropriate reimbursement. However, correct coding does not guarantee reimbursement; tests must still be considered reasonable and necessary by the payer. For

24

Waites

et al.

CUMITECH

Medicare in particular, knowledge of national coverage policies and local medical review policies is essential to ascertain if ICD-9-CM codes support medical necessity. It is also necessary to determine current Food and Drug Administration approval status since many payers consider investigational tests to be not reasonable or necessary. This appendix is provided as coding guidance for proce-

Table

A3.1.

CPT-4

codes

for Mycoplasma

dures and clinical conditions discussed in the text. Readers should confirm validity with individual payers and should review revisions of both UT-4 and ICD-9-CM codes as appropriate. Note that CPT-4 codes are a trademark of the American Medical Association and are updated annually. ICD-9-CM codes are in the public domain and are updated regularly by cooperating parties.

testing C PT-4 code

Test

Description

87109

Culture,

Isolate identification” Biochemical

87077

Aerobic isolate, additional methods required for definitive identification, each isolate Culture, typing; immunofluorescence method, each antiserum Culture, typing; immunologic method, other than immunofluorescence, per antiserum Culture, typing; immunologic method, other than immunofluorescence, per antiserum Culture, typing; immunologic method, other than immunofluorescence, per antiserum Culture, typing; immunologic method, other than immunofluorescence, per antiserum Culture, typing; immunologic method, other than immunofluorescence, per antiserum

Each isolate

For direct For direct

Epi-immunofluorescence

87140

In situ immunoperoxidase

87147

lmmunoblotting

87147

growth

inhibition

87147 87147

Metabolic Mycoplasmacidal

activity

87147

mycoplasma,

Comment

Culture

Agar

any source

PCRa M. pneumoniae Other mycoplasmas

87581 87798

M. pneumoniae, Infectious-agent not otherwise technique

Susceptibility testi w” Broth microdilut ion or agar dilution

87186

Sensitivity studies, antimicrobial agent; microdilution or agar dilution, each antimicrobial per plate Susceptibility studies, antimicrobial agent; agar dilution method, per agent (e.g., antibiotic gradient strip) Susceptibility studies, antimicrobial agent; microdilution or agar dilution, minimum lethal concentration

E test

87181

MBC

87187

Serologic testinga Cold agglutinins Complement fixation (CF) Indirect immuriofluorescence

Particle

Enzyme

agglutination

immunoassay

(IFA)

86156 86157 87171 86738

86738

(EIA)

34

86738

Cold agglutinin; Cold agglutinin; Complement Qualitative or immunoassays for antibodies M ycoplasma Qualitative or immunoassays for antibodies MycojAasma Qualitative or immunoassays for antibodies Mycoplasma

amplified detection specified,

probe technique by nucleic acid; amplified probe

Primary

code

Each antiserum Each antiserum Each antiserum Each antiserum Each antiserum Each antiserum

specimens specimens

Each multiantimicrobial plate Each antimicrobial

Use in conjunction with 87186

screen titer fixation tests; each antigen semiquantitative by multiple-step methods to infectious agents;

86156 reflex Each antiserum Each antigen and each antiserum or antibody class

semiquantitative by multiple-step methods to infectious agents;

Each antigen and each antiserum or antibody class

semiquantitative by multiple-step methods to infectious agents;

Each antigen and each antiserum or antibody class

CUMITECH

34

Mycoplasmal

ICD-9CM code

Condition Respiratory Tracheobronchitis Bronchitis

Asthma,

exacerbation

Coryza Sore throat Pneumonia

Interstitial pneumonia Hoarseness Cough Extrapulmonary Fever with or without Headache Skin rashes Malaise Pericarditis Hemolytic anemia

chills

Arthritis Meningoencephalitis Peripheral neuropathy Infection, NOS Wound infection

Urogenital Urethritis Urethral syndrome Epididymo-orchitis Urinary crystals Urinary calculi Acute pyelnonephrits Bacterial vaginosis Vaginal discharge Salpingitis Infertility

Bronchitis,

466.0 491.8 491 .I 491.21 493.xx

Acute bronchitis Other chronic bronchitis Mucopurulent chronic bronchitis Obstructive chronic bronchitis with Asthma

460 462 485 486 483.0 516.8 784.49 786.2

Acute nasopharyngitis Acute pharyngitis Bronchopneumonia, organism unspecified Pneumonia, organism unspecified Pneumonia due to other specified organisms, Other specified alveolar and parietoalveolar Voice disturbance, other Cough

780.6 784.0 782.1 780.79 420.9x 283.~~

Fever Headache Rash and other nonspecific skin eruption Other malaise and fatigue Other and unspecified acute pericarditis Acquired hemolytic anemia

716.9x 323.9 322.9 356.9 136.9 958.3 998.59

Arthropathy, unspecified Unspecified cause of encephalitis Meningitis, unspecified Hereditary and idiopathic peripheral neuropathy, Unspecified infectious and parasitic disease Posttraumatic wound infection not elsewhere Postoperative infection

099.40 597.80 597.81 604.90 791.9 592.x 590.80 616.10 623.5 614.x

Other nongonococcal urethritis, unspecified Urethritis, unspecified Urethral syndrome NOY Orchitis and epididymitis, unspecified Other nonspecific findings on examination of urine Calculus of kidney and ureter Pyelonephritis, unspecified Vaginitis and vulvovaginitis, unspecified Leukorrhea, not specified as infective Inflammatory disease of ovary, fallopian tube, pelvic tissue, and peritoneum Infertility, male Infertility, female

634.x 779.9x

Prematurity

765.0x 765.1x 762.7

not specified

as acute

or chronic

acute

exacerbation R equires 4th and 5th digits

M. pneumoniae pneumopathies

Requires 5th digit Requires 4th or 5th digit 5th digit for site

unspecified classified

672.0x 639.x

Postpartum infection Neonatal pneumonia

771.8x 77o.xx

Pyrexia of unknown origin during the puerperium Complications following abortion and ectopic or molar pregnancy Other infection specific to the perinatal period Other respiratory conditions of fetus and newborn

771.8x 77o.xx

Other Other

infection respiratory

764.0x

Slow

fetal growth

Neonatal bacteremia Chronic lung disease prematurity Low birth weight

of

cellular

Spontaneous abortion Other and ill-defined condition originating in the perinatal period Extreme immaturity Other preterm infants Fetus or newborn affected by chorioamionitis, amnionitis, membranitis, placentitis Other infection of amniotic cavity complicating pregnancy

Placental inflammation, infant effects Placental or other inflammation, maternal effects Postpartum or postabortal fever Postabortal infection

658.4x

specific to the perinatal period conditions of the fetus and newborn without

mention

25

Comment

490

606.x 628.x

Perinatal and neonatal Spontaneous abortion Stillbirth

Description

Infections

of fetal malnutrition

Requires

4th digit

Requires

4th digit

Requires Requires

4th digit 4th digit

Requires Requires

4th digit 4th digit

Requires Requires

5th digit 5th digit

Requires

5th digit

Requires Requires

5th digit 5th digit

Requires 5th Requires 4th 5th digits Requires 5th Requires 4th 5th digits Requires 5th

digit and digit and digit

26

Waites

CUMITECH

et al.

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46. Hirschberg, L., A. Krook, C. A. Pettersson, and T. Vikerfors. 198 8. Enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae specific immunoglobulin M. Eur. J. Clin. Microbial. Infect. Dis. 7~420 -423. 47. Horner,

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R. 0. M.

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