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If syphilis is suspected, material collected from the chancre can be examined by direct fluorescent-antibody assay for T. Bacterial agents known to cause pharyngitis are described in more detail in subsequent sections. Some agents were chosen because of their frequent occurrence, whereas others were included because of their important epidemiologic aspects. GAS pharyngitis is a common infection in the throat and skin, causing an estimated 4 to 5 million cases in the United States each year In addition to oropharyngeal infections and other autoimmune sequelae such as rheumatic fever and acute glomerulonephritis, the more serious deep-tissue GAS infections such as necrotizing fasciitis and streptococcal toxic shock syndrome have resurfaced over the past several years 33, Much of this organisms success as a human pathogen is owed to the M protein.

A comprehensive review of the pathogenesis of streptococcal disease provides details of the complex process of adherence, cell invasion, and toxin production in the pathogenesis of S. Some individuals who are carriers may harbor GAS in their upper respiratory tracts without clinical symptoms. These individuals do not exhibit complications from the colonization, nor do they seroconvert when paired sera are tested for streptococcal antibodies. Diagnosis from clinical signs and symptoms is difficult, and mere isolation of the organism from a throat culture is not diagnostic.

Infection may move beyond the pharynx to encompass the tonsils, uvula, and fauces. The scarlet fever variety of GAS throat infection is associated with a characteristic rash caused by the erythrogenic exotoxin. Streptococcus dysgalactiae subsp. Streptococcus equi subsp. Group C streptococci may be relatively common causes of acute pharyngitis among college students and adults 47 , and pharyngitis caused by both C and G groups has been associated with food-borne outbreaks The small-colonyforming member of the beta-hemolytic Streptococcus anginosus group Streptococcus constellatus subsp.

Group A non-S. Beta-hemolytic streptococci other than GAS are not associated with autoimmune sequelae such as rheumatic fever. There is no supportive evidence to suggest that streptococci in groups B and F are important causes of pharyngitis It is obvious from the above discussion that the Lancefield grouping system cannot be used alone for accurate identification of individual beta-hemolytic streptococcal species, but it can be a useful part of the overall identification procedure Complicating matters even further, non-beta-hemolytic variants of S.

Streptococcus mitis, a viridans group alpha-hemolytic streptococcus that is usually a commensal oral organism, can also cause severe pharyngitis often accompanied by toxic shocklike syndrome complications. One outbreak in China involved a single clone that produced a potent exo-.

Historically, culture has been the cornerstone for diagnosis of streptococcal pharyngitis. Gram stains of pharyngeal swabs are not performed because of the ubiquitous presence of commensal oral streptococci. Over the past several years, however, the importance of rapid antigen detection tests RADTs in the initial diagnosis has increased as the sensitivities and specificities of the many assays have improved.

Serology is not useful for diagnosis of acute streptococcal pharyngitis, but measurement of titers of antibodies to various GAS toxins such as streptolysin-O is valuable for confirmation of prior infections in persons suspected of having acute rheumatic fever or acute glomerulonephritis Specimen Collection, Transport, and Processing for Culture. The collection, processing, and culture identification methods described are suitable for all streptococci.

Dacron polyester or calcium alginate swabs are acceptable for specimen collection. The tongue is depressed, and the swab is rubbed vigorously over the tonsillar area and posterior pharynx and other inflamed areas. Take care to avoid touching the tongue and uvula. If processing will be delayed beyond 2 h after collection, place the swab into a suitable transport medium such as Amies gel and store for 24 h at room temperature Fifty years ago, Breese and Disney first established the culture of a throat swab on a sheep blood agar SBA plate as the diagnostic standard for GAS pharyngitis, and it has remained so to the present time Although a major disadvantage of culture is the time required for incubation 24 to 48 h , it is often used in conjunction with the newer RADTs for confirmation of infection.

Upon arrival in the laboratory, the swab is rolled over one-sixth of the surface of an SBA plate. A sterile loop is then used to streak for isolation in four quadrants. The loop is stabbed into the agar several times in an unstreaked area, and the remaining plate surface is streaked for isolation. The subsurface growth in the stabbed areas provides a more reliable indication of true hemolysis due to the activity of both oxygen-stable and oxygen-labile hemolysins.

Betahemolysis appears as a complete lysis of the red blood cells of the medium, especially in the areas of lowered oxygen tension. The basal medium must not contain a high dextrose concentration, as that would inhibit the production of hemolysins. SBA is preferred to media containing blood from other animals because it is less likely to support growth of beta-hemolytic Haemophilus spp.

Inoculated plates are incubated at 35 to 37C, examined after 18 to 24 h, and reincubated if negative, with a final reading at 48 h. Use of CO2 supplementation may enhance recovery of non-GAS beta-hemolytic streptococci and other organisms such as Arcanobacterium spp.

Clinical Practice and Pathological Principles

Bacterial Identification and Reporting Results. The presence of any beta-hemolytic streptococcus grown in a throat culture should be evaluated for possible clinical significance. Colonies may be opaque or transparent with a matte or smooth surface. They are surrounded by a wide area of betahemolysis which is more prominent in the areas of lowered oxygen tension. The bacitracin susceptibility test is sometimes used for presumptive differentiation of S.

After 18 to 24 h of incubation at 37C, any detectable zone of inhibition around the disk is interpreted to indicate susceptibility. This test should be performed only on pure cultures and not on primary inoculation plates. However, some strains of group B, C, and G streptococci also test as bacitracin susceptible. Consequently, an additional procedure using a disk containing 1. Detection of any inhibitory zone around the disk can be interpreted to indicate susceptibility. Alternatively, inhibitory SBA supplemented with SXT can be used as the primary inoculation medium, since growth of GAS will be enhanced as other organisms from the normal flora may be inhibited.

Use of this selective SXT-containing medi-. This test can be performed rapidly on pure cultures by using commercially available reagents and reacting bacterial colonies with the substrate incorporated into a paper disk to which a color developer is then added. Development of a pink area on the disk after a few minutes of incubation at room temperature constitutes a positive test. Other beta-hemolytic streptococci are PYR negative, including some strains of group A that are not S.

A number of companies market latex particles coated with antibody directed against groupspecific carbohydrate antigens. These reagents can be used to rapidly distinguish the major group A, B, C, F, and G beta-hemolytic streptococci causing infections in humans without overnight incubation or other means for determination of biochemical reactions.

From a practical standpoint, clinical laboratories may limit reporting of pharyngeal isolates of beta-hemolytic streptococci to the Lancefield groups based on reactions with group-specific-antibodycoated latex particles and not attempt to classify them further into individual species.

However, it is advisable to verify any small-colony beta-hemolytic streptococcus that belongs to group A as S. PYR-negative strains are considered part of the normal flora. Similarly, one can perform the Voges-Proskauer VP test on group C or G isolates to help differentiate commensal organisms in the small-colony S. Until more evidence accumulates to support a significant role for S. Despite the known association of beta-hemolytic streptococci in groups C and G with pharyngitis, some laboratories may choose to identify and report only the presence of GAS in throat cultures.

Any other beta-hemolytic streptococci are designated as beta-hemolytic streptococcusnot group A. This approach is economical since it eliminates the need to characterize non-GAS isolates by using latex-based reagents or other methods. Decisions regarding whether or not to identify non-GAS isolates in throat cultures should be made in consultation with clinicians who utilize the laboratorys services.

Reporting the presence or the absence of pathogenic beta-hemolytic streptococci without description of other organisms which may be commensals provides the clearest message for directing patient management. However, a positive culture for betahemolytic streptococci does not distinguish between acute infection and colonization. It is also helpful to characterize the numbers of pathogenic streptococci as few growth limited primarily to the first quadrant , moderate growth primarily in the first and second quadrants , or abundant growth in the third and fourth quadrants on the agar plate used for primary inoculation in the event that further differentiation beyond Lancefield grouping is desired.

Table 1 describes the major beta-hemolytic streptococcal species isolated from humans based on the organisms biochemical reactions. The alpha-hemolytic viridans group streptococci are rarely sought individually in pharyngeal cultures because of their infrequency of association with disease and their ubiquity in the oral commensal flora.

The taxonomy of these organisms has been undergoing revision which has made the identification of the organisms to the species level both complex and difficult. Although a number of commercially available biochemical systems, including automated microbiology instruments, can be employed to identify these streptococci to the species level, the performance of these systems in general has been less than ideal and there is no compelling reason to attempt to isolate, identify, or classify these organisms in pharyngeal cultures under normal circumstances.

References and 40 provide tabular information useful. Development of antimicrobial resistance in GAS is not widespread. Penicillin remains the treatment of choice, and there is no resistance to this agent or other comparable -lactams. Macrolides and clindamycin can be used for penicillin-allergic or intolerant patients. A recent large-scale in vitro surveillance study detected erythromycin resistance in only 4. Tetracycline resistance may also occur. Antimicrobial susceptibility testing AST of pharyngeal isolates of beta-hemolytic streptococci is not indicated except by special request in the event that drugs other than penicillin are needed.

Penicillin and erythromycin resistance is now rather common among the viridians group streptococci, however 40, Rapid Diagnosis of Streptococcal Pharyngitis. Commercial point-of-care RADTs for GAS were developed primarily because of the to h turnaround time required for bacterial culture and the need to initiate antimicrobial treatment in a timely manner. RADTs use acid extraction to solubilize the cell wall carbohydrate, followed by an immunologic reaction.

A latex agglutination test was the first type of rapid test to be developed, but this format has been replaced by a variety of enzyme immunoassays EIAs and optical immunoassays OIAs that have the ad-. Table 1. Laboratory identification of beta-hemolytic streptococci known to occur in humansa Species S. This table has been derived from information provided in references 40 and Only data for streptococcal species that have been isolated from humans are included.

They are also referred to as the S. There are insufficient data to know the percentage of each of these species that contain carbohydrate antigens. However, since the autoimmune sequelae of GAS infection do not occur to any extent with streptococci from these other groups and controlled clinical trials have not shown convincing evidence of a clinical response to antibiotics, missing the occasional pharyngitis case by the use of RADTs may not be clinically important The American Academy of Pediatrics AAP recommends that laboratory testing be performed in all cases of pharyngitis in children due to the nonspecific nature of the illness and the likelihood that GAS may be involved.

Despite this recommendation, a large study in a pediatric clinical practice found that only 2. The Infectious Disease Society of America IDSA clinical practice guidelines 13 also recommend that laboratory testing should be performed unless a clinician is able to exclude GAS pharyngitis on clinical and epidemiological grounds. Clinicians can choose whether to use RADTs or culture in the initial evaluation. For children and adolescents, the IDSA recommends that a negative RADT result be confirmed by culture, unless the clinician has ascertained directly that the RADT being used is of sensitivity comparable to that of culture.

These practice guidelines provide a different recommendation for adult pharyngitis because of a lower incidence of GAS disease and a lower risk for development of rheumatic fever in adults. A negative RADT in adults does not require confirmation by culture, and antibiotic therapy is not necessary.

Positive RADTs need not be confirmed by culture. This document goes a step further than the AAP. For patients meeting two or more criteria, recommended strategies include i testing patients meeting two to four criteria by RADTs and limiting antibiotic therapy to patients with positive test results or patients meeting four criteria and ii not performing any diagnostic tests and limiting antibiotic therapy to patients meeting three to four criteria. As with any type of microbiological tests, cost and reimbursement play an important role when clinicians and laboratory directors select diagnostic tests.

RADTs are more expensive than culture, but they provide more rapid results, allowing initiation of specific treatment and potentially shortening the duration of illness, and they may reduce the risk of the spread of infection within the community. The number of new RADTs has increased in recent years.

Many, but not all of them, are in the waived category in the Clinical Laboratory Improvement Amendment CLIA classification, meaning that physician office laboratories do not have to meet the more rigorous certification requirements of laboratories performing moderate- and high-complexity testing. Both assays are most suitable for batch testing of specimens in laboratories experienced in molecular biology-based testing. The cost of these advanced molecular biology-based assays and their instrumentation is considerable. Due to their complexity and the 1.

They underscored the need for studies based on standardized comparisons of RADTs with one another, including waived and nonwaived tests, and suggested that clinicians perform their own evaluations to determine which RADT works best in their clinical setting. Nonstreptococcal Pharyngitis The beta-hemolytic streptococci are the most important bacteria sought in throat cultures from patients with acute pharyngitis. However, some laboratories prefer to offer a variety of different categories for throat cultures to allow clinicians to choose the most appropriate and cost-effective test based on the patient presentation and history.

For example, a test ordered as strep culture would use techniques and provide results only for detecting the presence or absence of beta-hemolytic streptococci, whereas a GC culture would provide results only for N. A broader category of miscellaneous throat culture could include specific examination for other organisms that may cause throat infections, such as N. Laboratory methods for detecting some of these other bacterial pathogens and circumstances in which they should be considered are described below.

Arcanobacterium haemolyticum and Arcanobacterium pyogenes Arcanobacterium haemolyticum and Arcanobacterium pyogenes previously classified as Corynebacterium haemolyticum and Actinomyces pyogenes, respectively are rare causes of pharyngitis, and they have also been implicated in a wide variety of cutaneous and invasive infections including sinusitis, cellulitis, and septicemia Pharyngitis due to these bacteria is often associated with a rash similar to that observed with scarlet fever Understanding the true importance of the arcanobacteria in pharyngitis is complicated since they can be isolated from some individuals without disease and are often isolated in association with other potential pathogens The detection of arcanobacteria does not require any special specimen collection procedures or plating media beyond what have been described above for streptococci.

These bacteria should be suspected when streptococcal antigen tests are negative and gram-. Both species are catalase negative and nonmotile. This species may be distinguished by a CAMP inhibition reaction when incubated with a beta-hemolysin-producing strain of S. A summary of the medically relevant corynebacteria and other coryneform bacteria, including the arcanobacteria, and their biochemical reactions may be found in reference Laboratories should report the presence of Arcanobacterium spp.

AST is not standardized for arcanobacteria and other coryneforms, but MICs of -lactams, macrolides, tetracyclines, and rifampin are very low for these bacteria, suggesting the utility of these drugs for therapeutic purposes. Treatment failure with -lactams and response to macrolides may be due to intracellular localization of the bacteria since -lactams, in contrast to macrolides, penetrate host cells poorly, rendering them unable to kill the organisms Although this is uncommon, over cases of gonococcal pharyngitis have been described since Such cases are typically found in sexually active homosexual and bisexual men and heterosexual women who acquire the infection by engaging in orogenital sexual relations 4.

Most asymptomatic infections have been diagnosed by throat culture using appropriate media and growth conditions for detection of neisseriae, but newer molecular assays such as the ligase chain reaction LCR and other technologies have improved detection in pharyngeal specimens. In a sexually transmitted disease clinic in San Francisco, 4.

Regardless, N. Carriage can be transient, intermittent, or chronic. Despite the complexity of confirming N. Inspect plates at 24, 48, and 72 h for growth. Appropriate laboratory methods must be included if N. Gram stains are not appropriate for diagnosis of pharyngeal infection with N. Diagnosis of pharyngeal infections with neisseriae rests on detection by culture. Techniques described are also suitable for cultures screening for carriage of N. Dacron or rayon swabs are used to obtain oropharyngeal specimens for culture of Neisseria spp. If possible, plate specimens at the time of collection.

Otherwise, place the swab into transport medium and keep it at room temperature. Complete transport systems that include a sealed pouch and catalyst to generate an appropriate incubation atmosphere are available commercially from multiple manufacturers.

Plates should be warmed to room temperature, swabbed in a Z pattern, and then cross-streaked with an inoculating loop. There are a variety of commercially available enriched selective media that support the growth of neisseriae, including Thayer-Martin medium, Martin-Lewis medium, GC-Lect medium, and New York City medium. These media contain antimicrobial agents such as vancomycin, colistin, nystatin, and trimethoprim to inhibit normal flora. However, other organisms that are part of the normal flora of the oropharynx do grow on these selective media and must be differentiated from N.

A nonselective medium such as chocolate agar should also be inoculated because some strains of N. Subculture oxidase- and catalase-positive colonies consisting of gram-negative diplococci onto chocolate agar for further testing as necessary for species confirmation. There are several commercial biochemical and chromogenic enzyme substrate kits, products containing a combination of biochemicals and chromogenic substrates, and immunologic methods that are useful for identifying N.

These are described in more detail in reference texts e. In view of the important social and medicolegal consequences regarding diagnosis of any sexually transmitted disease, laboratories must be aware of the potential for erroneous results with respect to Neisseria sp. For example, the occasional maltose-negative N. Confirmation of species identification by two independent methods may sometimes be necessary, especially for nonurogenital sites in which the presence of N.

Colonies on chocolate agar are larger than those of N. Confirmation of species identity can be achieved by acid production or chromogenic enzyme substrate tests as described for N. The presence of N. However, whether or not N. In the reference laboratory, N. They may also be serotyped based on their outer membrane proteins. AST of neisseriae is currently not recommended or needed in hospital laboratories, even though methods and interpretive criteria for N. Treatment of N. Penicillin and cephalosporins remain the treatments of choice for meningococcal infections.

Changes in antimicrobial resistance in N. Antimicrobial resistance in N. In the event of a clinically significant case of documented pharyngeal infection with Neisseria spp. Molecular Biology-Based Tests. Several new DNA hybridization-amplification assays in a variety of formats are gaining popularity for detection of N. Findings of the study cited above using LCR for detection of pharyngeal gonorrhea 95 are encouraging regarding the potential use of these types of assays for diagnosis and screening.

However, non-culture-based molecular tests are not yet approved by the U. Food and Drug Administration for testing pharyngeal or rectal specimens and cannot be recommended for this purpose Moreover, the LCR system is no longer sold commercially for urogenital specimens. Its role in human disease has been recently reviewed Studies from Italy demonstrated that M. This association with recurrent infections is consistent with the organisms ability to cause chronic respiratory carrier states A significant proportion of pharyngitis cases associated with M. Sore throat associated with M.

Macrolides are effective against M. Thus, empiric treatment of acute pharyngitis is more complex if M. No studies comparable to those from Europe have been published from North America using appropriate diagnostic methods to quantitate the frequency of pharyngitis due to M. In light of the studies cited above, M. Cumitech 34 is devoted to a discussion of laboratory diagnosis of mycoplasmal infections.

Detailed descriptions of laboratory procedures, medium formulations, serologic tests, and molecular biology-based tests are provided there. The Clinical Microbiology Procedures Handbook, second edition , has step-by-step procedures for detection of. In view of the availability of this information from these other sources, treament of diagnostic aspects in this publication is limited to brief summaries.

The media are expensive and nutritionally complex, the culture process is labor-intensive, and the time from inoculation of clinical specimens to isolation can be several weeks. If culture is attempted, scrupulous attention to proper methodology and specimen handling is essential for success. Appropriate specimens for diagnosis of M. Take care to collect material from the nasopharyngeal area and not merely the anterior nares. Either Dacron or calcium alginate swabs are suitable. Avoid wooden-shaft cotton swabs that can be inhibitory.

The swab is then placed into a transport medium such as 2SP or into a culture medium such as SP4 broth with antibiotics Swabs should be swirled and pressed against the side of the tube before they are removed prior to submission to the laboratory. If there is lower respiratory tract involvement and the patient is able to produce sputum, it can also be inoculated into transport medium and submitted to the laboratory. Refrigerate specimens in transport media if they cannot be inoculated onto culture media immediately. If the specimen must be held more than 24 h, freeze it at 70C. If submission to a reference laboratory is required, specimens must be shipped on dry ice.

SP4 agar and broth are the best media for cultivation of M. The complete formulation and instructions for their preparation are provided in Cumitech 34 , and both are sold commercially in the United States by Remel Laboratories. Upon receipt in the laboratory, a specimen in transport medium is centrifuged at 8, to 10, g for 20 min and then the bottom l of transport medium containing the clinical specimen is transferred to 1. From this initial tube, serial fold dilutions of specimens are made to and then a portion of each dilution is subcultured onto agar.

The centrifugation step can be omitted if specimens are collected directly into culture medium such as SP4 broth. Broths are incubated at 37C under atmospheric conditions. Agar plates. However, M. Subculture any clear broth culture showing a color change to fresh broth and to agar. After 7 to 10 days of incubation, all original dilutions not showing a color change should be passaged into fresh SP4 broth and reincubated. Protocols at the CDC specify holding cultures for up to 12 weeks before designating them as negative, although most positive specimens are detected by 2 to 6 weeks. Agar plates are examined with a stereomicroscope at regular intervals for spherical colonies of up to m in diameter.

Tests to identify mycoplasmas to species level include hemadsorption of guinea pig erthrocytes, reduction of tetrazolium, agar growth inhibition with appropriate antisera, immunofluorescent assays or immunoperoxidase staining, monoclonal antibody tests, and PCR assays , Detection of M. AST of M. Methods for MIC determinations that provide reproducible results have been described but have not been reviewed or endorsed by the CLSI , Treatment trials evaluating the clinical response of M. If pharyngitis occurs in association with M. Therefore, specific treatment may be helpful in speeding recovery overall.

Development of testing modalities such as the PCR assay has lessened the importance of culture as a means for detecting M. Studies using. The same types of clinical specimens that can undergo culture can also be tested by PCR. The use of two different targets can maximize the ability to detect the organism. The CDC has also developed a real-time PCR using a unique internal control, which targets a different portion of this gene. Other sequences, primarily those of the P1 adhesin gene and conserved regions of 16S rRNA, have also been utilized as targets To date, there has not been formal standardization of the approach for validation of the published PCR methods as there has been for C.

Reznikov et al. Dilution of samples may sometimes overcome inhibition of PCR, but this may also diminish the sensitivity because the nucleic acid is diluted along with any inhibitors that may be present. There are also commercial reagents for nucleic acid purification that are effective in removing most inhibitors of amplification in PCR assays. Until PCR assays can be standardized, made available at a reasonable cost, and sold commercially as complete diagnostic kits, this method of diagnosis is unlikely to gain widespread use for detection of M.

Specimen collection and transport for PCR analysis are identical to those described above for culture. For culture and PCR from the same specimen, it is suggested that l be left, allowing l for culture and l for DNA extraction. There are numerous DNA extraction kits sold commercially, and most include the necessary enzymes and spin columns to collect the extracted material. The extracts are refrigerated or frozen at 70C if they are to be held more than 7 days prior to processing. Freezing and thawing of these specimens is strongly discouraged. Water should be extracted as a control from each kit, as it has been shown that some lots of commercial kits are contaminated with bacterial DNA and in-house water is often a source of contamination.

Any of the published PCR methods cited above may be used for analysis. Because inhibition is a major factor in creating false-negative results from PCR. This step may dilute inhibitors enough to allow detection of the target DNA. Proper controls should be run with each assay, including a low-copy-number positive control 5 gene copies per sample , depending on the sensitivity of the assay.

The real-time assays are much more sensitive than the conventional assays, and the inclusion of a specific probe increases specificity. Overall, PCR analyses have decreased the time required to diagnose infections, but it is suggested that serology or culture be used as an adjunct. In view of the enhanced analytical sensitivity of PCR over that of culture, a positive PCR result and a negative culture result can be easily explained. However, in a case with a negative PCR assay and a positive culture or serology , the presence of inhibitors or some other technical problem with the PCR assay must be considered.

Measurement of antibody remains the cornerstone for M. The complement fixation test was the standard for antibody detection for many years and is still used today in some state health laboratories. However, it is a laborious assay to perform and has inherent disadvantages such as nonspecific cross-reactions. Complement fixation has been largely replaced by commercial assays utilizing immunofluorescence, particle agglutination, or EIA formats EIAs offer several advantages over the other assay designs, including increased sensitivity, small volume demands, isotypic discrimination, and ease of use.

Commercially available serologic assays sold in the United States are described in reference texts A recent study comparing the commercial EIAs available in the United States showed that some perform significantly better than others and that paired sera are recommended for serodiagnosis of M.

Among the tests evaluated in that study are two qualitative rapid membrane-based EIAs with a moderate-complexity CLIA classification that can be performed as pointof-care procedures in a physicians office or in a clinical laboratory. These tests do not require any specialized equipment, and they are cost-effective when performed on single serum samples or small batches. Therefore, these tests may. However, it is important to understand that although specific IgM antibodies to M.

Duration of the IgM response is variable, and in some instances the response may persist for several weeks. This observation supports the need to test paired sera for optimum diagnosis of current or recent infection. The true incidence of pharyngitis due to this organism is unknown because relatively few cases have included successful isolation of the organism to accompany a serologic diagnosis Sore and scratchy throat with hoarseness is a very common initial manifestation of C.

Sinusitis and otitis may also occur. Some studies have found a low incidence of pharyngitis due to this organism and suggest its role in pharyngitis to be as more of a copathogen than a primary pathogen since it is often detected in the presence of other organisms known to produce the illness The true incidence is probably much greater because in many cases no attempt is made to obtain a microbiological diagnosis and patients are treated empirically.

This illness is usually acquired from inhalation of respiratory droplets from infected birds, but human-to-human transmission can occur in rare circumstances. Many cases of psittacosis begin with sore throat and pharyngitis before progressing to pneumonia. Their report was subsequently published To detect C. If the specimens are to be held longer than 24 h they must be frozen at 70C and transported on dry ice if not processed locally.

Most hospital-based laboratories cannot offer C. To process the specimens, swabs are mixed on a vortex mixer for 20 s and then pressed against the side of the tube to extract all the liquid. Tissue specimens are suspended in cell culture medium before homogenization. Both HEp-2 cells and HL cells support chlamydial growth and are primarily cultured in well plate or shell vial formats.

To inoculate cells, the specimens are centrifuged onto the monolayer at to 3, g for 60 min. After centrifugation, replace the medium with cycloheximide-supplemented medium. Diagnosis of Chlamydophila infections is highly variable among laboratories due to the lack of reference methods and the use of nonstandardized techniques. This interest was due to the importance of this organism in acute respiratory illnesses as well as a purported role in other chronic inflammatory.

Genus- and species-specific monoclonal antibodies can be used to identify C. It is recommended that an average of 1 inclusion per well or tube be considered a presumptive positive, and only if the strain is propagated by subsequent passage or confirmed by another test such as PCR should it be considered a confirmed positive. The use of serum-free media, multiple centrifugations, or pretreatment of cells is not warranted.

Further details on culture and suggested controls can be found in the summary by Dowell et al. When C. Culturing of C. Since Chlamydophila culture is seldom performed, AST is an even rarer procedure. Chlamydophila organisms are susceptible to the expected agents in the macrolide, ketolide, tetracycline, and fluoroquinolone classes. Methods for AST for C. MIF serologic assays have been developed for commercial use and are available through reference laboratories.

Diagnosis of acute laryngitis is usually made by history alone. Illness begins as a common cold with minimal or no associated fever. The patient complains of hoarseness, and vocal cords appear hyperemic, resulting from edema. Acute laryngotracheobronchitis, the croup syndrome, encompasses several types of infections of the larynx, with hoarseness and a barking cough and variable respiratory distress, and affects primarily young children.

Croup can be a serious infection which extends downward from the larynx to involve the trachea and sometimes the bronchi. The onset is gradual, also following upper respiratory tract infection. Severe respiratory distress, especially in young infants, and fever are common manifestations. Croup produces narrowing of the airway and similar signs and symptoms as seen in epiglottitis, but children with croup tend to have a longer course of illness, worsening at night, and a barking cough.

However, in children younger than 6 months, the presentation of croup and epiglottitis can be indistinguishable. The primary etiologic agents for both of these conditions are respiratory viruses However, bacterial respiratory tract infections due to B. Isolation of M. In many instances, initial infection is caused by various viruses, and bacteria play a role as superinfecting agents taking advantage of the damaged respiratory tract mucosa. Unusual causes described in case reports include Candida spp. Laryngitis can also be associated with pulmonary tuberculosis, blastomycosis, and histoplasmosis.

In view of the viral etiology for most cases of acute laryngitis and croup, bacterial or fungal cultures are needed only when there is no other apparent cause or when. Difficult-to-culture organisms such as Chlamydophila are readily adaptable to detection by PCR. Collection, transport, and processing of specimens for PCR are similar to those used for culture. One milliliter of the inoculated transport medium is centrifuged at 18, g for 15 min. The pellet is then processed for DNA extraction by using an efficient and reliable protocol or a commercial kit. Each run should include low-copy-number positive controls 1 inclusion-forming unit and water controls with every fifth extraction No PCR assays for detection of C.

There is a PCR assay for the detection of C. Other assays have been developed, but thus far there are no validation criteria for PCR diagnosis of C. An immunochromatographic assay to detect specific C. Serologic diagnosis of C. The microimmunofluorescence MIF assay is the only serologic method that has been evaluated with good results, and it is the only assay that can measure isotype-specific antibodies to C.

The MIF assay uses purified species-specific elementary bodies as the antigen and is therefore recommended as the standard for serologic diagnosis. Acute infection is defined by a fourfold rise in IgG or IgM titer to 16, and past exposure is defined by an IgG titer of In the interpretation of results, one has to consider the usu-. Epiglottitis Epiglottitis is an infectious process that produces inflammation and edema of the supraglottic structures, which include the epiglottis, uvula, base of the tongue, aryepiglottic folds, arytenoids, false vocal cords, and adjacent pharyngeal walls.

In contrast to laryngitis and croup, epiglottitis is primarily of bacterial etiology. Acute epiglottitis typically occurs in children between 2 and 6 years of age and characteristically presents with an acute onset of high fever, sore throat, and respiratory obstruction with stridor, dysphagia, drooling, and agitation. It is important to differentiate this condition from viral croup because of therapeutic implications Table 2.

Epiglottitis is uncommon in U. Adults with epiglottitis usually have a less acute presentation characterized by odynophagia and a change in voice. Less common manifestations in adults are dyspnea, drooling, stridor, pharyngitis, fever, cervical adenopathy, cough, and hemoptysis. Epiglottitis affects approximately 1 in , adults annually. Other bacterial species that have been associated with epiglottitis include nontypeable H.

Various respiratory viruses may also account for some cases. Even though the incidence of invasive disease in the pediatric population due to H. Endoscopy or indirect laryngoscopy can be performed to assess the supraglottic structures for evidence of supraglottitis in adults, but this should not be attempted in young children without support of an anesthesiologist because slight agitation may precipitate acute and life-threatening respiratory obstruction requiring intubation.

Alternatively, characteristic radiographic changes indicating an enlarged epiglottis and leukocytosis with a left shift are supportive of the diagnosis. Recovery of H. The high rates of asymptomatic carriage make it difficult to accurately assess the role of Haemophilus spp. Moreover, manipulation of the epiglottis may lead to respiratory obstruction. Blood is collected and processed according. Differential characteristics of common infectious laryngeal syndromes Epiglottitis Children 26 yr of age Laryngitis Older children, adolescents, and adults Influenza virus, adenovirus, rhinovirus, parainfluenza viruses, respiratory syncytial virus, papillomavirus, M.

Acute onset of fever, sore throat, drooling, agitation Blood cultures, radiographic imaging, laryngoscopy Artificial airway, systemic antibiotics. Fever, barking cough, wheezing, respiratory distress, stridor Radiographic imaging Mist therapy, racemic epinephrine, dexamethasome, systemic antibiotics if bacterial infection is suspected. In contrast to that in a case of otitis media, the tympanic membrane is mobile on insufflation.

Based on the appearance of the supraglottic structures and overall status of the patient, insertion of an artificial airway intubation or tracheostomy may be required. Administration of parenteral antibiotics such as an extended-spectrum cephalosporin should be initiated as soon as possible after the patient with epiglottitis presents for medical treatment. Hospitalization is usually indicated. OTITIS Otitis Externa Otitis externa is an infection of the external ear canal that is usually caused by excessive moisture that allows bacteria to multiply in the cerumen of the ear canal, leading to maceration and inflammation.

However, it can also be the result of trauma to the external auditory canal, sometimes induced by attempts to clean or scratch the itching ear, or a variety of dermatologic conditions such as eczema and psoriasis. Otitis externa is technically not a disease of the upper respiratory tract, but it is included here because of the importance in distinguishing it from otitis media with discharge secondary to a ruptured tympanic membrane. Otitis externa can be acute, chronic, localized, diffuse, or malignant Otitis externa can occur in persons of any age, most commonly affecting children aged 7 to 12 years.

It occurs most commonly in swimmers and individuals who have other types of exposure that allow contaminated water to be trapped in the external canal. Deep-seated infection malignant otitis externa occurs almost exclusively in the patients who are immunocompromised or have chronic diseases such as diabetes mellitus Untreated malignant otitis externa can lead to cellulitis and osteomyelitis. Otitis externa typically presents with a serosanguinous or purulent discharge from the erythematous and swollen external ear canal in association with ear pain and itching.

In some cases there may be furuncles in the ear canal. Severe cases may be associated with preauricular, postauricular, or cervical adenopathy and fever. Otitis externa is distinguished from purulent otitis media with perforation of the tympanic membrane and drainage by careful examination of. The most common causative organisms of acute otitis externa are Pseudomonas aeruginosa and S.

Other aerobic gram-negative bacilli and gram-positive cocci such as S. Vibrio alginolyticus has been implicated as a cause of otitis externa in persons who swim in salt water Commensal cutaneous organisms such as corynebacteria and coagulase-negative staphylococci may be isolated from the external ear canal, but they are not normally considered to be of clinical significance in this setting.

Chronic otitis externa may be secondary to a persistent, suppurative middle-ear infection accompanied by tympanic membrane perforation. Thus, the bacterial etiologies of this condition are reflective of those involved with the middle-ear disease Rare bacterial causes of chronic otitis externa include Mycobacterium and Nocardia spp.

These organisms are not normally sought in microbiological evaluations without prior consultation with the clinician or evidence of their presence based on Gram-stained smears of discharge from the ear canal. Anaerobic bacteria involved in otitis externa include Peptostreptococcus spp. Herpesviruses have also been implicated in some cases Fungal otitis externa can be the result of prolonged treatment of bacterial otitis externa that alters the flora of the ear canal, sometimes leading to mixed bacterial and fungal infections.

However, fungi are occasionally the primary pathogens. The clinical diagnosis of otitis externa can be made by direct examination of the affected ear. Since several different microorganisms can be involved, a microbiological diagnosis requires demonstration of the organisms by Gram staining and identification in cultures. However, from a practical standpoint, cases of only mild to moderate severity are usually managed empirically without microbial evaluation. Deepseated infections are likely due to P. Material can be collected for Gram staining and culture by swabbing of the ear canal, by needle aspiration of furuncles, or by surgical debridement.

Use of separate swabs for Gram staining and culture is preferred. Use of swab transport systems with supportive media such as Amies gel facilitates maintenance of organism viability until cultures can be inoculated. Fluid can be sent to the laboratory in a sterile cap syringe with the needle removed.

Tissue obtained by surgical debridement can be sent to the laboratory in a sterile screw-cap container. Fluids or tissue specimens collected without transport media must be refrigerated at 4C if they cannot be processed within 2 h of collection. If anaerobe cultures are specifically requested through direct consultation with the clinician, a swab, fluid, or tissue is placed into the appropriate anaerobic transport system, such as the Port-A-Cul Becton Dickinson , and sent to the laboratory as soon as possible. Anaerobe cultures are kept at room temperature until inoculated onto appropriate media.

Primary inoculation media for anaerobic bacteria include Brucella agar with sheep blood, phenyl-ethyl-alcohol agar with sheep blood, laked kanamycin-vancomycin blood agar, Bacteroides-bile esculin agar, thioglycolate broth with hemin and vitamin K, or chopped meat glucose broth. However, many laboratories prefer to use one of the numerous commercial biochemical or automated systems such as MicroScan Dade MicroScan, West Sacramento, Calif.

These products are described more completely in reference texts e. Rapid species-level identification of S. Discussion of streptococcal identification is included in the previous section on pharyngitis. Common anaerobes can also be identified biochemically or by Gram stain morphology supplemented by a variety of phenotypic tests, including high-potency antibiotic disk assays, as outlined in reference texts e.

An external ear specimen culture with growth of S. Growth of a predominant anaerobe alone is also likely to be of etiologic significance. However, growth of anaerobes such as P. Spores and hyphae may be observed with the Gram stain if the etiology is fungal, and they should be noted in the report. Otherwise, the Gram reaction, the cellular arrangement of bacteria, and the presence of inflammatory cells should be described. The normal flora of the external ear canal includes coagulasenegative staphylococci and Corynebacterium spp.

All other aerobic and facultative bacteria isolated are identified to the species level when possible unless there are mixed cultures of gram-negative rods. The most common aerobic bacterial agents of otitis externa can be readily identified by standard biochemical methods available in clinical laboratories.

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Simple tests such as the appearance of gram-pos-. AST should be performed on the predominant pathogenic bacteria isolated in culture except in cases caused by beta-hemolytic streptococci. However, administration of systemic antibiotics is not always necessary. Local measures, including gentle removal of debris and discharge and treatment with topical antimicrobial agents directed at the causative pathogens, often suffice. More severe cases in which infection extends beyond the skin of the ear canal and cases of malignant otitis externa may require systemic antimicrobials accompanied by analgesics, topical corticosteroids to reduce local inflammation, and surgical debridement of necrotic tissue.

Use of acidifying otic drops following exposure to water may be beneficial in reducing recurrences of otitis externa. Fungal Culture. Inoculation of media specific for fungal isolation and utilization of incubation conditions and identifi-. A general discussion of appropriate techniques for isolation and identification of Candida spp. Many Candida infections can be readily detected using SBA. Aspergillus spp.

Use of inhibitory mold agar which contains antimicrobials such as chloramphenicol and gentamicin to reduce bacterial overgrowth can be helpful to recover organisms such as Aspergillus. Otitis Media Acute otitis media is an infection of the middle ear with rapid onset, presence of middle-ear effusion, and signs of middle-ear inflammation. Otitis media with effusion is characterized by fluid collection in the middle ear without signs or symptoms of infection, and it is usually caused when the Eustachian tube is blocked and fluid becomes trapped in the middle ear.

Signs and symptoms of acute otitis media usually occur when fluid in the middle ear becomes infected, although microorganisms may be detected in some cases of otitis media with effusion when sensitive techniques such as PCR are used Otitis media with effusion is more common than acute otitis media, and it may be a prelude or sequelae of that condition.

Recurrent otitis media is defined as three episodes of acute otitis media within 6 months or four or more episodes within 1 year Some patients develop chronic suppurative otitis media. Infection of the middle ear usually arises as a complication of a preceding viral upper respiratory tract infection in which the acute inflammatory response caused swelling and occlusion of the Eustachian tubes.

Normally, air in the middle ear is absorbed by the ears mucosal lining. If the infection and the resultant inflammatory reaction persist, perforation of the tympanic membrane or extension into the adjacent mastoid air cells may occur, leading to mastoiditis. Acute otitis media is the most common reason antibiotics are prescribed for young children in the United States This condition occurs more often in boys than in girls, and its incidence has increased over the.

Children with anatomic malformations such as a cleft palate are especially prone to frequent episodes of acute otitis media. Even though acute otitis media is most common in young children, it can occur in persons of any age. The higher frequency in children is presumably related to immunologic factors, such as a lack of pneumococcal antibodies, and anatomic factors, including a lower angle of the Eustachian tube with relation to the nasopharynx, and the higher frequency in young children of viral respiratory tract infections that can lead to blockage of the Eustachian tubes.

Clinical diagnosis of otitis media can be made based on a typical history of fever, ear pain, and hearing loss and an inflamed, bulging, immobile, tympanic membrane assessed by otoscopy. Infants and very young children may often tug at their ears but can be asymptomatic otherwise except for irritability Some cases, when left untreated, progress to spontaneous perforation of the tympanic membrane with drainage of purulent material into the external ear canal.

Significant concerns in young children with severe otitis media with recurrence and tympanic membrane perforation are hearing deficit and speech delay. Otitis media is not considered a common source of bacteremia or meningeal seeding, but local brain abscesses and systemic spread can occur 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 Ureaplasmas require media with acidic pH 6. In contrast, M. 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.

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. 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 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.

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. Therefore, a 1:lOO dilution will generally yield the desired inoculum. Other species such as M. It is important to verify the inoculum by serially diluting an aliquot, typically 0. 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. The obvious drawback to this exercise is the added time and expense incurred. 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 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, Then l..

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.

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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. MICs for M. Turbidity or color change in broth control indicates bacterial contamination. 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. 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.

Agar Dilution Dilution of antimicrobials in agar has been adapted for use with several mycoplasmal species and ureaplasmas 6, 58, 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. The goal is to obtain 30 to 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. The E test has also been successfully used for testing the in vitro susceptibilities of Ureaplasma spp.

Susceptibility testing with the E test can be done by adapting the procedure from methods used for other techniques. 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 , 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.

For most antimicrobial agents of potential use against mollicutes, there will be a clear distinction between susceptible and resistant strains. Tetra cycline-resistant M. Ureaplasmas will often demonstra te erythromyci. 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 , Minimum Testing Mycoplasmacidal 34 color change and no colonies formed on subculture to agar. 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 At 0,2, 4, 8, and 24 h, samples are removed and diluted in control broth to lob4 and 0. 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.

Times for killing vary markedly: aminoglycosides kill within minutes, and quinolones show a 6-h latent period. Time-kill studies for M. Tetracyclines can be used as a control because they are mycoplasmastatic and viability is preserved for h. The mycoplasmacidal concentration of an antibiotic can also be determined by direct subculturing of 0. 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 small petri plates 60 mm used for agar require 7. The volume of broth is also dictated by the length of incubation required, e. Selective media to separate M. Ureaplasmas can be excluded by addition of erythromycin, but this is usually not necessary because they die more rapidly than M.

Urea is included in this medium to enhance the differentiation of U. Ingredients and Preparation To prepare 1 liter, mix the following ingredients in a flask in the order specified. Adjust the pH to 5. Autoclave for 15 min at OC. Base Ultrapurified water. CaCl, dihydride dissolve before adding other ingredients.

Trypticase soy broth Becton-Dickinson [BD]. Yeast extract Difco. Putrescine dihydrochloride.. Select Agar BD. Mix, add mixture to base agar, and adjust the pH to 6. Pour plates, invert after 2 h, and keep at room temperature overnight. Horse serum HyClone. GHL tripeptide solution CalbiochemNovabiochem. Ingredients useful for cultivation of and Preparation To prepare 1 liter, add the following ingredients to a flask.

Ultrapurified water Ingredients and Preparation To prepare 1 liter, add the following ingredients to a flask. Adjust the pH to 7. If agar is added, pour plates, invert after 2 h, and keep at room temperature overnight. Ingredients used M. Hayand sub- and Preparation To prepare 1 liter, add the following ingredients to a flask. Base for Broth Ultrapurified water Heart infusion broth BD. Heart infusion broth BD For Broth Heat-inactivated 56OC for 30 min fetal bovine or foal serum. 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. 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. If a full green color develops, only ureaplasmas are present. A blue-green color indicates the presence of ureaplasmas and M. Autoclave for 1.

Dissolve in a minimal volume of 1 N NaOH, and make up to volume with water. Supplements Prepare and filter sterilize 0. Sterile, pooled normal horse serum, preadjusted to pH 6. Greater acidity will reduce the ability of the medium to support growth. 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. Manassas, VA Phone: Website: http:llwww. However, correct coding does not guarantee reimbursement; tests must still be considered reasonable and necessary by the payer. For 24 Waites et al. 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. CPT-4 codes for Mycoplasma dures and clinical conditions discussed in the text. ICDCM codes are in the public domain and are updated regularly by cooperating parties. I Abele-Horn, M. Blendinger, C. Becher, I?. Emmerling, and G. Evaluation of commercial kits for quantitative identification and tests on antibiotic susceptibility of genital mycoplasmas.

Ainsworth, J. Hourshid, J. Clarke, D. Mitchell, J. Weber, and D. Detection of Mycoplasma fermentans in HIV-positive individuals undergoing bronchoscopy. IOM Lett. Alexander, T. Gray, J. Kraft, D. Leland, M. Nikaido, and D. Performance of Meridian ImmunoCard Mycoplasma test in a multicenter clinical tria1. Barker, C. Sillis, and T. Baseman, J. Mycoplasmas: sophisticated, reemerging, and burdened by their notoriety. Bebear, C. Determination of minimal inhibitory concentration, p. Tully and S. Razin ed. Academic Press, Inc. Bebear, H. Renaudin, and A. New developments in diagnosis and treatment of mycoplasma infections in humans.

Bove, C. Bebear, and J. Characterization of mutations involved in resistance to fluoroquinolones in MycopZasma hominis. Agents Chemother. Renaudin, A. Charron, J. Charron, H. Renaudin, B. Schaeverbeke, and C. Mutations in the gyrA, parC, and parE genes associated with fluoroquinolone resistance in clinical isolates of Mycoplasma hominis. Blanchard, A. Hentschel, L. Duffy, K. Baldus, and G. Detection of Ureaplasma ureaZyticum by polymerase chain reaction in the urogenital tract of adults, in amniotic fluid and in the respiratory tract of newborns.

AIDS-asso- ciated mycoplasmas. Broitman, N. Floyd, C. Johnson, L. Comparison of commercially available media for detection and isolation of Ureaplasma urealyticum and Mycoplasma hominis. Brown, M. Cassell, D. Taylor-Robinson, and M. Measurement of antibody to Ureaplasma urealyticum by an enzyme-linked immunosorbent assay and detection of antibody responses in patients with nongonococcal urethritis. Clin Microbial. Cassell, W. McCormack, and J.

Measurement of antibody to Mycoplasma hominis by an enzyme-linked immunosorbent assay and detection of antibody responses in women with postpartum fever. Cassell, G. Davis, K. Waites, I?. Marriott, M. Brown, and J. Isolation of Mycoplasma hominis and Ureaplasma urealyticum from amniotic fluid at weeks of gestation: potential effect on outcome of pregnancy.

Gambill, and L. ELISA in respiratory infections of humans, p. Waites, D. Crouse, P. Rudd, K. Canupp, S. Stagno, and G. Association of Ureaplasma urealyticum infection of the lower respiratory tract with chronic lung disease and death in very-low-birth-weight in infants. Lancet ii Waites, and D.

Mycoplasmal infections, p. Remington and J. Klein ed. The W. Saunders Co. Choppa, P. Vodjani, C. Tagle, R. Andrin, and L. Multiplex PCR for the detection of Mycoplasma fermentans, Probes An assessment of a new diagnostic indirect enzyme immunoassay for the detection of anti-MycopZasma pneumoniae IgM. Passey, M. Yoannes, and A. High rates of genital mycoplasma infections in the highlands of Papua New Guinea determined both by culture and a commercial detection kit.

Coombs, R. Easter, I?. Matejtschuk, and T. Fergusson, F. Davidson, A. Lyon, and I? Comparison of culture with the polymerase chain reaction for detection of Ureaplasma urealyticum in endotracheal aspirates of preterm infants. Berner-Poggi, F. Febrer, H.

Renaudin, M. Dupon, and C.

Fletcher's Media - For the cultivation of Leptospira spp.

Detection of Mycoplasma pneumoniae and Mycoplasma genitalium in clinical samples by polymerase chain reaction. Dupon, I?. Rodriguez, H. Renaudin, and C. Slike transposon carries the Tet M gene in tetracycline resistant strains of Bacteroides ureolyticus as well as Ureaplasma urealyticum but not Neisseria gonorrhoeae. Deguchi, T. Gilroy, and D. Failure to detect Mycoplasma fermentans, Mycoplasma penetrans, or Mycoplasma pirum in the urethra of patients with acute nongonococcal urethritis.

Ditty, S. Connolly, B. Li, and S. Mycoplasma orale has a sequence similar to the insertion-like sequence of M. Doble, A. Thomas, P. Furr, M. Walker, J. Harris, R. Witherow, and D. A search for infectious agents in chronic abacterial prostatitis utilizing ultrasound guided biopsy. Dosa, E. Nagy, W. Falk, I. Szoke, and U. Evaluation of the Etest for susceptibility testing of Mycoplasma hominis and Ureaplasma urealyticum.


Dowdle, W. An indirect hemagglutination test for diagnosis of Mycoplasma pneumoniae infections. Echevarria, J. Leon, P. Balfagon, J. Lopez, and M. Diagnosis of Mycoplasma pneumoniae infection by microparticle agglutination and antibody-capture enzyme-immunoassay. Fedorko, D. Emery, S. Franklin, and D. Evaluation of a rapid enzyme immunoassay system for serologic diagnosis of Mycoplasma pneumoniae infection. Foy, H. Infections caused by Mycoplasma pneumoniae and possible carrier state in different Mycoplasmal populations 1 Infections 27 Clin.

Freundt, E. Culture media for classic mycoplasmas. Methods Mycoplasmol. Furr, P. Microimmunofluorescence technique for detection of antibody to Mycoplasma genitalium. Longand ureaplas- D. Taylor-Robinson, and A. WebMycoplasmas and ureaplasmas in pahypogammagobulinaemia and their role microbiological observations over 20 Rheum. Gerstenecker, B.

Kahane and A. Adoni ed. Plenum Press, New York, N. Gilroy, C. Methods Granstriim, M. Holme, A. Sjogren, A. Kovacic, R. Griffais, V. Development of says for the detection of two human cies, Mycoplasma penetrans and M. Launay, and L. PCR-based asmollicute spehominis. Grenabo, L. Hedelin, and S. Urinary infection stones caused by Ureaplasma urealyticum: a review.

Puppe, A. Hoppe, I. Kiihne, J. Weidl, and H. Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study. Micro biol. Holme, and A. Human antibody response to the major adhesin of Mycoplasma pneumoniae: increase in titers against synthetic peptides in patients with pneumonia. APMIS Hirschberg, L. Krook, C. Pettersson, and T. Enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae specific immunoglobulin M.

Horner, P. Gilrov, B. Thomas, R. Naidoo, and D. Lancet Hussain, A. Robson, R. Kelley, T. Reid, and J. Mycoplasma penetrans and other mycoplasmas in urine of human immunodeficiency virus-positive children. Jacobs, E. Serological plasma pneumoniae infections: a critical review of current procedures. Vonski, G. Stemke, and J. Identification of Ureaplasma biotypes. Jalil, N. Doble, C. Gilchrist, and D.

Infection of the epididymis by Ureaplasma urealyticum. Jensen, J. Uldum, J. Sondergard-Andersen, J. Vuust, and K. Polymerase chain reaction for detection of Mycoplasma genitalium in clinical samples. Hansen, and K. Isolation of Mycoplasma genitalium strains from the male urethra. Kai, M. Kamiya, H. Yabe, I. Takakura, K. Shiozama, and A. Rapid detection of Mycoplasma pneumoniae in clinical samples by the polymerase chain reaction. Karppelin, M.

Hakkarainen, M. Kleemola, and A. Comparison of three serological methods for diagnosing Mycoplasma pneumoniae infection. Katseni, V. Gilroy, B. Ryait, K. Ariyoshi, I?. Bieniasz, J. Mycoplasma fermentans in individuals seropositive and seronegative for HIV-l. Kenny, G. Serodiagnosis, p.

Maniloff, R. McElhaney, L. Finch, and J. Baseman ed. American Society for Microbiology, Washington, D. Effect of pH, inoculum size, and incubation time on the susceptibility of Ureaplasma urealyticum to erythromytin in vitro. Eaton pleuropneumonia-like organism Mycoplasma pneumoniae complement-fixing antigen: extraction with organic solvents. Kaiser, M. Cooney, and H. Diagnosis of Mycoplasma pneumoniae pneumonia: sensitivities and specificities of serology with lipid antigen and isolation of the or- CUMITECH 34 ganism on soy peptone medium for identification infections.

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