SPECIMEN COLLECTION AND TRANSPORT

 The pathogenic Neisseria spp. described in this chapter are very sensitive to drying and temperature extremes. there are some special requirements for isolation of N. gonorrhoeae and N. meningitidis.

Swabs are acceptable for N. gonorrhoeae testing if the specimen will be plated within 6 hours; however, reduced recovery may result within 30 minutes of collection. If cotton swabs are used, the transport medium should contain charcoal (Ames medium) to inhibit toxic fatty acids present in the fibers. Calcium alginate has also been found to be inhibitory. Dacron or rayon fibers are recommended. N. gonorrhoeae should be inoculated to growth media immediately after specimen collection. The sample should then be placed in a container able to sustain an atmosphere of increased carbon dioxide (CO₂) during transport. Specially packaged media consisting of selective agar in plastic trays that contain a CO₂-generating system are commercially available (JEMBEC plates). The JEMBEC system (Figure1) is transported to the laboratory at room temperature. Upon receipt in the laboratory, the agar surface is cross-streaked to obtain isolated colonies, and the plate is incubated at 35°C in 3% to 5% CO₂. Additional commercial transport systems that may be useful, particularly when the collection site is separate from the diagnostic laboratory, are the Bio-Bag, Gono-Pak, and Transgrow.

Fig1. EMBEC system. Plate contains modified Thayer Martin medium. The CO₂-generating tablet is composed of sodium  bicarbonate and citric acid. After inoculation the tablet is placed in  the well, and the plate is closed and placed in the zip-lock plastic  pouch. The moisture in the agar activates the tablet, generating a  CO₂ atmosphere in the pouch.

Recovery of N. gonorrhoeae or N. meningitidis from normally sterile body fluids requires no special methods, except for blood cultures. Both organisms are sensitive to sodium polyanethol sulfonate (SPS), the preservative typically found in blood culture broths. If a blood culture broth is inoculated, the SPS content should not exceed 0.025%. In addition, if blood is first collected in Vacutainer tubes containing SPS (Becton Dickinson, Sparks, Maryland), the specimen must be transferred to the broth culture system within 1 hour of collection.

Nasopharyngeal swabs collected to detect N. meningitidis carriers should be plated immediately to the JEMBEC system, or they should be submitted on swabs placed in charcoal transport media.

SPECIMEN PROCESSING

 The JEMBEC system should be incubated at 35° to 37°C as soon as the plate is received in the laboratory. Body fluids (e.g., joint or cerebrospinal fluid [CSF]) should be stored until cultured at 37°C, because both gonococci and meningococci are sensitive to cold.

Any volume of clear fluid greater than 1 mL suspected of containing either of these pathogens should be centrifuged at room temperature at 1500× g for 15 minutes. The supernatant fluid should then be removed and the sediment should be vortexed and inoculated onto the appropriate media.

Any specimens or cultures in which N. meningitidis is a consideration should be handled in a biological safety cabinet to avoid laboratory-acquired infections.

DIRECT DETECTION METHODS

 Gram Stain

 The members of the genus Neisseria discussed in this chapter and M. catarrhalis appear as gram-negative diplococci (Figure 2) with adjacent sides flattened. They are often referred to as “kidney bean”–shaped diplo cocci. Direct Gram staining of urethral discharge from symptomatic males with urethritis is an important test for gonococcal disease. The appearance of gram-negative diplococci inside polymorphonuclear leukocytes is diagnostic in this situation. However, because the normal vaginal and rectal flora are composed of gram-negative coccobacilli, which can resemble Neisseria spp., direct examination of endocervical secretions in symptomatic women is only presumptive evidence of gonorrhea, and the diagnosis must be confirmed by culture. In addition, avirulent strains (i.e., pili types 3 to 5) may be present as extracellular diplococci; these are not pathogenic. Pharyngeal specimens should not be Gram stained, because nonpathogenic, commensal Neisseria spp. may be present, and these are not diagnostic of infection.

Fig2. Gram stain of Neisseria gonorrhoeae showing gram negative diplococci (arrows). 

The direct Gram stain of body fluids for either N. gonorrhoeae or N. meningitidis is best accomplished using a cytocentrifuge, which can concentrate small numbers of organisms 100-fold.

Commercial Molecular Assays

 Molecular assays have replaced old enzyme-linked immunosorbent assay systems for rapid diagnosis of N. gonorrhoeae. The U.S. Food and Drug Administration (FDA) has cleared a number of amplified and nonamplified tests. The nonamplified DNA probe assay, PACE 2 (Hologic, Inc., Bedford, MA), has a chemiluminescent detection system for direct detection of gonococcal ribosomal RNA (rRNA) in genital and conjunctival specimens. This test performs well in high-risk patients, is rapid (results are available in 2 hours), and is suitable for screening many patients simultaneously. The Gen-Probe Accuprobe test targets rRNA after lysis of bacteria; the rRNA is detected using a single-strand chemiluminescent DNA probe. The hybrids are then detected in a luminometer. In addition, the Digene CT/GC Dual ID HC2 (HC2; Qiagen) detects RNA-DNA hybrids using antibody mediated recognition of the hybrids and visualization of a chemiluminescent substrate.

Amplified assays, which are more sensitive than the nonamplified assay, are commercially available from Roche Diagnostic Systems. They include the AMPLICOR and COBAS AMPLICOR PCR (Branchburg, New Jersey) and the Hologic Gen-Probe Aptima Combo 2 transcription-mediated amplification (Bedford, MA) and other tests produced by other manufacturers. The Pro-beTec ET (Becton Dickinson, Sparks, Maryland) also is available. These tests are suitable for large-scale screening programs, but none are admissible as evidence in medicolegal cases. An advantage of all molecular assays is the ability to test for Chlamydia trachomatis from the same specimen at the same time. N. gonorrhoeae DNA can be found in a specimen for up to 3 weeks after successful treatment, so amplified molecular assays should not be used to assess cure.

Molecular assays have also been developed to detect N. meningitidis. Sequence-based typing methods combined with serologic typing are currently recommended. Molecular targets for identification include PCR and sequencing of a variety of genes, including PorA, PorB, FetA (associated with PorA), global housekeeping genes, penA (penicillin susceptibility) and Factor H binding protein.

Following the manufacturer’s recommendations is important in evaluating molecular diagnostic tests to identify Neisseria spp. Some of the assays have limitations with regard to the type of specimen that may be used, cross-reactivity with nonpathogenic species, and assay inhibition and false-negative results caused by substances present in patient samples.

Antigen Detection

The detection of Neisseria meningitidis capsular polysaccharide antigen in body fluids (e.g., urine, serum, CSF) is no longer recommended in the United States.

CULTIVATION

 Media of Choice

 N. meningitidis, M. catarrhalis, and saprophytic Neisseria spp. grow well on 5% sheep blood and chocolate agars. N. gonorrhoeae is more fastidious and requires an enriched chocolate agar for growth on primary culture. Because gonococci and sometimes meningococci must be isolated from sites that contain large numbers of normal flora (e.g., genital tract or upper respiratory tract), selective media have been developed to facilitate their recovery. The first of these was Thayer-Martin medium, a chocolate agar with an enrichment supplement (IsoVi taleX) and the antimicrobials colistin (to inhibit gram negative bacilli), nystatin (to inhibit yeast), and vancomycin (to inhibit gram-positive bacteria). This original medium was subsequently modified to include trimethoprim (to inhibit swarming Proteus spp.), and its name was changed to modified Thayer-Martin medium (MTM). Martin-Lewis (ML) medium is similar to MTM except that anisomycin, an antifungal agent, is substituted for nystatin and the concentration of vancomycin is increased. GC-LECT agar is a selective medium that contains additional antimicrobials to inhibit bacteria found in oropharyngeal specimens; it includes vancomycin and lincomycin (to inhibit gram-positive bacteria), colistin (to inhibit gram-negative bacteria), amphotericin B (to inhibit yeast), and trimethoprim (to inhibit swarming Proteus spp. and Capnocytophaga spp.).

New York City (NYC) medium, a transparent medium containing lysed horse blood, horse plasma, yeast dialysate, and the same antibiotics as MTM, also has been used. The advantage of NYC medium is that genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum) also grow on this agar. Some strains of N. gonorrhoeae are inhibited by the concentration of vancomycin in the selective media, so the addition of nonselective chocolate agar is recommended, especially in suspect cases that are culture negative or for sterile specimens (e.g., joint fluid).

Unlike the pathogenic species, some of the saprophytic Neisseria spp. (N. flavescens, N. mucosa, N. sicca, and N. subflava) may grow on MacConkey agar, although poorly. N. gonorrhoeae and N. meningitidis will grow in most broth blood culture media but grow poorly in common nutrient broths such as thioglycollate and brain-heart infusion. M. catarrhalis and the other Neisseria spp. grow well in almost any broth medium.

Incubation Conditions and Duration

 Agar plates should be incubated at 35° to 37°C for 72 hours in a CO2-enriched, humid atmosphere. N. gonorrhoeae, N. meningitidis, and M. catarrhalis grow best under conditions of increased CO2 (3% to 7%). This atmosphere can be achieved using a candle jar, CO2-generating pouch, or CO2 incubator. Only white, unscented candles should be used in candle jars, because other types may be toxic to N. gonorrhoeae and N. meningitidis.

Humidity can be provided by placing a pan with water in the bottom of a CO₂ incubator or by placing a sterile gauze pad soaked with sterile water in the bottom of a candle jar (Figure 3).

Fig3. Candle jar. 

Colonial Appearance

 Table 1 describes the colonial appearance and other distinguishing characteristics (e.g., pigment) of M. catarrhalis and the Neisseria spp. on chocolate agar.

Table1. Colonial Appearance and Other Characteristics on  Chocolate Agar*

APPROACH TO IDENTIFICATION

Various commercial systems are available for the rapid identification of the coccoid Neisseria spp. and M. catarrhalis. These systems employ biochemical or enzymatic substrates and work very well for the pathogenic species (N. gonorrhoeae, N. meningitidis, and M. catarrhalis). A heavy inoculum of the organism is required, but because these systems detect the activity of preformed enzymes, viability of the organisms in the inoculum is not essential. Manufacturers’ instructions should be followed exactly; several systems have been developed only for strains isolated on selective media and should not be used to test other gram-negative diplococci.

Biochemical Identification

Table 2 presents some conventional biochemical tests that traditionally have been used to identify these organ isms definitively. The extent to which identification of isolates is carried out depends on the source of the specimen and the suspected species of the organism involved.

Table2. Biochemical and Physiologic Characteristics of Moraxella catarrhalis and Coccoid Neisseria spp.

An isolate from a child or a person involved in a case of sexual abuse must be identified unequivocally because of the medicolegal ramifications of these results. It is recommended that these organisms be identified using at least two different types of tests; that is, biochemical, immunologic, enzymatic, or the nonamplified DNA probe previously discussed. Isolates from normally sterile body fluids should also be completely identified. However, isolates from genital sites in adults at risk of sexually transmitted disease (STD) can be identified presumptively; that is, oxidase-positive, gram-negative diplococci that grow on gonococcal selective agar. Likewise, an oxidase-positive, gram-negative diplococcus that hydrolyzes tributyrin from an eye or ear culture can be identified as M. catarrhalis.

Comments About Specific Organisms

 Determination of carbohydrate utilization patterns historically has been performed in cysteine trypticase soy agar (CTA) with 1% dextrose, maltose, lactose, and sucrose. This medium is no longer widely used, because it does not work well for oxidative Neisseria spp., specifically N. gonorrhoeae and N. meningitidis. Therefore, carbohydrate utilization patterns are currently determined by inoculating an extremely heavy suspension of the organism to be tested in a small volume of buffered, low-peptone substrate with the appropriate carbohydrate. These methods do not require subculture or growth, and results are available in approximately 4 hours. Commercially available methods include the Rim-Neisseria Test (Rapid Identification Method–Neisseria) (Remel Laboratories), the Neisseria Kwik Test (Micro-Biologics) and the Gonobio Test (I.A.F Production).

The saprophytic Neisseria spp. are not routinely identified in the clinical laboratory. N. cinerea may be misidentified as N. gonorrhoeae if the isolate produces a weak positive glucose reaction. However, it grows on nutrient agar at 35°C, whereas the gonococcus does not. Moreover, N. cinerea is inhibited by colistin, whereas N. gonorrhoeae is not.

M. catarrhalis can be differentiated from the gonococci and meningococci based on its growth on blood agar at 22°C and on nutrient agar at 35°C, the reduction of nitrate to nitrite, its inability to utilize carbohydrates, and its production of DNase. M. catarrhalis is the only member of this group of organisms that hydrolyzes DNA.

Chromogenic substrate enzyme tests for beta galactosidase, gamma-glutamyl aminopeptidase, and prolyl-hydroxylprolyl aminopeptidase are available for the differentiation of N. gonorrhoeae, N. meningitidis, N. lactamica, and M. catarrhalis. M. catarrhalis lacks all three of these enzymes. The presence of prolyl-hydroxylprolyl aminopeptidase alone identifies an organism as N. gonorrhoeae. The presence of beta-galactosidase and gamma glutamyl aminopeptidase indicates N. meningitidis. Two commercial chromogenic substrate kits are the Gono check II (EY Laboratories, San Mateo, California) and BactiCard Neisseria (Remel Laboratories, Lenexa, Kansas). A limitation of these methods is misidentification of various nonpathogenic strains of Neisseria spp. In addition, isolate colonies on selective media should be used to avoid misidentification of contaminants as a Neisseria spp. Modified chromogenic substrate kits, such as the BactiCard Neisseria, can be used to identify and speciate Neisseria and Haemophilus organisms from selective and nonselective media. These modified tests use a com bination of enzyme substrate tests and additional bio chemical tests.

N. lactamica may grow on selective media and may be confused with N. meningitidis. The ONPG test is used to determine an organism’s ability to produce beta-galactosidase, which is an indicator of lactose utilization. N. lactamica is ONPG positive, and N. gonorrhoeae is ONPG negative.

The eugonic fermenter N. animaloris propagates well on routine laboratory media and ferments glucose; this distinguishes it from dysgonic fermenters that grow poorly on blood and chocolate agars. N. animaloris ferments no carbohydrates other than glucose and is indole negative and arginine dihydrolase positive.

Immunoserologic Identification

 Particle agglutination methods are available for immunoserologic identification of N. gonorrhoeae. They include the Phadebact GC OMNI test (Karo Bio Diagnostics AB, Huddinge, Sweden), the MicroTrak Culture Confirmation test (Trinity Biotech, Bray, Ireland), and the GonoGen II test (Becton Dickinson, Sparks, Maryland). These tests can be performed from colonies growing on primary plates; isolates are typed with specific monoclonal anti bodies. The Phadebact GC OMNI test is a coagglutination assay that contains inactivated Staphylococcus aureus cells coated with antibodies to staphylococcal protein A via the Fc region. The GonoGen II is a colorimetric test using antibodies adsorbed to metal sol particles. The MicroTrak assay uses fluorescein isothiocyanate-labeled antibodies (FITC) for confirmation of N. gonorrhoeae using a fluorescent microscope.

Serotyping

Twelve different serogroups are distinguishable for N. meningitidis. Antisera are commercially available for identifying N. meningitidis serogroups A, B, C, H, I , K, L, X, Y, Z, W135, and 29E. Serologic identification is usually performed by slide agglutination. A, B, C, Y, and W135 are the serotypes that most frequently cause systemic disease in the United States. Serotyping has been replaced in many laboratories by DNA sequence typing methods related to the hypervariable outer membrane proteins. Information is available at http://neisseria.org.

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مواضيع ذات صلة

Antimicrobial Susceptibility Testing and Therapy of Neisseria and Moraxella catarrhalis

Spirillum Minus

Streptobacillus moniliformis

Myositis and Myonecrosis

Necrotizing Fasciitis

Cellulitis

Laboratory Diagnosis of Francisella

Laboratory Diagnosis of Bordetella pertussis, Bordetella parapertussis, and Related Species

Epidemiology and Pathogenesis of Bordetella pertussis, Bordetella parapertussis, and Related Species

Group B Streptococcal Disease

Streptococcal Toxic Shock Syndrome

Signs and Symptoms of Streptococcal Infections