Urethritis Cervicitis and Vaginitis

Specimen Collection. This discussion focuses only on those specimens submitted for culture and/or direct examination. Procedures for the collection and transport of specimens for detection of agents by other non-cultural methods (e.g., detection of Chlamydia tracho-

matis by amplification) should be followed according to the respective manufacturer's instructions. Refer to Table 5-1 for a review of collection, transport, and processing of genital tract specimens.

Urethral. Urethral discharge may occur in both males and females infected with pathogens such as Neisseria gonorrhoeae and Trichomonas vaginalis. The presence of infection is more likely to be asymptomatic in females because the discharge is usually less profuse and may be masked by normal vaginal secretions. Urea-plasma ureafyticum can also be isolated from male urethral discharge.

A urogenital swab designed expressly for collection of such specimens should be used. These swabs are made of cotton or rayon that has been treated with charcoal to adsorb material toxic to gonococci and wrapped tightly over one end of a thin wire shaft. Cotton- or rayon-tipped swabs on a thin wire may also be used to collect specimens for isolation of mycoplasmas and chlamydiae. Calcium alginate swabs are generally more toxic for HSV, gonococci, chlamydiae, and mycoplasmas than are treated cotton swabs. Because Dacron swabs are least toxic, they are recommended for viral specimens. Dacron-tipped swabs on plastic shafts are also acceptable for chlamydiae and genital mycoplasmas.

To obtain a urethral specimen, a swab is inserted approximately 2 cm into the urethra and rotated gently before withdrawing. Because chlamydiae are intracellular pathogens, it is important to remove epithelial cells (with the swab) from the urethral mucosa. Separate swabs for cultivation of gonococci, chlamydiae, and ureaplasma are required. When profuse urethral discharge is present, particularly in males, the discharge may be collected externally without inserting a sampling device into the urethra. However, a urethral swab for chlamydia must be collected on males. A few drops of first-voided mine has also been used successfully to detect gonococci in males.

Because T. vaginalis may be present in urethral discharge, material for culture should be collected by swab as just described and another specimen collected on a swab and placed into a tube containing 0.5 mL of sterile physiologic saline. This specimen should be hand delivered to the laboratory immediately. Direct wet mounts and cultures for T. vaginalis can be performed from this second specimen. Commercial media for culture of Trichomonas are available. The first few drops of voided urine may also be a suitable specimen for recovery of Trichomonas from infected males, if it is inoculated into culture media immediately. Alternatively, material may be smeared onto a slide for later performance of a fluorescent antibody stain. Plastic envelopes for direct examination and subsequent culture are also available

Besmeared Materials The Room

Figure 58-4 InFouch TV diagnostic system for wet mount examination and culture of Trichomonas vaginalis. The swab collected from the patient is inserted into liquid medium in the upper chamber of the plastic pouch and swirled. The top of the pouch is folded over and then sealed with the tabs. Once received in the laboratory, the upper chamber is examined for motile organisms; if no motile organisms are seen by microscopy, the upper chamber material is inoculated into the lower chamber and then incubated for up to 5 days. The lower chamber is similarly examined daily by microscopy for the presence of motile T. vaginalis. (Coiutesy BioMed Diagnostics, White City, Ore.)

Figure 58-4 InFouch TV diagnostic system for wet mount examination and culture of Trichomonas vaginalis. The swab collected from the patient is inserted into liquid medium in the upper chamber of the plastic pouch and swirled. The top of the pouch is folded over and then sealed with the tabs. Once received in the laboratory, the upper chamber is examined for motile organisms; if no motile organisms are seen by microscopy, the upper chamber material is inoculated into the lower chamber and then incubated for up to 5 days. The lower chamber is similarly examined daily by microscopy for the presence of motile T. vaginalis. (Coiutesy BioMed Diagnostics, White City, Ore.)

(InPouch TV, BIOMED, White City, Ore); sensitivity of this system is superior to other available methods and organism viability is maintained up to 48 hours (Figure 58-4). In addition, several other techniques are available including enzyme immunoassay, latex agglutination tests, and the Affirm VPm probe (Becton Dickinson, Cockeysville, Md); polymerase chain reaction (PCR) has also been used to detect T. vaginalis directly in clinical specimens.

Cervical/Vaginal. Organisms that cause purulent vaginal discharge (vaginitis) include T. vaginalis, gonococci, and, rarely, beta-hemolytic streptococci. The same organisms that cause purulent infections in the urethra may also infect the epithelial cells in the cervical opening (os), as can HSV. Mucus is removed by gendy rubbing the area with a cotton ball. The urethral swab just described is

Vaginal Discharge Epithelial

Figure 58-5 Gram-negative intracellular diplococd, which are diagnostic for gonorrhea in urethral discharge and presumptive for gonorrhea in vaginal discharge.

inserted into the cervical canal and rotated and moved from side to side for 30 seconds before removaL

Swabs are handled as described for urethral swabs for isolation of Trichomonas and gonococci. Chlamydiae cause a mucopurulent cervicitis with discharge. Endo-cervical specimens are obtained after the cervix has been exposed with a speculum, which allows visualization of vaginal and cervical architecture, and after ectocervical mucus has been adequately removed. The speculum is moistened with warm water, because many lubricants contain antibacterial agents. Because normal vaginal secretions contain great quantities of bacteria, care must be taken to avoid or minimize contaminating swabs for culture by contact with these secretions. A small, nylon-brisded cytology brush, or cytobrush, may be used to ensure that cellular material is collected, but its use is associated with discomfort and bleeding. Some controversy exists over whether the cytobrush results in better specimens, at least for detection of Chlamydia trachomatis?

In addition to cervical specimens, which are particularly useful for isolating herpes, gonococci, mycoplasmas, and chlamydiae, vaginal discharge specimens may be collected. Organisms likely to cause vaginal discharge include Trichomonas, yeast, and the agents of BV. Swabs for diagnosis of BV are dipped into the fluid that collects in the posterior fornix of the vagina.

Genital tract infections caused by sexually transmitted agents in children (preadolescents) are most often the result of sexual abuse. Because of medico-legal implications, the laboratory should treat specimens from such patients with extreme care, carefully identifying and documenting all isolates. At the time of this writing, cultures should be obtained, especially for Chlamydia trachomatis, because amplification detection methods are still not admissible evidence in most states.

Because it is impossible to exclude contamination with vaginal flora, obtaining swabs of Bartholin gland exudate are not recommended. Infected Bartholin glands should be aspirated with needle and syringe after careful skin preparation, and cultures should be evaluated for anaerobes and aerobes.

Transport. Swabs collected for isolation of gonococci may be transported to the laboratory in modified Stuart's or Amie's charcoal transport media and held at room temperature until inoculated to culture media. Good recovery of gonococci is possible if swabs are cultured within 12 hours of collection. Material that must be held longer than 12 hours should be inoculated directly to one of the commercial systems designed for recovery of gonococci, described later in this chapter.

Swabs for isolation of chlamydiae and mycoplasmas are best transported in specific transport media that have antibiotics and other essential components. Specimens for chlamydiae culture should be transported on ice. (Specimens transported at room temperature should be inoculated within 15 minutes of collection.) Specimens can be stored at 4° C for up to 24 hours. If culture inoculation will be delayed more than 24 hours, specimens should be quick-frozen in a dry ice and 95% ethanol bath and stored at -70° C until cultured. If collected and transported in specific transport media, specimens for genital mycoplasma culture may be transported on ice or at room temperature. If not in genital mycoplasma transport media, specimens should be transported on ice to suppress the growth of contaminating flora.

Direct Microscopic Examination. In addition to culture, urethral discharge may be examined by Gram stain for the presence of gram-negative intracellular diplococci (Figure 58-5), usually indicative of gonor

Clue Cells
Figure 58-6 Clue cells ill vaginal discharge suggestive of bacterial vaginosis.

rhea in males. After inoculation to culture media, the swab is rolled over the surface of a glass slide, covering an area of at least 1 cm2. If the Gram stain is characteristic, cultures of urethral discharge need not be performed. Urethral smears from females may also be examined. However, presumptive diagnosis of gonorrhea from these smears is reliable only if the microscopist is experienced, because normal vaginal flora, such as Veillonella or occasional gram-negative coccobadlli, may resemble gonococci. If extracellular organisms resembling N. gonorrhoeae are seen, the microscopist should continue to examine the smear for intracellular diplococci. Presumptive diagnosis can be useful when decisions are to be made regarding immediate therapy, but confirmatory cultures or an alternative nonculture method should always be performed on specimens from females. Some strains of N. gonorrhoeae are sensitive to the amount of vancomycin present in selective media. If suspicious organisms seen on smear fail to grow in culture, reculture on chocolate agar without antibiotics may be warranted.

Fluorescein-conjugated monoclonal antibody reagents are sensitive and specific for visualization of the inclusions of Chlamydia trachomatis in cell cultures or elementary bodies in urethral and cervical specimens containing cells. Reagents for direct staining of specimens are available commercially in complete collection and test systems, but the relatively greater technologist time required for this method limits its usefulness for laboratories that receive many specimens, except as a confirmatory test for other antigen detection systems with borderline results. In some studies, the sensitivity of visual detection of chlamydiae with these newer reagents has been similar to that of culture, although such comparative results are obtained only by techno logists experienced in fluorescent techniques and when the slides are examined thoroughly. False-positive results should not occur if at least 10 morphologically compatible fluorescing elementary bodies are seen on the entire smear. No direct visual methods exist for detection of mycoplasmas at this time, but molecular assays have been evaluated.

Direct microscopic examination of a wet preparation of vaginal discharge provides the simplest rapid diagnostic test for Trichomonas vaginalis when such a specimen is available and can be examined immediately. The plastic envelope method combines direct visualization with culture. Motile trophozoites of Trichomonas can be visualized in a routine wet preparation performed by a proficient technologist in two thirds of cases or a direct fluorescent antibody (DBA) stain (Meridian Diagnostics, Cincinnati, Ohio) may be used.

Budding cells and pseudohyphae of yeast can also be easily identified in wet preparations by adding 10% potassium hydroxide (KOH) to a separate preparation, thereby dissolving host cell protein and enhancing the visibility of fungal elements.

BV, characterized by a foul-smelling discharge, can be diagnosed microscopically or clinically. The discharge is primarily sloughed epithelial cells, many of which are completely covered by tiny, gram-variable rods and coccobadlli. These cells are called due cells (Figure 58-6). The absence of inflammatory cells in the vaginal discharge is another sign of BV. Although Gardnerella vaginalis has been historically assodated with the syndrome and can be cultured on a human blood bilayer plate, culture is not recommended for diagnosis of BV.13 A clinical diagnosis of BV is best made using three or more of the following criteria: homogeneous, gray discharge; due cells seen on wet mount or Gram stain; a pH greater

Figure 58-7 A, Predominance of Lactobacilli in Gram stain from healthy vagina. B, Absence of lactobacilli and presence of Gardnerella vaginalis (A arrows) and Mobiluncus spp. (B arrows) morphologies.

than 4.5; and an amine or fishy odor elicited by the addition of a drop of 10% KOH to the discharge on a slide or on the speculum.12

Probably the best way to differentiate BV from other vaginal infections is by Gram stain (Figure 58-7). Nugent and colleagues has developed a grading system for Gram stains of vaginal discharge (Procedure 58-1).8 This system is based on the presence or absence of certain bacterial morphologies. Typically, in patients with BV, lactobacilli are either absent or few in number, whereas curved, gram-variable rods (Mobiluncus spp.) and/or G. vaginalis and Bacteroides moiphotypes predominate. The Gram stain is more sensitive and specific than either the wet mount for detection of clue cells or culture for G. vaginalis, and the smear can be saved and reexamined later.

Culture. Samples for isolation of gonococci may be inoculated direcdy to culture media, obviating the need for transport medium. Commercially produced systems have been developed for this puipose, and many clinicians inoculate standard plates direcdy if convenient access to an incubator is avadable. Modified Thayer-Martin medium is most often used, although New York City (NYC) medium has the added advantage of supporting the growth of mycoplasmas and gonococci. Excellent recovery of gonococci is the rule when specimens are inoculated direcdy to any of these media in self-contained incubadon systems such as JEMBEC plates (Figure 58-8). The specimen swab containing material is rolled across the agar with constant turning to expose all surfaces to the medium. The JEMBEC plate, which generates its own increased carbon dioxide atmosphere by means of a sodium bicarbonate tablet, is inoculated ip a W pattern. The plate may be cross-streaked with a sterile loop in the laboratory (Figure 58-9).

Figure 58-8 JEMBEC plate containing modified Thayer-Martin medium in a plastic, snap-top box with a self-contained C02-generating tablet, all sealed inside a Zip-lock plastic envelope after inoculation.

Specimens must be inoculated to additional media for isolation of yeast, streptococci, and mycoplasmas. Yeast grows well on Columbia agar base with 5% sheep blood and colistin and nalidixic acid (CNA), although more selective media are available. Most yeast and streptococci also grow on standard blood agar; thus, adding special fungal media such as Sabouraud brain-heart infusion agar (SABHI) is unwarranted.

A specimen from the lower vagina followed by the rectum using the same swab at 35 to 37 weeks' gestation reliably predicts the presence of group B streptococci at delivery.11 The swab should be transported to the laboratory in a nonnutritive transport medium such as Amies or Stuart's without charcoal and then inoculated into a recommended selective broth medium such as Todd-Hewitt broth supplemented with either gentami-dn and nalidixic acid or with colistin and nalidixic acid.

Urethritis
Figure 58-S Method of cross-streaking JBMBEC plate after original specimen has been inoculated by rolling the swab over the surface of the agar in a W pattern.

Selective enrichment broths are subcultured to agar the next day to isolate and identify group B streptococci. In addition, the presence of group B streptococci in urine in any concentration from a pregnant woman is a marker for heavy genital tract colonization.11 Thus, any quantity of group B streptococci in urine from pregnant women should be worked up in the laboratory (see Chapter 57).

T. vaginalis may be cultured in Diamond's medium (available commercially) or plastic envelopes inoculated with discharge material. Culture techniques are most sensitive. A commercially available biphasic genital mycoplasma culture system (Mycotrim-GU, Irvine Scientific, Santa Ana, Calif) can be used to culture Mycoplasma hominis and Ureaplasma ureatyticum, although commercially prepared media are not as sensitive as fresh media.18 Mycoplasma genitalium may not grow on commercial media because of the presence of thallium acetate.14

Nonculture Methods. Various noncuhure methods may be used to diagnose genital tract diseases, including serology, latex agglutination, nucleic acid hybridization and amplification assays, and enzyme immunoassays. Most assays detect a single or possibly two genital tract pathogens, and most are commercially available.6,15 These methods are described in more detail in chapters relating to individual pathogens.

As previously discussed, BV involves several organisms. Besides the Gram stain, BV can be diagnosed by using the Amsel criteria, which include pH measurement, performance of an amine test, and wet mount microscopy of vaginal secretions.12 However, this approach has been considered unreliable because of the lack of microscopy-related skills and availability of pH paper in most doctors' offices.112 Although the Gram stain offers high sensitivity and specificity, it is not immediately available. Currendy, commercial laboratory tests are available to aid in the diagnosis of BV but are not all available in the United States; a test for sialidase (OSOM BVBLUE, Genzyme Diagnostics, Cambridge, Mass) in conjunction with measuring pH has been reported to be a rapid, highly sensitive and specific means to diagnose BV.12 (Sialidases are secreted from anaerobic gram-negative rods such as Bacteroides and Prevotetta as well as Gardnerella and play a role in bacterial nutrition, cellular interactions, and immune response evasion, which in turn improves the ability of bacteria to adhere, invade, and destroy mucosal tissue.) Of note, a hybridization assay (Affirm VP in Microbial Identification Test; Becton Dickinson Microbiology Systems, Sparks, Md) is commercially available to diagnose BV, as well as genital tract infections caused by Candida spp. and Trichomonas vaginalis. Once the appropriate reagents and specimen are added to spécial trays, the entire hybridization assays are then performed by instrumentation (Figure 58-10). Evaluations indicate this system is sensitive and specific.

Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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