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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
open this folder and view contentsBlood
open this folder and view contentsCerebrospinal fluid
open this folder and view contentsUrine
open this folder and view contentsStool
open this folder and view contentsLower respiratory tract infections
open this folder and view contentsUpper respiratory tract infections
close this folderSexually transmitted diseases
View the documentIntroduction
View the documentUrethritis in men
View the documentGenital specimens from women
View the documentSpecimens from genital ulcers
open this folder and view contentsPurulent exudates, wounds, and abscesses
open this folder and view contentsAnaerobic bacteriology
open this folder and view contentsAntimicrobial susceptibility testing
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover
 

Genital specimens from women

The vaginal flora of premenopausal women normally consists predominantly of lactobacilli, and of a wide variety of facultative aerobic and anaerobic bacteria.

Abnormal vaginal discharge may be due to:

 

• vaginitis: Trichomonas vaginalis, Candida albicans;
• bacterial vaginosis: overgrowth of anaerobes and Gardnerella vaginalis;
• cervicitis: Neisseria gonorrhoeae, Chlamydia trachomatis.

Other bacteria, such as Enterobacteriaceae, are not proven causes of vaginitis. Vaginitis in prepubertal girls may be due to N. gonorrhoeae or C. trachomatis.

Bacterial vaginosis (nonspecific vaginitis) is a condition characterized by an excessive, malodorous, vaginal discharge associated with a significant increase of Gardnerella vaginalis and various obligate anaerobes, and a decrease in the number of vaginal lactobacilli. A minimum diagnostic requirement for bacterial vaginosis is the presence of at least three of the following signs: abnormal vaginal discharge, vaginal pH > 4.5, clue cells (epithelial cells with so many bacteria attached that the cell border becomes obscured), and a fishy, amine-like odour when a drop of 10% potassium hydroxide is added to the vaginal secretions.

Urethritis in women is also often caused by N. gonorrhoeae and C. trachomatis.

Ascending infections with N. gonorrhoeae, C. trachomatis, vaginal anaerobes, and facultative anaerobic bacteria can cause pelvic inflammatory disease, with infertility or ectopic pregnancy as late sequelae.

Genital infections with bacterial agents, including N. gonorrhoeae and C. trachomatis, during pregnancy may result in complications such as premature delivery, prolonged rupture of membranes, chorio-amnionitis, and postpartum endometritis in the mother, and conjunctivitis, pneumonia, and amniotic infection syndrome in the newborn.

On special request, cervicovaginal specimens may be cultured for bacterial species, such as S. aureus (toxic shock syndrome), S. agalactiae (group B streptococci, neonatal infection), Listeria monocytogenes (neonatal infection), and Clostridium spp (septic abortion).

Although infections with C. trachomatis and with human herpesvirus are common, and can cause significant morbidity, their laboratory diagnosis requires expensive equipment and reagents and will not be discussed here.

Collection and transport of specimens

All specimens should be collected during a pelvic examination using a speculum. The speculum may be moistened with warm water before use, but antiseptics or gynaecological exploration cream should not be used, since these may be lethal to gonococci.

For examination for yeasts, T. vaginalis, and bacterial vaginosis, samples of vaginal discharge may be obtained with a swab from the posterior fornix of the vagina. Samples for gonococcal and chlamydial culture should be collected in the endocervix. After inserting the speculum, cervical mucus should be wiped off with a cotton wool ball. A sampling swab should then be introduced into the cervical canal and rotated for at least 10 seconds before withdrawal.

Urethral, anorectal, and oropharyngeal specimens for gonococci may be obtained in a similar manner as from males.

In all cases of pelvic inflammatory disease (PID), as a minimum, the cervix should be sampled for N. gonorrhoeae. Sampling from the fallopian tubes is more reliable, but in most areas a cul-de-sac aspirate is the best sample available.

In infants with ophthalmia neonatorum, conjunctival exudate should be collected with a swab or a loop.

Amies and Stuart transport media are convenient for transport of cervical and vaginal samples, with the exception of specimens to be tested for C. trachomatis.

Direct examination and interpretation

Direct examination of vaginal secretions is the method of choice for the etiological diagnosis of vaginitis, but is much less useful for the diagnosis of cervicitis.

A wet mount is prepared by mixing the vaginal sample with saline on a glass slide, after which a cover slip is added. A diluted preparation is preferred to ensure the separation of the cells, which may otherwise be clumped together. Examine at a magnification of x 400 for the presence of T. vaginalis with typical movement, budding yeasts, and clue cells. C. albicans may form pseudomycelia, which may be observed occasionally in vaginal material. Clue cells are found in most women with bacterial vaginosis. A granular or dirty appearance of the epithelial cell cytoplasm is a less objective criterion than the loss of the cell border. Microscopic examination of a wet mount of cervical specimens is not recommended.

Preparation of a Gram-stained smear is the method of choice for the diagnosis of bacterial vaginosis. The smear should be prepared by gently rolling, rather than smearing, a swab over the glass slide. A normal vaginal smear contains predominantly lactobacilli (large Gram-positive rods) and fewer than 5 leukocytes per field. In typical smears from women with bacterial vaginosis, clue cells covered with small Gram-negative rods are accompanied by a mixed flora consisting of very large numbers of small Gram-negative and Gram-variable rods and coccobacilli, and often Gram-negative curved rods, in the absence of larger Gram-positive rods. Only a few (<5) leukocytes are found per field. This picture is a sensitive and specific diagnostic indicator for bacterial vaginosis caused by G. vaginalis.

A large number of white blood cells (> 10 per field) on the Gram-stained vaginal smear suggests trichomoniasis or cervicitis.

Gram-staining is not particularly helpful for the diagnosis of gonococcal infection in female patients. The examination of Gram-stained smears of endocervical secretions for intracellular Gram-negative diplococci has a sensitivity of 50 - 70%, and a specificity of 50 - 90% for the diagnosis of gonococcal infection, resulting in a poor predictive value of a positive test in populations with a low prevalence of gonorrhoea. Gram-negative intracellular diplococci in cervical smears should be reported as such, and not as N. gonorrhoeae or gonococci. Over-interpretation of cervical smears, which often contain Gram-negative coccobacilli and bipolar stained rods, must be avoided.

The main interest in a cervical smear is its validity for the diagnosis of mucopurulent cervicitis: the presence of more than 10 polymorphonuclear leukocytes per oil-immersion field is a reasonably good indication of mucopurulent cervicitis, most often due to N. gonorrhoeae and/or C. trachomatis.

The examination of a Gram-stained conjunctival smear is a sensitive and specific technique for the diagnosis of gonococcal conjunctivitis. The presence of intracellular Gram-negative diplococci is diagnostic for gonococcal conjunctivitis.

Culture

Cervical, rectal, urethral, conjunctival, and cul-de-sac specimens may be cultured for N. gonorrhoeae using the methods specified on page 54. Specimens should be processed as soon as they arrive in the laboratory or, preferably, in the clinic itself. Unlike in males, culture is essential for the diagnosis of gonococcal infection in females. The sensitivity of a single cervical culture for the diagnosis of gonorrhoea in women is 80 - 90%. The sensitivity is lower for specimens taken during the peripartum period.

Cultures for G. vaginalis or anaerobes are not recommended for the diagnosis of bacterial vaginosis, since the organisms are recovered from 20 - 40% of women without vaginal infection. The presence of G. vaginalis in vaginal discharge is in itself not an indication for treatment, and only patients fulfilling the diagnostic criteria for bacterial vaginosis should be treated for this condition.

As compared with microscopy, cultures increase the detection of C. albicans by 50 - 100%. Culture methods are usually more efficient when the number of organisms is low. However, low numbers of C. albicans can be found in the vagina of 10 - 30% of women without signs or symptoms of vaginitis, and only large numbers of C. albicans should be considered as evidence of vaginal candidiasis. Consequently, culture is not recommended. Cultures for T. vaginalis will mainly detect asymptomatic carriers when performed in addition to a wet mount, and should not be performed.

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