Clinical Laboratory - Microbiology

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Microbiologic Testing

Genital Tract infections

Symptoms of urethritis in a sexually active person should raise the suspicion for a sexually transmitted disease. However, some persons may be asymptomatic. The two most likely agents are Neisseria gonorrheae and Chlamydia trachomatis. Though the latter is more frequent, the former is more likely to be symptomatic. Other common sexually transmitted diseases (STDs) include Syphillis (Treponema pallidum), Herpes simplex virus (HSV 1 and 2), and Human papillomavirus (HPV).

Collection and culture of organisms associated with STDs can be challenging. Routine gram-stain and culture can be difficult to perform or negative. The preferred test for Chlamydia trachomatis and Neisseria gonorrhoeae, the two most common STDs, is by Transcription-Mediated Amplification (TMA).

Neisseria gonorrheae

A gram stain of the urethral exudate can detect the characteristic gram-negative intracellular diplococci of Neisseria. There are usually many neutrophils present. If a culture is obtained, a swab should be inoculated directly onto gonococcal selective media such as modified Thayer-Martin media. The samples should be inoculated and transported immediately to the lab, because the gonococcal organisms dry out readily and are sensitive to oxygen. N. gonorrheae may be detected in the sample by nucleic acid amplification testing.

Chlamydia trachomatis

Chlamydia culture is difficult. Therefore, diagnosis is usually made by obtaining a swab from the putative source of infection and testing fluid obtained from the swab for antigens to C. trachomatis with a serologic assay, such as an ELISA test, or nucleic acid amplification test (NAAT).

Syphillis (Treponema pallidum)

Diagnostic testing is determined by the length of time for the infection, as primary (weeks), secondary (months), or tertiary (years to decades). A diagnostic test for syphillis is done by scraping of a primary lesion and darkfield microscopy, but this test is raarely done outside of an STD clinic. More often, serologic testing is the mainstay of syphilis diagnosis. The serologic tests for syphilis (STS) include the rapid plasma reagin (RPR) screening test. The venereal disease research laboratory (VDRL) test can also be used for screening, particularly on cerebrospinal fluid. Since false positives occur at least 1% of the time, particularly with underlying infections or immunologic diseases, confirmatory tests are employed. The most common confirmatory tests are the fluorescent treponemal antibody-absorbed (FTA-ABS) test and the agglutination assays for antibodies to T. pallidum (MHA-TP, or TP-PA).

Herpes simplex virus (HSV)

The cytopathic effect of HSV can aid in diagnosis. A scraping of the epithelium in the region of the vesicle or ulcer can be performed, and the cells examined cytologically for the appearance of intranuclear inclusions. Immunohistochemical staining can confirm that HSV is present in the inclusions.

Serologic assays by ELISA of a blood sample for antibodies to HSV types I and II can be performed. A positive test does not prove that the acute infection is due to HSV, since many persons have had past infection and, therefore, circulating antibodies. Cross reactivity between HSV I and II is common, so distinguishing the two is not always possible. Tests for IgG and IgM antibodies can be done to establish diagnosis of acute infection, with IgM being prominent in acute infection.

Human papillomavirus (HPV)

HPV is associated with squamous epithelial dysplasias, carcinomas, and condyloma accuminatum. HPV can be sub-typed into over a hundred types, with HPV types 16 and 18 having the greatest potential for producing cervical squamous dysplasias and carcinomas. HPV types 6 and 11 are most often linked to condyloma accuminatum in the anogenital region. A vaccine is available that is polyvalent for these four types.

HPV cytopathic effects are diagnosed cytologically in Pap smears and in biopsies. Lesions may begin as low-grade squamous intraepithelial lesions (LSIL), also classified as cervical intraepithelial neoplasia I (CINI). Over time such lesions can progress to high-grade squamous epithelial lesions (HSIL), which encompass both CINII and CINIII marked by more marked and more extensive dysplastic changes. HSIL, if not treated, may progress to invasive cervical carcinoma. Thus, Pap smear screening is important for prevention of cervical cancer.

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