Sexually Transmitted Disease Cultures

Definition

Sexually transmitted diseases are infections spread from person to person through sexual contact. A culture is a test in which a laboratory attempts to grow and identify the microorganism causing an infection. Laboratory culture is performed to isolate and identify the causes of several sexually transmitted infections.

Purpose

Sexually transmitted diseases (STDs) produce symptoms such as genital discharge, pain during urination, bleeding, pelvic pain, skin ulcers, or urethritis. Often, however, they produce no immediate symptoms. Therefore, the decision to test for these diseases must be based not only on the presence of symptoms, but on whether or not a person is at risk of having one or more of the diseases. Activities such as drug use and sex with more than one partner put a person at high risk for these diseases. STD cultures are necessary to diagnose certain types of STDs. Only after the infection is diagnosed can it be treated and further spread of the infection prevented. Left untreated, consequences of these diseases range from discomfort to infertility to death. In addition, these diseases in a pregnant woman can be passed from mother to fetus.

Precautions

Some infections, particularly gonorrhea, can be difficult to culture. It may be necessary to culture other sites that may be infected, such as the anus and mouth if the patient has corresponding sexual habits that may put him or her at risk. Also, health care workers should be aware that testing of anyone who mentions a sexual assault must be done very carefully, following a protocol which is usually best carried out in the emergency room. The physician, nurse, or physician assistant performing sample collection should observe universal precautions for the prevention of transmission of bloodborne pathogens.

Description

Gonorrhea, bacterial vaginosis, candidiasis, chancroid, chlamydiosis, herpes, and mycoplasma are common sexually transmitted diseases that can be cultured. The organisms that cause the first three conditions are cultured routinely while those that cause the last four are more difficult to grow and are more frequently identified immunologically or by DNA amplification. Syphilis, human immunodeficiency virus, and trichomoniasis are sexually transmitted diseases that usually are not cultured because they do not grow on artificial culture medium.

The female patient will be in the dorsal lithotomy position (lying on the back with legs raised and bent) typical for Pap testing. A speculum is moistened with warm water (no lubricant should be used) and inserted into the vagina to secure good visualization of the cervix. Any excess cervical mucous should be removed with a cotton ball (held by ring forceps). A sterile swab is inserted just inside the opening of the cervix (the os) and rotated gently for 30 seconds. Genital swabs are usually placed in a transport medium that contains charcoal to absorb toxins that inhibit the growth of gonococcus.

Care should be taken not to touch the vaginal surfaces with the swab in order to avoid the transfer of normal vaginal flora. For culture, the sample is placed in Stuart or Amies transport medium with charcoal added and delivered to the laboratory at room temperature. Since Neisseria gonorrhoeae are very sensitive to drying and temperature changes, plating is performed as soon as possible. For DNA probe or immunological testing (in which organisms are not cultured), the swab is broken off at the top of the sterile tube provided, and the tube is capped and sent to the laboratory. For immediate viewing, a swab sample may be placed in normal saline. One drop can then be placed between a slide and coverslip, and viewed beneath the microscope. This is called a "wet prep." A wet prep is useful for diagnosing yeast infection and trichomoniasis. Pelvic inflammatory disease samples and samples from genital lesions such as chancres are collected by aspiration. Plating for H. ducreyi should be done from the chancre aspirate and performed immediately because the organism is fastidious.

In the male patient, a smaller sterile swab is used to remove cells and any discharge from the last 0.75 inch (2 cm) of the urethra, and the swab is transported for culture (or DNA probe or immunological testing) as described for the female patient. If visible discharge is present on the surface of the penis, this should be swabbed, and it is unnecessary to enter the urethra. For anal specimens the physician inserts a sterile, cotton-tipped swab about 1 inch (2.5 cm) into the anus and rotates the swab for 30 seconds. Stool must not contaminate the swab. For oropharynx (throat) specimens the person's tongue is held down with a tongue depressor, as a healthcare worker moves a sterile, cotton-tipped swab across the back of the throat and tonsilar region.

Gonorrhea

Neisseria gonorrhoeae, also called gonococcus or GC, causes gonorrhea. It infects the mucosal surfaces of the genitourinary tract, primarily the urethra in males and the cervix in females. When seen on Gram stain, Neisseria gonorrhoeae are gram-negative diplococci (pairs of round or bean-shaped bacteria ) often located inside white blood cells. The best specimen from which to culture Neisseria gonorrhoeae is a swab of the urethra in a male or the cervix in a female. Other possible specimens include the mouth, anus, or a swab of a genital lesion. All swabs are plated on modified Thayer-Martin (MTM) agar or New York City (NYC) agar. These media are selective for the growth of N. gonorrhoeae. MTM is chocolate agar (heated sheep blood agar) containing colistin to inhibit the growth of gram negative bacilli, nystatin or anisomycin to inhibit yeast, vancomycin to inhibit growth of gram-positive bacteria, and trimethoprim to inhibit Proteus spp. NYC agar contains amphotericin B instead of nystatin and consists of clear proteosepeptone supplemented agar. In addition, the sample is plated on either 5% sheep blood agar or Columbia agar with 5% sheep blood and colistin and nalidixic acid (CNA) to isolate Candida albicans which causes a yeast infection in the vagina and Gardnerella vaginalis which causes vaginosis as well. Plates are incubated at 96.8°F (36°C). in 5-10% carbon dioxide. MTM or NYC agar are examined for growth at 24 hours and if negative again at 48 hours. After 24 hours, any suspicious colonies are Gram-stained and tested for oxidase which provides presumptive identification of Neisseria if positive. The physician can be notified at this point by a preliminary report that gonococcus has been identified presumptively. Further biochemical testing may be performed to differentiate N. gonorrhoeae from N. meningitides which is sometimes isolated from homosexual males. Isolated colonies should also be tested for penicillin resistance. Plates may be discarded at 48 hours if no growth is seen. Rapid nonculture DNA amplification and enzyme immunoassay tests are available to test for Neisseria gonorrhoeae and provide results on the same day.

Microscopic analysis should always be included with genital culture. Wet preparations can identify yeast, Trichomonas vaginalis, and G. vaginalis. The latter can be seen as rods attached to large squamous epithelial cells called "clue cells." A Gram stain of the swab material can identify gram-negative diplococci, which is presumptive evidence of gonococcal infection. In males, a positive finding on the Gram stain obviates the need for culture and the patient can begin antibiotic treatment. In females, the diplocicci must be located intracellularly in order to make a presumptive diagnosis of gonorrhea infection, and culture must be performed to confirm the diagnosis. The presence of clue cells, epithelial cells containing gram-negative or gram-variable coccobacilli, can signal the presence of Gardnerella vaginalis.

Chancroid

Chancroid is caused by Haemophilus ducreyi. It is characterized by genital ulcers with nearby swollen lymph nodes. The specimen is collected by swabbing one of these pus-filled ulcers. The Gram stain cannot differentiate Haemophilus ducreyi from other Haemophilus species. The physician must request a specific culture for a person who has symptoms of chancroid. Even using special culture, Haemophilus ducreyi is isolated from less than 80% of the ulcers it infects. If a culture is negative, the physician must diagnose chancroid based on the person's symptoms, and by ruling out other possible causes of these symptoms, such as syphilis (which is diagnosed by a blood test for antibodies).

H. ducreyi is fastidious and culture media should be inoculated within 10 minutes of sample collection. The swab should be spread over a chocolate (heated sheep blood agar) plate and incubated at 96.8°F (36°C) in 5-10% carbon dioxide. Isolated colonies are Gram-stained to identify the bacteria as small gram-negative bacilli, a colony is transferred to trypticase soy broth and a suspension is made. This is plated onto Mueller-Hinton or trypticase soy agar and strips of factor X (hemin) and factor V (NAD) are applied. Haemophilus ducreyi requires X factor but not V factor for growth. Like other Haemophilus species the organism is oxidase positive and reduces nitrate. Unlike most other Haemophilus species it does not produce catalase and does not ferment glucose, and these tests can be used for positive identification.

Mycoplasma and Ureaplasma

Three types of mycoplasmal organisms cause sexually transmitted disease: Mycoplasma hominis, Mycoplasma gentialium, and Ureaplasma urealyticum. M. hominis causes pelvic inflammatory disease (PID) and pyelonephritis in females but does not cause urethritis, vaginitis, or cervicitis. Ureaplasma urealyticum can cause urethritis in males and may cause PID in females but does not cause vaginitis or cervicitis. M. gentialium has been implicated as a cause of urethritis and PID. Samples are collected from the cervix in a female, and from the urethra (or urine) in a male. Swabs must be immediately placed in sucrose-phosphate or other acceptable transport medium and transported to the lab immediately. These organisms will grow on New York City agar and M. hominis will also grow on CNA plates, but swabs should be inoculated onto a selective agar or broth such as SP-4, which differentiates Mycoplasma from Ureaplasma based upon the ability of the latter to hydrolyze urea. Cultures are incubated aerobically at 96.8°F (36°C) and grow for two to four days. Colonies are very small and difficult to see with the unaided eye. When growth is seen, a portion of the agar is removed and stained with Dienes stain. The colonies are examined under a microscope for their characteristic fried egg appearance. They will have a dark blue center and light blue periphery. These organisms cannot be seen with the Gram stain.

Chlamydiosis

Chlamydiosis is caused by the gram-negative bacterium Chlamydia trachomatis. It is one of the most common STDs in the United States (approximately three million cases occur each year), and generally appears in sexually active adolescents and young adults. While chlamydiosis often does not have any initial symptoms, it can if left untreated lead to PID and sterility. Samples are collected from one or more of these infection sites: cervix in a female, urethra in a male, or the rectum. Swabs must be immediately placed in sucrose-phosphate or other acceptable transport medium and transported to the lab immediately. Culture is successful in recovering Chlamydia trachomatis about 80% of the time. The organism is inoculated onto monolayers of malignant tissue culture cells such as HeLa cells or McCoy cells in shell vials. The cultures are incubated for two to three days at 96.8°F (36°C) in 5-10% carbon dioxide. Following this they are stained with flourescent-labeled monoclonal antibodies to the major outer membrane protein (MOMP) to identify the characteristic chlamydial inclusions. This technique is expensive and requires a high level of tissue culture expertise. Consequently most labs use non-culture tests such as enzyme immunoassay or DNA amplication methods to diagnose chlamydial infections.

Genital herpes

Herpes is generally diagnosed based on the patient's symptoms and the physical exam. Approximately two-thirds of genital herpes is caused by herpes simplex 2 (HSV-2) and the remainder by herpes simplex 1 (HSV-1). Extremely painful blisters around the genital area are classic for initial herpes presentation. However, if questions remain, the herpes virus can be cultured from a vesicle (blister) which has been "unroofed" carefully with a scalpel blade. The base of the vesicle is swabbed with a sterile cotton applicator, and the virus taken to the laboratory in a tube of viral transport medium. Herpes can be cultured in several cell lines including human diploid fibroblasts (HDF), HEp2 cells (epithelial cancer cells from the larynx), primary monkey kidney cells (PMK), and rabbit kidney cells (RK). Cell cultures are inoculated and allowed to grow for one to three days at 96.8°F (36°C) in 5-10% carbon dioxide. Usually by the end of the first day of culture the cytopathic effect (CPE) can be seen by observing the cells under a microscope. Herpes induces the formation of giant cells.

Antibiotic susceptibility testing

Antibiotic susceptibility is not usually required for organisms isolated from a genital culture. Gonorrhea is treated with penicillin or related drugs. Chlamydiosis and mycoplasmal infections are treated with erythromycin. Herpes is treated with acyclovir or related antivirals. Candida is treated with clotrimazole or other antifungal. Bacterial vaginosis is treated with metronidazole, Haemophilus ducryi is treated with ceftriaxone or erythromycin.

For both male and female patients, urine tests for the DNA of Chlamydia trachomatis and Neisseria gonorrhoeae are available. In recent years, use of these nucleic acid amplification tests (NAATs) has increased, particularly for screening and where urethral and vervical culture samples are not possible because patients do not accept the procedure or because of logistics. These tests measure bacterial DNA that is amplified either by the ligase chain reaction (LCR) or the polymerase chain reaction (PCR). Both methods can detect the organisms within four hours, affording more rapid treatment. However, the tests do not detect any other genital tract pathogens that might be present in the patient.

Preparation

Cultures should always be collected before the person begins taking antibiotics. Men should not urinate within one hour before collection of a urethral specimen. Women should not douche or take a bath within 24 hours of collection of a cervical or vaginal culture.

Aftercare

Patients should be instructed to have no sexual contacts until test results are reported.

Complications

The minor discomforts of genital testing are short lived, and no significant complications are common.

Results

With the exception of Mycoplasma and Ureaplasma, these microorganisms are not found under normal conditions, so tests should be negative. M. hominis can be found in the male urethra and Ureaplasma urealyticum can be found in the female genital tract in the absence of disease. Therefore positive cultures for these organisms may indicate colonization without infection and the physician must differentiate these conditions on the basis of the physical examination and symptoms. Therefore, these organisms are treated at the discretion of the physician. If a person has a positive culture for any other of these microorganisms, antibiotic treatment is started and his or her sexual partners should be notified and tested. After treatment is completed, the physician may request a follow-up culture to confirm that the infection is cured.

Health care team roles

Genital cultures are ordered by a physician and collected by a physician, nurse, or physician assistant. Culture, microscopic analysis, immunoassay, and DNA testing are performed by clinical laboratory scientists/medical technologists. Wet preparations may also be performed by the physician or physician assistant or nurse practitioner with appropriate training. Nursing staff have a very important task in educating the patient in what to expect, assisting with obtaining samples, and helping to explain test results to patients. Many patients undergoing genital testing are in need of counseling regarding the risks of careless sexual behavior, and the opportunity should be used by staff for education to reduce risks in the future.

Resources

BOOKS

Fishbach, Frances Talaska. A Manual of Laboratory and Diagnostic Tests, 6th ed. Lippincott: 2000: 563-565.

Malarky, Louise M., and Mary Ellen McMorrow. Nurses Manual of Laboratory Tests and Diagnostic Procedures. WB Saunders Co., 2000: 177-179.

Tierney, Lawrence M., Stephen J. McPhee and Maxine A. Papadakis. Current Medical Diagnosis and Treatment 2001. Lange Medical Books/McGraw-Hill, 2001: 1309.

PERIODICALS

Campos-Outcalt, Doug. "Sexually Transmitted Disease: Easier Screening Tests, Single-dose Therapies." The Journal of Family Practice (December 2003): 965-969.

ORGANIZATIONS

Centers for Disease Control and Prevention. National Center for HIV, STD, and TB Prevention. 1600 Clifton Road NE, Atlanta, GA 30333. (404) 639-8000. 〈http://www.cdc.gov/nchstp/od/nchstp.html〉.