Pediatric Annals

Laboratory Evaluation of Sexually Transmitted Diseases

Mychelle Y Farmer, MD; Edward W Hook, III, MD; Felix P Heald, MD

Abstract

During the last 20 years, there has been a dramatic increase in our knowledge of sexually transmitted diseases (STDs) and their consequences. Although gonorrhea rates have declined by 17% in the US, over 900,000 cases were reported in 1984. Age specific gonorrhea rates reveal that 26% of all reported gonorrhea occurs in people less than 19 years old, with women in this group sustaining the highest morbidity from this infection.1 In addition, other STDs such as condyloma, herpes, and HTLV-III infections, which were previously unfamiliar to the pediatrician, are now discussed as important health concerns for sexually active adolescents.

The diagnosis and management of STDs have also changed in the last decade. The 1985 Treatment Guidelines of the Centers for Disease Control2 reflect the changes in management but do not address laboratory procedures helpful in determining appropriate diagnosis.

A number of relatively simple office laboratory procedures are essential to the initial management of a patient with a (possible) sexually transmitted disease. Many pediatricians see adolescent patients for whom an STD evaluation becomes necessary. The purpose of this article is to review commonly used office laboratory procedures that may aid in the diagnosis and differentiation of sexually transmitted diseases. These tests may be critical in the initial management of a patient, when results of cultures and more specialized laboratory tests are still unavailable.

SPECIMEN COLLECTION FROM THE FEMALE PATIENT

In women, an adequate pelvic examination is the first step in obtaining appropriate samples for office laboratory procedures. The vagina must be inspected carefully, and a sample of any vaginal discharge can be obtained using a cotton swab. A single swab of the vaginal discharge can be used for the KOH and the wet preps.

There are several methods used for the wet prep, but the preferred method is to place a swab containing vaginal secretions in a test tube containing a small amount of normal saline. This method optimizes one's ability to identify motile organisms such as trichomonas. One can also prepare a wet prep by applying a vaginal swab to a glass slide on thich a small amount of normal saline has been placed. Such preparations must be evaluated immediately, because they dry out quickly.

The KOH preparation can be prepared by placing a portion of the vaginal secretions on a glass slide then applying several drops of 10% KOH on the slide. This technique is also used for the whiff test, which helps to diagnose bacterial vaginosis (formerly called nonspecific vaginitis or NSV). The initial cervical sample is utilized for gonorrhea culture. Studies have shown that an adequate gonorrhea culture may be obtained without prior removal of any cervical secretions.3 A second endocervical sample is used for the pap smear. The exocervical component of the pap smear is obtained using the Ayre wooden spatula. Checklist 1 outlines a suggested order of cervical sampling in the sexually transmitted disease evaluation.

SPECIMEN COLLECTION FROM MALE PATIENTS

Urethral specimens for culture are usually obtained with a calcium atginate swab; however, a small plastic loop can be used for this purpose in men with urethral discharge. In male patients without discharge but with symptoms of dysuria or pain, a calcium alginate swab is necessary for accurate culture and Gram's stain. Urinalysts in males may be a useful STD screening procedure when urethritis is suspected. Several studies have confirmed that the presence of pyuria correlates with urethral culture for gonorrhea and chlamydia.4'8 One study of adolescent males showed that more than 10 white blood cells per high-power field (WBC/hpf) in urine sediment of the first voided fraction correctly identified 91% (21 out of 23)…

During the last 20 years, there has been a dramatic increase in our knowledge of sexually transmitted diseases (STDs) and their consequences. Although gonorrhea rates have declined by 17% in the US, over 900,000 cases were reported in 1984. Age specific gonorrhea rates reveal that 26% of all reported gonorrhea occurs in people less than 19 years old, with women in this group sustaining the highest morbidity from this infection.1 In addition, other STDs such as condyloma, herpes, and HTLV-III infections, which were previously unfamiliar to the pediatrician, are now discussed as important health concerns for sexually active adolescents.

The diagnosis and management of STDs have also changed in the last decade. The 1985 Treatment Guidelines of the Centers for Disease Control2 reflect the changes in management but do not address laboratory procedures helpful in determining appropriate diagnosis.

A number of relatively simple office laboratory procedures are essential to the initial management of a patient with a (possible) sexually transmitted disease. Many pediatricians see adolescent patients for whom an STD evaluation becomes necessary. The purpose of this article is to review commonly used office laboratory procedures that may aid in the diagnosis and differentiation of sexually transmitted diseases. These tests may be critical in the initial management of a patient, when results of cultures and more specialized laboratory tests are still unavailable.

SPECIMEN COLLECTION FROM THE FEMALE PATIENT

In women, an adequate pelvic examination is the first step in obtaining appropriate samples for office laboratory procedures. The vagina must be inspected carefully, and a sample of any vaginal discharge can be obtained using a cotton swab. A single swab of the vaginal discharge can be used for the KOH and the wet preps.

There are several methods used for the wet prep, but the preferred method is to place a swab containing vaginal secretions in a test tube containing a small amount of normal saline. This method optimizes one's ability to identify motile organisms such as trichomonas. One can also prepare a wet prep by applying a vaginal swab to a glass slide on thich a small amount of normal saline has been placed. Such preparations must be evaluated immediately, because they dry out quickly.

The KOH preparation can be prepared by placing a portion of the vaginal secretions on a glass slide then applying several drops of 10% KOH on the slide. This technique is also used for the whiff test, which helps to diagnose bacterial vaginosis (formerly called nonspecific vaginitis or NSV). The initial cervical sample is utilized for gonorrhea culture. Studies have shown that an adequate gonorrhea culture may be obtained without prior removal of any cervical secretions.3 A second endocervical sample is used for the pap smear. The exocervical component of the pap smear is obtained using the Ayre wooden spatula. Checklist 1 outlines a suggested order of cervical sampling in the sexually transmitted disease evaluation.

SPECIMEN COLLECTION FROM MALE PATIENTS

Urethral specimens for culture are usually obtained with a calcium atginate swab; however, a small plastic loop can be used for this purpose in men with urethral discharge. In male patients without discharge but with symptoms of dysuria or pain, a calcium alginate swab is necessary for accurate culture and Gram's stain. Urinalysts in males may be a useful STD screening procedure when urethritis is suspected. Several studies have confirmed that the presence of pyuria correlates with urethral culture for gonorrhea and chlamydia.4'8 One study of adolescent males showed that more than 10 white blood cells per high-power field (WBC/hpf) in urine sediment of the first voided fraction correctly identified 91% (21 out of 23) of patients who were culture-positive for chlamydia or gonorrhea, thus giving this test a 95% sensitivity and a 93% specificity.*

EVALUATION OF SPECIMENS

The wet prep is essential for the evaluation of a patient with vaginal discharge. Most forms of vaginitis can be diagnosed using a wet prep, although it is most useful in conjunction with other laboratory tests such as KOH preps, vaginal pH, and the Pap smear.

Trichomonas vaginitis is commonly diagnosed using the wet prep. These organisms may be visualized using the low- (10x ) or the high-power dry (40x ) objectives (Figure 1). The wet prep has a sensitivity of only 75% when compared to culture. A Pap smear diagnoses 80% of all Trichomonas infections, it is therefore a good non-culture technique to diagnose Trichomonas.

Figure 1. Trichomatis vaginalis. as pictured above, demonstrates the difficulty in distinguishing the immotile organisms from WBCs and epithelial cells, which are also included in this slide. The presence of trichomonads is confirmed by visualization of moving, flagellated organisms.

Figure 1. Trichomatis vaginalis. as pictured above, demonstrates the difficulty in distinguishing the immotile organisms from WBCs and epithelial cells, which are also included in this slide. The presence of trichomonads is confirmed by visualization of moving, flagellated organisms.

Figure 2. The clue cell in this photograph is easily identified on normal saline wet mounts by its irregular cell borders and the coarse appearance of the cytoplasm. The presence of four or more clue cells per high power field suggests the presence of bacterial vaginosis.

Figure 2. The clue cell in this photograph is easily identified on normal saline wet mounts by its irregular cell borders and the coarse appearance of the cytoplasm. The presence of four or more clue cells per high power field suggests the presence of bacterial vaginosis.

The clue cell, a characteristic feature of bacterial vaginosis, is also identified by the wet prep. The clue cell is a vaginal epithelial cell whose borders and cytoplasm are distorted by the adherence of multiple bacterial organisms. This gives the epithelial cell a lacy, granular appearance with an irregular border (Figure 2).

Frequently, many WBCs are present on the wet prep of patients with vaginitis. This finding is nonspecific and is not diagnostic for any single condition. In fact, there are noninfectious causes of vaginal leukorrhea.

Figure 3. Candida appears as branched hyphae with buds on the KOH preparation of vaginal secretions in patients having candidai infections.

Figure 3. Candida appears as branched hyphae with buds on the KOH preparation of vaginal secretions in patients having candidai infections.

KOH Preps of the Vagina

One of the primary usages of the KOH prep is to identify the presence of candidai species. This test is performed by applying a few drops of KOH to a sample of vaginal secretions that have been placed on a glass slide. The buds and hypae of a yeast infection are seen by low and high magnifications on the microscope. KOH lyses the epithelial cells, which enhances the visualization of the Candida (Figure 3). By heating the KOH preparation, the epithelial cell lysis is further enhanced and visualization of the candidai forms is improved. The KOH prep is also used to perform the "whiff test" for bacterial vaginosis (NSV). It is one of four office procedures used to determine the presence of bacterial vaginosis.9'11 This test involves smelling the slide immediately after the KOH has been applied to the vaginal secretions. The presence of a "fishy" odor is considered to be a positive whiff test. This fishlike odor is caused by release of aromatic amines, like putrescine and cadaverine, from the vaginal secretions of patients with bacterial vaginosis.12·13 Checklist 2 gives additional office laboratory procedures used for the diagnosis of bacterial vaginosis.

Gram's Stain

The Gram's stain has long been recognized as an essential office laboratory procedure that rapidly provides a wealth of valuable information regarding an infection. It is a simple procedure (see above), but some common errors must be avoided in order to optimize one 's assessment of the stain. First, the smear should be prepared using a thin coating of the material to be stained. This can be accomplished by using the plastic loops for obtaining a sample from the male urethra or by rolling the endocervical swab gently over the glass slide. A thick smear is a common error that impairs the slide's interpretation.

The slide should be air-dried, then fixed by heating. Failure to do so may cause specimen distortion or destruction. Underdecolorization is perhaps the most common error that renders the Gram's stain uninterpretable.

There are several methods used to perform Gram's stain. A rapid and longer version are provided at left.

Urethral Gram*s Stain. The urethral Gram's stain is essential to office management of urethritis when culture results are pending. A properly prepared Gram's stain has a sensitivity of 95% and a specificity of 97% in symptomatic men with gonorrhea urethritis.14 The diagnosis of urethritis is confirmed by the presence of five or more WBC/hpf on Gram's stain.15 Gonococcal urethritis is distinguished from nongonococcal urethritis by the presence of gram -negati ve intracellular diplococci (Figure 4). In cases of an early urethritis, these microorganisms may not be easily visualized, but in 95% of cases there is correlation of Gram's stain and culture results.

Figure 4. AJ Gonorrhea appears as gram-negative intracellular diplococci in the urethral Gram's stain of male patients with gonococcai urethritis. B) Nongonococcal urethritis is diagnosed by visualizing five or more WBCs per high power field on urethral Gram's stain.

Figure 4. AJ Gonorrhea appears as gram-negative intracellular diplococci in the urethral Gram's stain of male patients with gonococcai urethritis. B) Nongonococcal urethritis is diagnosed by visualizing five or more WBCs per high power field on urethral Gram's stain.

Figure 5. The cervical Grams stain suggestive of mucopurulent cervicitis will typically include pink-stained cervical mucus and ten or more WBCs per high power field.

Figure 5. The cervical Grams stain suggestive of mucopurulent cervicitis will typically include pink-stained cervical mucus and ten or more WBCs per high power field.

Figure 6. The Gram's stain of the clue cell has the typical appearance of the cell featured above. Gram negative pleomorphic organisms surround the cell membrane, thus obscuring its borders and the cytoplasm.

Figure 6. The Gram's stain of the clue cell has the typical appearance of the cell featured above. Gram negative pleomorphic organisms surround the cell membrane, thus obscuring its borders and the cytoplasm.

Gram's Stain of the Vagina. Although the vaginal to the urethral Gram's stain is the cervical Gram's stain. While practitioners infrequently use the cervical Gram's stain, studies have shown that it can be a helpful procedure in the evaluation of a patient with cervicitis. The cervical Gram's stain is most useful for identifying patients with mucopurulent cervicitis. This condition is defined as a cervical infection with the presence of a mucopurulent discharge from the cervix and greater than 10 WBC/hpf on cervical Gram's stain. Studies have shown that cervical Gram's stain showing greater than 10 WBC/hpf correlates most closely with chlamydial cervicitis.15 One series revealed a mean of 30 WBCs/hpf in endocervical secretions obtained from patients with chlamydial infections (Figure 5).

The cervical Gram's stain may also be used to detect gonorrhea cervicitis. Several early studies showed poor correlation of cervical Gram's stain and cervical culture, 16'17 although others have shown more encouraging results.18-20 Like the male urethral Gram's stain, the cervical Gram's stain with polys containing gramnegative diplococci suggests the presence of gonorrhea. One study shows that Gram's stain has a specificity of 97% and a sensitivity of 70%, when used in an STD clinic setting.21

Gram's Stain of the Vagina. Although the vaginal Gram's stain is also useful in the evaluation of a female patient for STDs, the wet prep is an excellent means of diagnosing bacterial vaginosis. The vaginal Gram's stain may provide supplemental information in patients with suspected vaginitis. The clue cell is also easily identified by Gram's stain (Figure 6). The appearance of this epithelial cell with gram-negative pleomorphic organisms supports this diagnosis. Candida! species are also identified on Gram's stain, and they ate generally seen as septated hyphae. Buds are more difficult to identify but also can be seen. They may be large, circular, or oval shaped organisms. Fortyseven percent of patients with candida can be identified by Gram's stain.22

Certain conditions are not identified by the vaginal Gram's stain. Trichomonads are poorly visualized by Gram's stain, thus it is not recommended for diagnosing this condition. The vaginal flora may normally contain neisserial species; thus, vaginal secretions are not helpful in the evaluation of patients for gonorrhea.

Inspection of the pubic region is an important component of the STD evaluation. Should one suspect pediculosis pubis, adult lice and eggs (nits) may be grossly visible. A magnifying glass may also aid in the search for pubic lice if the lice are not visible with the naked eye. In patients with multiple papules but without its visible lice, the diagnosis of pediculosis may be confirmed by applying oil to the intact lesion and then scraping with a sterile scalpel blade. The oil and scrapings can be examined under a microscope following application of a cover slip to a glass slide.

Other tests have also been used successfully to diagnose pediculosis. They include the Burrow ink test23 and needle excision of the mite.

Tzanck Preparations. Patients with vesicular or ulcerative lesions suggestive of genital herpes should be cultured to confirm the clinical diagnosis. Herpes cultures are performed using cotton or Dacron-tipped swabs. While this test is expensive, it is the best one available to diagnose herpes. Other widely used tests are not sensitive enough to replace culture.

Tzanck preparation is an office procedure that can be used to give rapid information regarding the presence of herpes. The intact vesicle gives the best results when performing this test. The lesions are opened and drained. Subsequently, the blunt edge of a scalpel blade is used to scrape the base of the lesions, and these scrapings are placed on a glass slide. The Tzanck test is performed by placing the air-dried slide in Giemsa or Wright's stain for 60 seconds. A positive slide contains mult inucleated giant cells.

Papanicolaou smears are also helpful in diagnosing herpes cervicitis. Herpes tends to affect squamous cells; thus, the ectocervix is most likely to be affected. Both multinuclear giant cells and nuclear inclusions having a ground glass appearance are seen by this technique. The Papanicolaou smear carries a sensitivity of 57% and the Tzanck preparation a sensitivity of 30%, based on one study. Thus, a large number of patients will be misdiagnosed if culture is not obtained.

These office procedures offer the possibility of diagnosing genital herpes infections using rapid, inexpensive staining techniques. However, these procedures are not sensitive tests when used on patients with ulcerated herpetic lesions.

Rapid Diagnostic Tests

Although the new rapid diagnostic tests generally require specialized laboratory conditions that are not available in an office laboratory, many practitioners may have access to such tests in the near future. Since some of these tests can provide valuable information in a short period, practitioners should know some of the basic principles operative in the laboratory technique for each rapid diagnostic test.

An enzyme immunoassay has been developed for the detection of gonorrhea. This test takes approximately 5 hours to complete. It is performed on urethra! or cervical discharge and is based on the direct detection of gonorrhea antigens within such specimens.

The gonorrhea enzyme immunoassay test has a high sensitivity and specificity in male patients with urethritis (99% and 92.8%, respectively) but performs less well in the female population (88% sensitivity and 89% specificity).24 This test is useful in populations with a high prevalence of gonorrhea when rapid (same-day) diagnosis is desired. However, it should not be used to replace cultures of gonorrhea because of the potential for false-positive results.

Immunofluorescent techniques using monoclonal antibodies have been a significant innovation as a nonculture method of detecting the presence of such STDs as Chlamydia trachomatis. These antibodies are specific for a protein on the outer membrane of Chianrydia and is a highly specific antibody for this organism. Thus, antibodies to Chlamydia are used to identify the presence of chiamydial elementary bodies in cervical or urethral samples. If the urethral sample contains chiamydial elementary bodies, the fluorescein conjugated antibodies will adhere to the slide. They will appear as green fluorescent particles under fluorescent microscopy. The detection of chlamydia by monoclonal antibodies offers some advantages over culture. This test can be completed in 1 hour, so results can be obtained the same day that the patient is seen. It is less expensive than culture and can be more widely used because of its lower cost. The monoclonal antibody test may improve one's ability to detect Chlamydia in patients living in remote areas.25 If culture cannot be processed within 48 hours, the sensitivity of culture appears to be significantly compromised. In such instances, the monoclonal antibody tests are preferred. This immunofluorescent non-culture technique has a sensitivity of 93% and a specificity of 96% when compared to culture.26

Chiamydial enzyme immunoassays have also been developed as an additional means of rapid diagnosis. This test takes 4 hours to complete; thus, same-day diagnosis of a chiamydial infection is possible with this new procedure as well. Studies indicate that this enzyme assay has a similar sensitivity and specificity to the direct monoclonal antibody test.27

Because these tests offer rapid results, they may become a helpful addition to STD evaluation in adolescent patients. They may be particulatly helpful in the assessment of the female patient for whom few accurate clinical or laboratory criteria are available for rapid diagnosis of cervicitis.

REFERENCES

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2. Centers for Disease Control: Treatment GtadeUnes. Atlanta, CDC, 1985.

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4. Adget H. Shafer MA, et al: Screening for Chlamydia cracnamatu and Neaifria fpntmkea in adolescent males: Values of fiisr-catch urine examination. Lancet 1984; ii:1944.

5. Holmes K, Hansfcld H, et al: Etiology oí nongonococcal urethritis. N Eng! i Med 1975; 292:1199.

6. Bowie W: Comparison of Gr am s stain and first-voided urine sediment in the diagnosis of urethritis. Sci Transm Dis 1978, 5:39.

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9. Pheifer T, et al: Nonspecific vaginitis: Role of H. vagmalis and treatment of metronidazole, N Engl J Med 1978; 298:1429.

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11 . Amsel R. Tinten P, et al: Nonspecific vaginitis: Diagnostic criteria and microbial and epidemiologie associations. AmJ Med 198); 74.14.

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13. Chen K, Amsel R. et al: Biochemical diagnosis of vaginitis: Determination of diamines in vaginal fluid. ; infect Da 1982; 145:3)7.

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17. Pederson A, Btinm P: Screening females for asymptomatic S0110TbCa infection. Nordwest;v Med April 1971; 255.

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19. Wald E: Gonorrhea diagnosis by Gram's stain in the female adolescent. AmJ Dis Child 1977;131:1094.

20. Judson F: A clinic-based system lor monitoring the quality of techniques for the diagnosis of gonorrhea- Sex Transm Du 1978; 5:141.

21. Lossick J, Smeltzer, Curran J; The value ni the cervical Gram's srain in (he diagnosis and treatment of gonorrhea in women in a venereal disease clinic. Sei Transm Du 1982; 9:124.

22. Oriel D: Genital yeast infections. Br MedJ 1972; 4:761.

23. Woodley D, Sautai JH: The Burrow ink test and the scabies mite. J Am Acmi Dermiuu!3 1981; 4:715.

24. Nasello MA, étal: A solid phase enzyme immunoassay (Gonoiyme) test for detection of Neisseria gonorrhea antigen m urogenital specimens from patients at a sexually transmitted disease clinic. Sii Tronsm Dis 1985; 12:198.

25. Williams T: Identifie aiiiin of (.',hlamydia irac/KHnuiis by direct immunofluurescence applied in specimens originating in remote areas. J CIm Microbio! 1985; 22:1053.

26. Tamm M. et al: Culture independent diagnosis of Chlamtdut irachommu using monoclonal antibodies. N Engf } Med 1984; 310:1146.

27. Alget L, Lovchik J, Farmer M: Diagnosis of cervical Chtamydia tróchameos colonization by rwu direct specimen tests. Abstract presented ai District 4 American College of Obstetrics and Gynecology. San Juan, Puerto Rico. November 1986.

10.3928/0090-4481-19861001-10

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