Diagnosis Of Trichomoniasis By Wet Mount

Trichomoniasis is a sexually transmitted disease caused by the protozoan organism Trichomonas vaginalis (T. vaginlis). It is the most common sexually transmitted pathogen accounting for 180 million infections annually.

The classical symptoms associated with the T. vaginalis infection include a yellowish-green frothy discharge; pruritus, dysuria, and the “strawberry” cervix which is characterized by punctuate hemorrhagic lesions. In Saudi Arabia, out of 39049 sexually transmitted infections reported to the ministry of health during 1995-1999, trichomoniasis infections comprised 28.1%. T. vaginalis is site specific for the genitourinary tract and has been isolated from virtually all genitourinary structures. Asymptomatic disease is common in both men and women, thus screening for disease is important. Women and men are infected with comparable frequency, but in men symptoms are normally mild and infections are cleared by the host’s immune system within weeks. In women, trichomonaisis infections can persist for many years, and symptoms.

Diagnosis cannot be made solely on the basis of clinical presentation for several reasons as the clinical symptom may resemble those of other STDs and frothy discharge is seen only in 12% of women with T. vaginalis. Various laboratory methods have been employed for the detection of T. vaginalis in vaginal discharge which varies in their sensitivity and specificity. In this respect, culture is still considered the “gold standard” for diagnosis of trichomoniasis. The wet mount preparation of vaginal discharge has been the most commonly rapid method used for diagnosis of trichomoniasis in women as it enables detection of viable, motile trophozoites. However, the sensitivity of the wet mount declines substantially with time delays between collection of samples and examination.

In set up lacking immediate microscopic facilities, staining techniques like Giemsa and Gram`s stains are very useful because prepared and fixed smears can be transported to the laboratory for diagnosis without affecting the reliability for diagnosis.

The study population subjects were women in Jazan area who have clinical manifestations suggestive of trichomoniasis. We used the non-probability sampling method to select the study subjects (the available responders to participate in the study). Each woman was asked to fill a questionnaire covering the personal data, clinical symptoms, marital status and obstetric and gynecological history. Vaginal swab samples were obtained from women fulfilling the following inclusion and exclusion criteria using sterile vaginal swabs provided to the patient. Inclusion criteria: ü Female married patient ≥18 years old. ü Complaining of one or more trichomoniasis symptoms (itching, yellowish or green color vaginal discharge, foul smell vaginal discharge, lower abdominal colic, pain in urination, pain in intercourse).

Sample collection: Vaginal secretions were collected using patients` self-obtained vaginal swabs according to Van Der Pol et al. The method of obtaining vaginal secretions was explained to each patient. The patient was informed to insert a single swab into the vagina and to rotate the swab three times. Each swab was suspended in 2 ml of 0.85% normal saline supplemented with two drops of 5% glucose. Each sample was labeled using a serial number and was quickly transported to the college parasitology laboratory for complete testing. Before preparation, the tubes with the swabs were vigorously shaken to displace all the parasites into the saline solution.

Procedures

Each vaginal specimen was subjected to the following:

Wet mount Examination

A drop of saline with suspended vaginal secretions was placed on a 22 × 40 glass slide and covered with a cover slip. The preparation was examined microscopically for motile T. vaginalis under × 10 and × 40 objectives. The vaginal secretion was characterized by the presence of the squamous epithelial cells while the trichomonads were identified by their size (10-20 μm), round or oval shape, and characteristic quivering or twitching motility.

Giemsa staining

A drop of saline with vaginal secretions was smeared on a 22 × 40 glass slide and allowed to air- dry then fixed by immersion in methanol for one minute. The slides were stained with Giemsa diluted 1:9 with phosphate buffer solution (pH 7.2) for 10 min. Slides were washed carefully with running tap water and air dried. Each slide was scanned for parasites at 10 × 100 magnification. At least 30 fields were examined before a negative finding was recorded. Trophozoite nucleus is round or oval, the flagella and the undulating membrane may appear.

Gram's staining

The vaginal smear was prepared and fixed as for the Giemsa staining then subjected to gram`s staining method as follows:

1. Flood the crystal violet for one minute.
2. Pour off excess dye and wash gently in tap water and drain the slide against a paper towel.
3. Expose the smears to Gram's iodine for one minute by washing with iodine, then adding more iodine and leaving it on the smear until the minute is over.
4. Wash with tap water and drain carefully.
5. Wash with 95% ethanol. Wash with tap water at the end of the 30 seconds to stop the decolorization.
6. Counterstain with 0.25% safranin for 30 seconds.
7. Wash, drain, blot, and examine under microscope. Under the microscope T. vaginalis trophozoites appears as pear shape with a round or oval nucleus, the flagella and the undulating membrane may appear and axostyle can be seen, the cytoplasm may be lacy (21).

D-Modified thioglycolate culture

I- Preparation of Modified thioglycolate culture medium: The medium is used at a concentration of 29.5g in every liter of distilled water. Dispense the well mixed medium in 10 ml amounts in glass tubes fitted with screw-caps. Sterilize by autoclaving (with caps loosened) at 121°C for 15 minutes. When cool, tighten the bottle caps. Cover each bottle top with a foil cap and Store in a cool dark place (refrigerator). When used, take the media from the refrigerator and let it take the temperature of the room after adding horse serum, Amphotericin B, Penicillin G, Gentamicin and sample and mix (19). II- Culture inoculation and examination: Cultures were inoculated directly from the same site as those for microscopy. Culture tubes with tight caps were incubated in an aerobic atmosphere at 37 °C for the first 24 h after which a wet preparation was done by taking a drop of the sample from the bottom of each tube with the use of a pasture pipette and examined for trophozoites of T. vaginalis. This is repeated at two days and continued up to the seventh day before negative culture was discarded.