Mycoplasma infection in genital tract with special reference to immunofluorescence.RR Bhatt, AA Gogate, LL Deodhar
The aim of the study was to find out the incidence of mycoplasma in female genital tract infections. Out of 225 patients mycoplasma was isolated in 52.88%. The isolated strains were confirmed by fluorescent antibody technique (FAT).
Keywords: Adolescent, Adult, Female, Fluorescent Antibody Technique, Genital Diseases, Female, diagnosis,epidemiology,Human, Incidence, India, epidemiology,Mycoplasma Infections, diagnosis,epidemiology,
In the past few years, mycoplasma infection has been found to be associated with the cases of unexplained infertility, NGU, salpingitis, amnionitis, prematurity etc., Extensive studies have been conducted to develop fluorescent antibody technique (FAT) and define their role as a serological tool in the diagnosis of various diseases. The aim of the present study was to isolate mycoplasma and identify them by FAT.
Two hundred and twenty-five patients in the age group of 18 to 35 year attending the Gynaecology Department of LTMMC were screened. The patients were clinically diagnosed as having vaginitis, cervicitis, repeated abortions, sterility, PID, erosion on cervix. From each patient two swabs from cervix/vagina were collected in Stuart's transport medium. Processing was done as described in the previous work. In short, wet preparations were examined for T. vagnitis; Gram staining for bacterial flora and yeast cells, Giemsa staining for the detection of inclusion bodies of Chlam. trachomatis. Culturing of the material was done on (1) PPLO agar/broth supplemented with arginine for (M. hominis). (2) PPLO agar/broth supplemented with urea (for U. urealyticum) (3) Sabouraud's agar (4) MacConkey's agar (5) Blood agar.
The plates were observed for the growth. After the large colony mycoplasmas were observed under inverted microscope, the colonies were confirmed to be that of M. hominis by FAT. The procedure followed in our laboratory was the modification of the procedure followed by Rosendal and Black. The procedure carried out was as follows.
(1) A colony bearing agar plate was taken and teflon rings were pushed gently into it.
(2) The rings were filled with 1 ml of IFA buffer. It was allowed to react for 20 min.
NaCl (0.85%) 4.25 gm
NaN3 (0.02%) 0.10 gm
KH2PO4 (0.05M) 3.40 gm
Fetal Calf serum (FCS) 25.0 ml
Distilled water 475.00 ml
pH 7.5 ml
(3) The buffer was aspirated. It was followed by adding 200 µl of rabbit antiserum raised against PG 21 standard strain (1:32). It was allowed to react for 15 min and then aspirated.
(4) Three washings were carried out, each washing kept for 5 min, 10 min and 10 min respectively.
(5) After the washings were over, 200 µl of (1:275) diluted fluorescein isothiocyanate conjugated serum (Pastour institute, Paris) raised in sheep was overlayed for 15 min. The conjugate was then aspirated.
(6) This step was followed by three washings, each washing kept for 5 min, 20 min and 10 min respectively.
The plates were finally viewed under the fluorescence microscope using KP490 excitation filter and G 247 barrier filter.
[Table - 1]shows the correlation of mycoplasma isolates and clinical diagnosis. The maximum number of mycoplasma strains isolated were from the cases of PID i.e. 9/11 giving an isolation rate of 81.81%. The next group was that of repeated abortion, where the isolation rate was 70%. The third highest isolation rate was seen in the cases of vaginitis i.e. 55.55%. This was followed by patients with sterility, cervicitis and with cervical erosion showing an isolation rate of 53.84%, 42.10% and 33.33% in that order.
Of 225 patients screened, mycoplasmas were recovered in 119 cases, giving an isolation rate of 52.88%. Of these 119 isolates. 32 isolates were of large colony mycoplasmas and 87 were of U. urealyticum.
Isolates of large colony mycoplasmas were confirmed to be M. hominis by FAT. The fluorescing colonies [Figure - 1] of M. hominis appeared apple green in colour.
In the present study, FAT helps in detecting M. hominis isolates growing on primary isolation plates. The use of teflon rings provides an essential saving of antisera and conjugates. Rosendal and Blacks concluded that indirect immunofluorescence of unfixed colonies is well suited for rapid serological identification of mycoplasmas, Barile et al showed a 48% positivity by the FAT from tissue cultures and 47% positivity by the cultural technique.
In conclusion, it can be said that FAT is a satisfactory method for detection of M. hominis from a mixture of mycoplasma growing on primary isolation plates. The method can be applied as soon as the growth is observed on agar plates.
[Figure - 1][Table - 1], [Table - 2]