|Year : 2002 | Volume
| Issue : 1 | Page : 37-39
Comparison of PCR method with the culture method for identification of gonococci from endocervical swabs
A Alam , Md RA Miah , M Rahman , H Sattar , AA Saleh
Department of Microbiology, MAG Osmani Medical College, Sylhet, Bangladesh
Gonococcal infection remains still a major cause of morbidity among sexually active individuals. Diagnosis of the infection in a female case is more difficult than that in a male. This was a prospective study among 269 female commercial sex workers (CSWs) to screen them for gonococcal infection, comparing the rapid method of identification of gonococci by polymerase chain reaction (PCR) with the selective culture method. A total of 92 (34.2%) CSWs were identified positive for Neisseria gonorrhoeae by combination of the two methods. The PCR method identified 87 of the specimens to harbour cppB gene of N. gonorrhoeae, whereas culture method identified 83 specimens showing colonies of gonococci. Taking into consideration of the total positive cases (92), the PCR method showed a sensitivity of 94.57%, whereas sensitivity of culture method was 90.22%. The selective culture method appears to be the most applicable in the identification of gonococci from clinical specimens, particularly in the less resourceful countries like Bangladesh.
|How to cite this article:|
Alam A, Miah MR, Rahman M, Sattar H, Saleh A A. Comparison of PCR method with the culture method for identification of gonococci from endocervical swabs. Indian J Med Microbiol 2002;20:37-9
|How to cite this URL:|
Alam A, Miah MR, Rahman M, Sattar H, Saleh A A. Comparison of PCR method with the culture method for identification of gonococci from endocervical swabs. Indian J Med Microbiol [serial online] 2002 [cited 2020 Aug 8];20:37-9. Available from: http://www.ijmm.org/text.asp?2002/20/1/37/8338
Gonococcal infection is one of the oldest known bacterial disease, but still it causes a significant morbidity among the sexually active individuals. Control of the disease is important because of the higher incidence of acute asymptomatic cases, complications and sequelae, and its proven role in the transmission of HIV infections. For the control of the disease, rapid diagnosis is very important. Polymerase chain reaction (PCR) method is one such rapid method applicable in the reference laboratories. Identification is possible in a day by this method and has a high sensitivity of 100% with specificity of 88.9%.1 Though rapid, the PCR method has several limitations: it needs costly equipment and reagents, experienced technicians, and moreover, organisms cannot be isolated in this method for further investigations. On the other hand, culture is a very widely practiced method, requires at least 3 days for confirmation of N. gonorrhoeae. The method is available in almost all tertiary level government healthcare setups in Bangladesh. It has a lower rate of sensitivity (85-95%),2 but organisms can be isolated in pure state for further investigation. The present study compares the sensitivity of the two methods in identifying gonococci from specimens of endocervical swabs collected from female commercial sex workers (CSWs).
| ~ Materials and Methods|| |
Two hundred and sixty nine floating female CSWs were examined consecutively at a Government vagrant home in Dhaka, Bangladesh. The CSWs who were menstruating and/or had taken antibiotics in the preceding two weeks were excluded from the study.
Before sample collection, the CSWs were registered with an identification number and specimens of endocervical swabs were taken. Two such swabs were taken: one for preservation into phosphate buffered saline (PBS) tubes at -20°C for PCR identification and the other to inoculate on culture media.
One of the endocervical swabs was inoculated on Modified Thayer Martin (MTM) medium plate and incubated at 37°C with CO2 and moisture for next 24 hours. CO2 was provided by candle extinction jar. Colonies of N. gonorrhoeae were identified by using standard laboratory tests [Table - 1].
The endocervical swabs preserved for PCR identification were brought to room temperature and then vortexed for one minute to release the material contained in the swab. The swabs were then discarded and the suspension was centrifuged for five minutes at 3000 revolutions per minute to pellet the cells. After removing the supernatant by aspiration, the cells were resuspended in 100 mL of K-buffer (1x PCR buffer with 0.5% non-ionic detergent Tween-20 and 200 mL/mL Proteinase K). The cell suspension was incubated at 550C for one hour and then heated to 950C for 10 minutes to inactivate the Proteinase K.
The sequence data on the cppB gene carried on chromosome as well as on 4.2 kb cryptic plasmid of N. gonorrhoeae was used to select two 20-mer oligonucleotide primers as designated below:
Sense 5' GCT ACG CAT ACC CGC GTT GC 3
Antisense 5' CGA AGA CCT TCG AGC AGA CA 3
The expected length of the amplified product of the target sequence with these primers was 390 bp. Amplification was performed in 25 mL reaction volume containing: 1 mL of each of the primers, 1 mL 10 mM dNTPs, 2.5 mL 1x PCR buffer, 2.5 mL 50 mM MgCl2 in 9.3 mL deionized water, 7.5 mL template DNA, and finally 0.2 mL super Taq DNA polymerase. All the reagents were taken in a 0.5 mL PCR tube and mixed by gentle vortexing before overlaying with a drop of mineral oil. Thirty cycles of amplification were performed in a DNA thermal cycler (Perkin-Elmer Cetus). Each cycle consisted of a one-minute denaturation step at 94°C, a one-minute annealing step at 48°C, and another one-minute amplification step at 72°C. Each reaction cycle was preceded by a five-minute denaturation and followed by an eight-minute extension step. Amplified product (7.5 mL) was analyzed by electrophoresis in a 1% agarose gel using standard methods. The ethidium bromide-stained gel was examined in ultraviolet light to see the presence of a 390 bp fragment.
| ~ Results|| |
Among 269 endocervical swabs examined, N. gonorrhoeae was identified in 92 (34.2%) cases by combination of both culture and PCR methods. Of these 92 cases, 78 (84.8%) specimens were positive by both culture and PCR methods, 5 (5.4%) were positive by only culture method, and the rest 9 (9.8%) were positive only by PCR method [Table - 2]. The sensitivity of PCR and culture methods were 94.57% and 90.22% respectively.
| ~ Discussion|| |
Rapid and specific means of diagnosis of the cases of gonococcal infection is very important for its control. This study was done to compare the PCR method of identification of N. gonorrhoeae from endocervical swabs with the culture method, and the PCR had higher sensitivity (94.57%) than the culture method (90.22%). The PCR method of detection of gonococci from clinical specimen has been reported to have further more sensitivity (100%) as mentioned previously.1 In comparison, lower sensitivity of PCR in this study may be because we were unable to standardize the optimum reaction volume that might yield lower or no detectable product in some of the specimens. Thus, PCR appears to be a highly demanding method. On the other hand, culture method, though more time consuming and has lower sensitivity, is a method of choice in the identification of N. gonorrhoeae, particularly in a developing country like Bangladesh. In addition, isolation of gonococci by culture method is very important in testing the susceptibility pattern that is another key factor in the successful treatment and control of the disease.
| ~ Acknowledgement|| |
The study was partially supported by the International Centre for Diarrhoeal Disease Control in Bangladesh (ICDDC, B).
| ~ References|| |
|1.||Ho BSW, Feng WG, Wong BKC, Egglestone SI. Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples. J Clin Pathol 1992; 45: 439-442. |
|2.||Stamm WE. Diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis using antigen detection methods. Diagn Microbiol Infect Dis 1985; 4: 985-995. |
|3.||Van Dyck E, Piot P, Meheus A. Bench-level Laboratory |
|4.||Manual for sexually transmitted diseases. WHO/VDT 1989; 443: 6-24. |
|5.||Hagblom P, Korch C, Jonsson A, Normark S. Intragenic variation by site-specific recombination in cryptic plasmid of Neisseria gonorrhoeae. J Bacteriol 1986; 167: 231-237. |
|6.||Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor laboratory 1989: 6.1-6.59. |