Polymerase chain reaction Archives - Revista Brasileira de Ginecologia e Obstetrícia
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2006;28(5):285-291
DOI 10.1590/S0100-72032006000500004
PURPOSE: to identify risk factors for cervical intraepithelial neoplasia (CIN) and human papillomavirus (HPV) types among women with CIN, and to compare with HPV types among patients with normal cervix. METHODS: a total of 228 patients were studied, of whom 132 with CIN (cases) and 96 with normal cervix (controls). In the two groups consisting of women selected among outpatients attended in the same hospital, living near the place of the research, mean ages were similar (34.0±8.3 years) and there was a predominance of married women. Possible risk factors for CIN were investigated with the application of a questionnaire surveying age, marital status, level of schooling, age at first coitus, number of pregnancies, number of sexual partners, method of used contraception, reference of previously sexually transmitted diseases (STDs) and smoking habits, with a comparison between the studied groups. Samples were collected for oncologic colpocytology and HPV search through polymerase chain reaction (PCR), using MY09/MY11 primers; then colposcopic and histopathological examinations were performed. For statistical analysis of the association between risk factors and CIN, odds ratio with 95% confidence interval and chi2 and Fisher tests were used at a significance level of 0.05. The logistic regression method with the significance expressed by the p value with maximum likelihood was also applied. RESULTS: the following variables remained in the logistic regression model: HPV infection of high oncogenic risk (OR=12.32; CI 95%: 3.79-40.08), reference of previous STDs (OR=8.23; CI 95%: 2.82-24.04), early age at first coitus (OR=4.00; CI 95%: 1.70-9.39) and smoking habit (OR=3.94; CI 95%: 1.73-8.98). PCR was positive in 48.5 and 14.6% in the case and control groups, respectively. CONCLUSIONS: the main risk factor for CIN was oncogenic HPV infection, with types 16, 18, 33, 35, 51, 52, 58, and 83. Among patients with a high-degree lesion, there was a predominance of HPV-16 or type 16 variant. In patients with normal cervix oncogenic, HPV types 51, 58, and 51 variant were also identified.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2006;28(5):285-291
DOI 10.1590/S0100-72032006000500004
PURPOSE: to identify risk factors for cervical intraepithelial neoplasia (CIN) and human papillomavirus (HPV) types among women with CIN, and to compare with HPV types among patients with normal cervix. METHODS: a total of 228 patients were studied, of whom 132 with CIN (cases) and 96 with normal cervix (controls). In the two groups consisting of women selected among outpatients attended in the same hospital, living near the place of the research, mean ages were similar (34.0±8.3 years) and there was a predominance of married women. Possible risk factors for CIN were investigated with the application of a questionnaire surveying age, marital status, level of schooling, age at first coitus, number of pregnancies, number of sexual partners, method of used contraception, reference of previously sexually transmitted diseases (STDs) and smoking habits, with a comparison between the studied groups. Samples were collected for oncologic colpocytology and HPV search through polymerase chain reaction (PCR), using MY09/MY11 primers; then colposcopic and histopathological examinations were performed. For statistical analysis of the association between risk factors and CIN, odds ratio with 95% confidence interval and chi2 and Fisher tests were used at a significance level of 0.05. The logistic regression method with the significance expressed by the p value with maximum likelihood was also applied. RESULTS: the following variables remained in the logistic regression model: HPV infection of high oncogenic risk (OR=12.32; CI 95%: 3.79-40.08), reference of previous STDs (OR=8.23; CI 95%: 2.82-24.04), early age at first coitus (OR=4.00; CI 95%: 1.70-9.39) and smoking habit (OR=3.94; CI 95%: 1.73-8.98). PCR was positive in 48.5 and 14.6% in the case and control groups, respectively. CONCLUSIONS: the main risk factor for CIN was oncogenic HPV infection, with types 16, 18, 33, 35, 51, 52, 58, and 83. Among patients with a high-degree lesion, there was a predominance of HPV-16 or type 16 variant. In patients with normal cervix oncogenic, HPV types 51, 58, and 51 variant were also identified.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2006;28(3):190-194
DOI 10.1590/S0100-72032006000300009
PURPOSE: to verify the viability of early diagnosis of fetal gender in maternal plasma by the real-time polymerase chain reaction (real-time PCR) starting at the 5th week of pregnancy. METHODS: peripheral blood was collected from pregnant women with single fetus starting at the 5th week of gestation. After centrifugation, 0.4 mL plasma was separated for fetal DNA extraction. The DNA was analyzed in duplicate by real-time PCR for two genomic regions, one of the Y chromosome and the other common to both sexes, through the TaqMan® method, which uses a pair of primers and a fluorescent probe. Patients who aborted were excluded. RESULTS: a total of 79 determinations of fetal DNA in maternal plasma were performed in 52 pregnant women. The results of the determinations were compared to fetal gender after delivery. Accuracy according to gestational age was 92.6% (25 of 27 cases) at 5 weeks with 87% sensitivity, and 95.6% (22 of 23 cases) at 6 weeks with 92% sensitivity. Starting at the 7th week of pregnancy, accuracy was 100% (29 of 29 cases). Specificity was 100% regardless of gestational age. CONCLUSION: real-time PCR for the detection of fetal gender in maternal plasma starting at the 5th week of gestation has good sensitivity and excellent specificity. There was agreement of the results in 100% of the cases in which male gender was diagnosed, regardless of gestational age, and from the 7th week of gestation for female gender diagnosis.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2006;28(3):190-194
DOI 10.1590/S0100-72032006000300009
PURPOSE: to verify the viability of early diagnosis of fetal gender in maternal plasma by the real-time polymerase chain reaction (real-time PCR) starting at the 5th week of pregnancy. METHODS: peripheral blood was collected from pregnant women with single fetus starting at the 5th week of gestation. After centrifugation, 0.4 mL plasma was separated for fetal DNA extraction. The DNA was analyzed in duplicate by real-time PCR for two genomic regions, one of the Y chromosome and the other common to both sexes, through the TaqMan® method, which uses a pair of primers and a fluorescent probe. Patients who aborted were excluded. RESULTS: a total of 79 determinations of fetal DNA in maternal plasma were performed in 52 pregnant women. The results of the determinations were compared to fetal gender after delivery. Accuracy according to gestational age was 92.6% (25 of 27 cases) at 5 weeks with 87% sensitivity, and 95.6% (22 of 23 cases) at 6 weeks with 92% sensitivity. Starting at the 7th week of pregnancy, accuracy was 100% (29 of 29 cases). Specificity was 100% regardless of gestational age. CONCLUSION: real-time PCR for the detection of fetal gender in maternal plasma starting at the 5th week of gestation has good sensitivity and excellent specificity. There was agreement of the results in 100% of the cases in which male gender was diagnosed, regardless of gestational age, and from the 7th week of gestation for female gender diagnosis.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2005;27(9):554-560
DOI 10.1590/S0100-72032005000900009
PURPOSE: to evaluate the performance of cytogenetic analysis, fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) in the study of numerical chromosomal anomalies and in fetal sex determination of spontaneous abortion material. METHODS: cytogenetic analysis was performed on 219 spontaneous abortion specimens. Forty of these cases were also submitted to fetal sex determination using nested-PCR. Thirty-two of these cases were selected due to failed cytogenetic culture and the other eight were selected randomly. Twenty samples were submitted to the FISH technique, using probes for chromosomes 13, 18, 21, X and Y. Thirteen of these samples were selected due to failed cytogenetic culture and the other seven were randomly selected. The success rates of each technique were compared using the chi2 test and an established p<0.05 level of significance. The results of samples submitted to more than one test were evaluated for accuracy, using the cytogenetic result as the gold standard. RESULTS: cytogenetic analysis was successful in 84.9% of the samples and in 51.1% of them the results were abnormal: 65.2% trisomy, 17.9% triploidy, 9.4% tetraploidy, 4.2% chromosome X monosomy, and 1.1% each for double trisomy, tetrasomy and structural abnormality. The most frequent trisomy was that of chromosome 16 (39%). The success rate of FISH and PCR techniques (90%) did nod differ significantly from the cytogenetic analysis. In all cases submitted to more than one test, the results were identical to those obtained through cytogenetic analysis. Samples that failed to grow on cytogenetic test and that were submitted to other techniques of molecular biology had a success rate of 87.5 and 84.6% for PCR and FISH, respectively. CONCLUSION: cytogenetic analysis of spontaneous abortions had a high success rate and chromosomal anomalies were identified in over half of the cases. Molecular biology techniques (PCR and FISH) complemented the cytogenetic study and proved to be reliable in the detection of numerical chromosomal anomalies and in fetal sex determination.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2005;27(9):554-560
DOI 10.1590/S0100-72032005000900009
PURPOSE: to evaluate the performance of cytogenetic analysis, fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) in the study of numerical chromosomal anomalies and in fetal sex determination of spontaneous abortion material. METHODS: cytogenetic analysis was performed on 219 spontaneous abortion specimens. Forty of these cases were also submitted to fetal sex determination using nested-PCR. Thirty-two of these cases were selected due to failed cytogenetic culture and the other eight were selected randomly. Twenty samples were submitted to the FISH technique, using probes for chromosomes 13, 18, 21, X and Y. Thirteen of these samples were selected due to failed cytogenetic culture and the other seven were randomly selected. The success rates of each technique were compared using the chi2 test and an established p<0.05 level of significance. The results of samples submitted to more than one test were evaluated for accuracy, using the cytogenetic result as the gold standard. RESULTS: cytogenetic analysis was successful in 84.9% of the samples and in 51.1% of them the results were abnormal: 65.2% trisomy, 17.9% triploidy, 9.4% tetraploidy, 4.2% chromosome X monosomy, and 1.1% each for double trisomy, tetrasomy and structural abnormality. The most frequent trisomy was that of chromosome 16 (39%). The success rate of FISH and PCR techniques (90%) did nod differ significantly from the cytogenetic analysis. In all cases submitted to more than one test, the results were identical to those obtained through cytogenetic analysis. Samples that failed to grow on cytogenetic test and that were submitted to other techniques of molecular biology had a success rate of 87.5 and 84.6% for PCR and FISH, respectively. CONCLUSION: cytogenetic analysis of spontaneous abortions had a high success rate and chromosomal anomalies were identified in over half of the cases. Molecular biology techniques (PCR and FISH) complemented the cytogenetic study and proved to be reliable in the detection of numerical chromosomal anomalies and in fetal sex determination.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2005;27(5):248-256
DOI 10.1590/S0100-72032005000500004
PURPOSE: to estimate the prevalence of HPV and its genotypes in HIV-infected and non-infected women, using the Polymerase Chain Reaction (PCR) technique. METHODS: a sectional study with 79 enrolled women: a study group, with 41 HIV-infected women, and a control group, with 38 non-infected women attended at a Basic Health Unit. All were submitted to a serologic test for the detection of HIV and spontaneously looked for gynecological attendance at those units, for the first time. They answered a standard questionnaire and were submitted to a gynecological examination with a cervical swab and specimen for the detection of DNA-HPV and its genotypes. Statistical analysis was performed using Kruskal-Wallis, chi2 or Fisher's exact tests. Statistical significance was considered at p<0.05. RESULTS: the demographic characteristics, obstetric and gynecological previous history were similar in both groups except for previous STD, but different as to the gynecological examination and cervical cytological analysis. The presence of DNA-HPV was significantly different (p<0.05) in the two groups. Among HIV-infected patients, 73.2% presented DNA-HPV positive results, as compared with 23.8% of non-infected women (OR=8.79; 95% IC: 2.83 28.37). Concerning HPV genotypes, there was no clear predominance of a specific HPV subtype in the HIV-infected or in the HIV non-infected groups, and the frequency of unidentified types was similar in both groups. Non-significant predominance of HPV multiple infections (p>0.05) was detected in the HIV-infected women (50.0%) and the most frequently found combination was of types 6, 11 and 16. HPV simple infection occurred in 66.6% of HIV-non-infected patients. The most frequent type found in both groups was 16, representing 44% of all the simple infections in both groups. CONCLUSIONS: HIV-infected women showed higher DNA-HPV prevalence in the uterine cervix, as compared to non-infected women. There was no difference in the predominance of specific types of HPV when both groups were compared. There was a tendency to HPV multiple infections in the HIV-infected women, whereas simple infection predominated in the non-infected patients.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2005;27(5):248-256
DOI 10.1590/S0100-72032005000500004
PURPOSE: to estimate the prevalence of HPV and its genotypes in HIV-infected and non-infected women, using the Polymerase Chain Reaction (PCR) technique. METHODS: a sectional study with 79 enrolled women: a study group, with 41 HIV-infected women, and a control group, with 38 non-infected women attended at a Basic Health Unit. All were submitted to a serologic test for the detection of HIV and spontaneously looked for gynecological attendance at those units, for the first time. They answered a standard questionnaire and were submitted to a gynecological examination with a cervical swab and specimen for the detection of DNA-HPV and its genotypes. Statistical analysis was performed using Kruskal-Wallis, chi2 or Fisher's exact tests. Statistical significance was considered at p<0.05. RESULTS: the demographic characteristics, obstetric and gynecological previous history were similar in both groups except for previous STD, but different as to the gynecological examination and cervical cytological analysis. The presence of DNA-HPV was significantly different (p<0.05) in the two groups. Among HIV-infected patients, 73.2% presented DNA-HPV positive results, as compared with 23.8% of non-infected women (OR=8.79; 95% IC: 2.83 28.37). Concerning HPV genotypes, there was no clear predominance of a specific HPV subtype in the HIV-infected or in the HIV non-infected groups, and the frequency of unidentified types was similar in both groups. Non-significant predominance of HPV multiple infections (p>0.05) was detected in the HIV-infected women (50.0%) and the most frequently found combination was of types 6, 11 and 16. HPV simple infection occurred in 66.6% of HIV-non-infected patients. The most frequent type found in both groups was 16, representing 44% of all the simple infections in both groups. CONCLUSIONS: HIV-infected women showed higher DNA-HPV prevalence in the uterine cervix, as compared to non-infected women. There was no difference in the predominance of specific types of HPV when both groups were compared. There was a tendency to HPV multiple infections in the HIV-infected women, whereas simple infection predominated in the non-infected patients.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2003;25(9):661-666
DOI 10.1590/S0100-72032003000900007
PURPOSE: to present the most frequent gynecologic results in a cohort of 300 outpatient HIV-infected women. METHODS: this is a prospective and descriptive study of HIV-infected women that have been followed up at the gynecological clinic from November 1996 to December 2002. These patients were subjected to a specific protocol which included an interview, a gynecological evaluation and a collecting cervical sample for Pap smear, research of HPV (PCR) and colposcopy. Cervical biopsy was performed when necessary. Data were stored and analyzed by Epi-Info, version 6.0. RESULTS: the mean age was 34.5 years. The small number of sexual partners, average of three partners, and the predominance of heterosexual contagion should be mentioned: 271 (90.6%) patients were contaminated through sexual contact with their partners. There was a high prevalence of cervical intraepithelial neoplasia (CIN) representing 21.7% of the total group. Of 109 patients subjected to PCR, 89 (81.7%) were found to have some HPV genotype. An inflammatory smear was present in 69% of the patients. CONCLUSIONS: there is a high frequency of CIN and genital infectious diseases among HIV-infected women, mainly HPV.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2003;25(9):661-666
DOI 10.1590/S0100-72032003000900007
PURPOSE: to present the most frequent gynecologic results in a cohort of 300 outpatient HIV-infected women. METHODS: this is a prospective and descriptive study of HIV-infected women that have been followed up at the gynecological clinic from November 1996 to December 2002. These patients were subjected to a specific protocol which included an interview, a gynecological evaluation and a collecting cervical sample for Pap smear, research of HPV (PCR) and colposcopy. Cervical biopsy was performed when necessary. Data were stored and analyzed by Epi-Info, version 6.0. RESULTS: the mean age was 34.5 years. The small number of sexual partners, average of three partners, and the predominance of heterosexual contagion should be mentioned: 271 (90.6%) patients were contaminated through sexual contact with their partners. There was a high prevalence of cervical intraepithelial neoplasia (CIN) representing 21.7% of the total group. Of 109 patients subjected to PCR, 89 (81.7%) were found to have some HPV genotype. An inflammatory smear was present in 69% of the patients. CONCLUSIONS: there is a high frequency of CIN and genital infectious diseases among HIV-infected women, mainly HPV.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2001;23(6):355-364
DOI 10.1590/S0100-72032001000600003
Purpose: to compare histopathology and polymerase chain reaction (PCR) for the diagnosis of human papillomavirus (HPV) in cervical lesions of human immunodeficiency virus (HIV)-seropositive women. Methods: fifty-two HIV-seropositive women with suspected HPV cervical lesions were studied. Cervical scrapes were collected for PCR and colposcopy-guided biopsy was made for the histopathologic study. Three samples were disqualified for PCR, leaving a study population of 49 women. Results: the prevalence of HPV was 53% by histopathology and 85.7% by PCR. Among the 42 patients in whom HPV was detected by PCR, 26 were confirmed by histopathology (sensitivity = 61.9%). This method gave no false-positives (specificity = 100%), with 100% of positive prediction. Compared to PCR, the histopathology had: positive predcitive value = 100% and negative predcitive value = 30.4%. Among the 26 patients with HPV-positive biopsy, 15 (57.7%) had cervical intraepithelial neoplasia (CIN); relative risk = 13.3. Conclusion: histopathology was 100% correct for HPV-infection diagnosis. It means that when the biopsy is positive, HPV will be present, confirming the clinical suspicion. However, the low sensitivity excludes histopathology as a screening examination in this group of women.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2001;23(6):355-364
DOI 10.1590/S0100-72032001000600003
Purpose: to compare histopathology and polymerase chain reaction (PCR) for the diagnosis of human papillomavirus (HPV) in cervical lesions of human immunodeficiency virus (HIV)-seropositive women. Methods: fifty-two HIV-seropositive women with suspected HPV cervical lesions were studied. Cervical scrapes were collected for PCR and colposcopy-guided biopsy was made for the histopathologic study. Three samples were disqualified for PCR, leaving a study population of 49 women. Results: the prevalence of HPV was 53% by histopathology and 85.7% by PCR. Among the 42 patients in whom HPV was detected by PCR, 26 were confirmed by histopathology (sensitivity = 61.9%). This method gave no false-positives (specificity = 100%), with 100% of positive prediction. Compared to PCR, the histopathology had: positive predcitive value = 100% and negative predcitive value = 30.4%. Among the 26 patients with HPV-positive biopsy, 15 (57.7%) had cervical intraepithelial neoplasia (CIN); relative risk = 13.3. Conclusion: histopathology was 100% correct for HPV-infection diagnosis. It means that when the biopsy is positive, HPV will be present, confirming the clinical suspicion. However, the low sensitivity excludes histopathology as a screening examination in this group of women.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2001;23(5):277-282
DOI 10.1590/S0100-72032001000500002
Objective: to test the effectiveness of the polymerase chain reaction (PCR) in the amniotic fluid for the detection of fetal contamination due to Toxoplasma gondii in pregnant women with acute infection and to correlate it with the inoculation technique and the histology of the placenta. Methods: thirty-seven patients were prospectively studied and the diagnosis was based on the identification of maternal acute infection followed by amniocentesis guided by ultrasound to obtain amniotic fluid for PCR and mice inoculation. The mothers were treated with spiramycin throughout pregnancy; when fetal infection was demonstrated, pyrimethamine and sulfadiazine were added to the regimen. The placentas were processed for histologic examination. The infants were followed for a period that varied from three to 23 months for the confirmation or exclusion of congenital toxoplasmosis. Results: association measures such as sensitivity, specificity and predictive values were calculated for PCR in the amniotic fluid, detection of the parasite through mice inoculation and placental histology and showed the following results: PCR values of sensitivity = 66.7% and specificity = 87.1%; the respective values for mice inoculation were 50 and 100% and for the placental histology were 80 and 66.7%. Conclusion: although PCR should not be used alone for the prenatal diagnosis of congenital toxoplasmosis, it is a promising method and deserves more studies to improve its efficacy.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2001;23(5):277-282
DOI 10.1590/S0100-72032001000500002
Objective: to test the effectiveness of the polymerase chain reaction (PCR) in the amniotic fluid for the detection of fetal contamination due to Toxoplasma gondii in pregnant women with acute infection and to correlate it with the inoculation technique and the histology of the placenta. Methods: thirty-seven patients were prospectively studied and the diagnosis was based on the identification of maternal acute infection followed by amniocentesis guided by ultrasound to obtain amniotic fluid for PCR and mice inoculation. The mothers were treated with spiramycin throughout pregnancy; when fetal infection was demonstrated, pyrimethamine and sulfadiazine were added to the regimen. The placentas were processed for histologic examination. The infants were followed for a period that varied from three to 23 months for the confirmation or exclusion of congenital toxoplasmosis. Results: association measures such as sensitivity, specificity and predictive values were calculated for PCR in the amniotic fluid, detection of the parasite through mice inoculation and placental histology and showed the following results: PCR values of sensitivity = 66.7% and specificity = 87.1%; the respective values for mice inoculation were 50 and 100% and for the placental histology were 80 and 66.7%. Conclusion: although PCR should not be used alone for the prenatal diagnosis of congenital toxoplasmosis, it is a promising method and deserves more studies to improve its efficacy.