Polymerase chain reaction Archives - Revista Brasileira de Ginecologia e Obstetrícia
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2012;34(12):544-549
DOI 10.1590/S0100-72032012001200003
PURPOSE: To describe the epidemiological cases and microbiological profile of Streptococcus agalactiae serotypes isolated from infected newborns of a Women's Health Reference Centre of Campinas, São Paulo, Brazil. METHODS: Cross-sectional laboratory survey conducted from January 2007 to December 2011. The newborns' strains, isolated from blood and cerebrospinal fluid samples, were screened by hemolysis on blood ágar plates, Gram stain, catalase test, CAMP test, hippurate hydrolysis or by microbiological automation: Vitek 2 BioMerieux®. They were typed by PCR, successively using specific primers for species and nine serotypes of S. agalactiae. RESULTS: Seven blood samples, one cerebrospinal fluid sample and an ocular sample, were isolated from nine newborns with infections caused by S. agalactiae, including seven cases of early onset and two of late onset. Only one of these cases was positive for paired mother-child samples. Considering that 13,749 deliveries were performed during the study period, the incidence was 0.5 cases of GBS infections of early onset per 1 thousand live births (or 0.6 per 1 thousand, including two cases of late onset) with 1, 3, 2, zero and 3 cases (one early and two late onset cases), respectively, for the years from 2007 to 2011. It was possible to apply PCR to seven of nine samples, two each of serotypes Ia and V and three of serotype III, one from a newborn and the other two from a paired mother-child sample. CONCLUSIONS: Although the sample was limited, the serotypes found are the most prevalent in the literature, but different from the other few Brazilian studies available, except for type Ia.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2012;34(12):544-549
DOI 10.1590/S0100-72032012001200003
PURPOSE: To describe the epidemiological cases and microbiological profile of Streptococcus agalactiae serotypes isolated from infected newborns of a Women's Health Reference Centre of Campinas, São Paulo, Brazil. METHODS: Cross-sectional laboratory survey conducted from January 2007 to December 2011. The newborns' strains, isolated from blood and cerebrospinal fluid samples, were screened by hemolysis on blood ágar plates, Gram stain, catalase test, CAMP test, hippurate hydrolysis or by microbiological automation: Vitek 2 BioMerieux®. They were typed by PCR, successively using specific primers for species and nine serotypes of S. agalactiae. RESULTS: Seven blood samples, one cerebrospinal fluid sample and an ocular sample, were isolated from nine newborns with infections caused by S. agalactiae, including seven cases of early onset and two of late onset. Only one of these cases was positive for paired mother-child samples. Considering that 13,749 deliveries were performed during the study period, the incidence was 0.5 cases of GBS infections of early onset per 1 thousand live births (or 0.6 per 1 thousand, including two cases of late onset) with 1, 3, 2, zero and 3 cases (one early and two late onset cases), respectively, for the years from 2007 to 2011. It was possible to apply PCR to seven of nine samples, two each of serotypes Ia and V and three of serotype III, one from a newborn and the other two from a paired mother-child sample. CONCLUSIONS: Although the sample was limited, the serotypes found are the most prevalent in the literature, but different from the other few Brazilian studies available, except for type Ia.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2012;34(9):425-431
DOI 10.1590/S0100-72032012000900007
PURPOSE: To evaluate the prevalence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) among women candidates to in vitro fertilization (IVF) in a reference public service in southeastern Brazil. METHODS: Women who were referred for IVF from April 1st, 2008 to December 31st, 2009 were enrolled sequentially in the study. A ginecological-obstetrical background questionnaire was applied and endocervical swab samples were obtained to search for CT and NG using hybrid capture and PCR. The variables studied were: age, color, education, duration of infertility, number of pregnancies and living children, history of miscarriage, ectopic pregnancy, number of sex partners, pelvic inflammatory disease (PID), pelvic surgery, manipulation of the uterine cavity, smoking, and illicit drug use. The women were distributed according to the presence/absence of confirmed chlamydia infection and descriptive analysis was employed. RESULTS: Among 176 women tested the prevalence of CT infection was 1.1% and there was no NG infection. Two thirds of the women were >30 years old, with schooling >8 years and <5 years of infertility, and 56.2% had no children. The main background data were pelvic surgery (77.8%), manipulation of the uterine cavity (62.5%) and PID (27.8%). The tubal factor was the most prevalent, 73.3% of women (from 129), 37.5% had been sterilized, 35.8% had not been sterilized, and other factors had a prevalence <30%. CONCLUSIONS: CT and NG infections had a low prevalence in this sample. Studies at other centers in the country are needed to confirm the prevalence of infection in this particular group of infertile women.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2012;34(9):425-431
DOI 10.1590/S0100-72032012000900007
PURPOSE: To evaluate the prevalence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) among women candidates to in vitro fertilization (IVF) in a reference public service in southeastern Brazil. METHODS: Women who were referred for IVF from April 1st, 2008 to December 31st, 2009 were enrolled sequentially in the study. A ginecological-obstetrical background questionnaire was applied and endocervical swab samples were obtained to search for CT and NG using hybrid capture and PCR. The variables studied were: age, color, education, duration of infertility, number of pregnancies and living children, history of miscarriage, ectopic pregnancy, number of sex partners, pelvic inflammatory disease (PID), pelvic surgery, manipulation of the uterine cavity, smoking, and illicit drug use. The women were distributed according to the presence/absence of confirmed chlamydia infection and descriptive analysis was employed. RESULTS: Among 176 women tested the prevalence of CT infection was 1.1% and there was no NG infection. Two thirds of the women were >30 years old, with schooling >8 years and <5 years of infertility, and 56.2% had no children. The main background data were pelvic surgery (77.8%), manipulation of the uterine cavity (62.5%) and PID (27.8%). The tubal factor was the most prevalent, 73.3% of women (from 129), 37.5% had been sterilized, 35.8% had not been sterilized, and other factors had a prevalence <30%. CONCLUSIONS: CT and NG infections had a low prevalence in this sample. Studies at other centers in the country are needed to confirm the prevalence of infection in this particular group of infertile women.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2011;33(2):65-69
DOI 10.1590/S0100-72032011000200002
PURPOSE: to evaluate the frequency of VDR gene polymorphism Fok1 in infertile women with endometriosis and Control and its relation to the disease. METHODS: a case-control study that included 147 infertile women with endometriosis and 154 fertile women without endometriosis as Control. Fok1 polymorphism (rs10735810, T2C), which promotes a T/C exchange in exon 2 of the VDR gene, was identified by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), that involves the combination of amplification by PCR and digestion with restriction endonuclease. The χ2 test was used to compare allele and genotype frequencies between groups. All p-values were two-tailed and a p-value < 0.05 was considered statistically significant. RESULTS: the TT, TC and CC genotype frequencies of VDR Fok1 polymorphism were 44.2%, 46.9% and 8.9% in infertile women with endometriosis and 41.6%, 50% and 8.4% in the Control Group. No significant difference was found (p=0.8), even when the patients were subdivided according to the stage of endometriosis (p=0.3 for minimal and mild endometriosis and p=0.2 for moderate and severe endometriosis). Alleles T and C were present, respectively, in 67.6% and 32.3% of infertile women with endometriosis (p=0.8), in 63.5% and 36.5% of women with minimal/mild endometriosis (p=0.5), in 72.5% and 27.5% of women with moderate/severe endometriosis (p=0.2), and in 66.6% and 33.4% of the Control Group. No statistically significant difference was found among any groups and the Control. CONCLUSION: the results suggest that VDR gene polymorphism Fok1 does not confer genetic susceptibility to endometriosis-associated infertility in the Brazilian population.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2011;33(2):65-69
DOI 10.1590/S0100-72032011000200002
PURPOSE: to evaluate the frequency of VDR gene polymorphism Fok1 in infertile women with endometriosis and Control and its relation to the disease. METHODS: a case-control study that included 147 infertile women with endometriosis and 154 fertile women without endometriosis as Control. Fok1 polymorphism (rs10735810, T2C), which promotes a T/C exchange in exon 2 of the VDR gene, was identified by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), that involves the combination of amplification by PCR and digestion with restriction endonuclease. The χ2 test was used to compare allele and genotype frequencies between groups. All p-values were two-tailed and a p-value < 0.05 was considered statistically significant. RESULTS: the TT, TC and CC genotype frequencies of VDR Fok1 polymorphism were 44.2%, 46.9% and 8.9% in infertile women with endometriosis and 41.6%, 50% and 8.4% in the Control Group. No significant difference was found (p=0.8), even when the patients were subdivided according to the stage of endometriosis (p=0.3 for minimal and mild endometriosis and p=0.2 for moderate and severe endometriosis). Alleles T and C were present, respectively, in 67.6% and 32.3% of infertile women with endometriosis (p=0.8), in 63.5% and 36.5% of women with minimal/mild endometriosis (p=0.5), in 72.5% and 27.5% of women with moderate/severe endometriosis (p=0.2), and in 66.6% and 33.4% of the Control Group. No statistically significant difference was found among any groups and the Control. CONCLUSION: the results suggest that VDR gene polymorphism Fok1 does not confer genetic susceptibility to endometriosis-associated infertility in the Brazilian population.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2011;33(1):37-42
DOI 10.1590/S0100-72032011000100006
PURPOSE: to evaluate the frequency of TP53 codon 72 polymorphism in infertile women with endometriosis, women with idiopathic infertility, controls and its relation to the disease. METHODS: a case-control study that included 198 infertile women with endometriosis, 70 women with idiopathic infertility and 169 fertile women without endometriosis as control. Detection of TP53 codon 72 gene polymorphism (rs1042522, Arg/C:Pro/G), that promotes a C/G exchange in the coding region of the gene, was performed by real time Polymerase Chain Reaction (PCR), using the TaqMan system of primers, that flank the implicated region and probes labeled with different fluorescent dyes, one for allele C and other for allele G. When two dyes were observed, the patient was considered to be heterozygous CG. In the presence of only one dye, the individual was considered to be homozygous CC or GG. The χ2 test was used to compare allele and genotype frequencies between groups. All p-values were two-tailed and a p-value <0.05 was considered to be statistically significant. RESULTS: we found no statistically significant difference in the distribution of TP53 codon 72 polymorphism genotypes CC, CG or GG (p=0.7) and alleles C or G (p=0.4) between infertile patients with endometriosis and controls (p=0.4), regardless of the stage of the disease. In relation to infertility, no statistically significant difference in the genotype or allele distribution (p=1.0 and p=0.9, respectively) was observed between idiopathic infertile women and controls. Considering the dominant inheritance model, again, no statistically significant difference was found even in the endometriosis (p=0.5) or the idiopathic infertility group (p=0.9) when compared to controls. Regarding the recessive inheritance model no statistically significant difference was found, with p=0.6 and p=1.0, respectively, for the endometriosis and idiopathic infertility groups. CONCLUSION: the results suggest that the TP53 codon 72 polymorphism does not confer genetic susceptibility to endometriosis and/or infertility in the Brazilian population, not even the severe form of the disease.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2011;33(1):37-42
DOI 10.1590/S0100-72032011000100006
PURPOSE: to evaluate the frequency of TP53 codon 72 polymorphism in infertile women with endometriosis, women with idiopathic infertility, controls and its relation to the disease. METHODS: a case-control study that included 198 infertile women with endometriosis, 70 women with idiopathic infertility and 169 fertile women without endometriosis as control. Detection of TP53 codon 72 gene polymorphism (rs1042522, Arg/C:Pro/G), that promotes a C/G exchange in the coding region of the gene, was performed by real time Polymerase Chain Reaction (PCR), using the TaqMan system of primers, that flank the implicated region and probes labeled with different fluorescent dyes, one for allele C and other for allele G. When two dyes were observed, the patient was considered to be heterozygous CG. In the presence of only one dye, the individual was considered to be homozygous CC or GG. The χ2 test was used to compare allele and genotype frequencies between groups. All p-values were two-tailed and a p-value <0.05 was considered to be statistically significant. RESULTS: we found no statistically significant difference in the distribution of TP53 codon 72 polymorphism genotypes CC, CG or GG (p=0.7) and alleles C or G (p=0.4) between infertile patients with endometriosis and controls (p=0.4), regardless of the stage of the disease. In relation to infertility, no statistically significant difference in the genotype or allele distribution (p=1.0 and p=0.9, respectively) was observed between idiopathic infertile women and controls. Considering the dominant inheritance model, again, no statistically significant difference was found even in the endometriosis (p=0.5) or the idiopathic infertility group (p=0.9) when compared to controls. Regarding the recessive inheritance model no statistically significant difference was found, with p=0.6 and p=1.0, respectively, for the endometriosis and idiopathic infertility groups. CONCLUSION: the results suggest that the TP53 codon 72 polymorphism does not confer genetic susceptibility to endometriosis and/or infertility in the Brazilian population, not even the severe form of the disease.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2010;32(7):315-320
DOI 10.1590/S0100-72032010000700002
PURPOSE: to determine the prevalence of HPV 16, 18, 31 and 45 in cervical screening samples of women with cellular changes and/or colposcopy suggestive of persistent high grade or low grade lesion who were submitted to conization. METHODS: a total of 120 women were included in the study. Histological analysis of the cervical cones revealed 7 cases of cervicitis, 22 of CIN1, 31 of CIN2, 54 of CIN3, and 6 invasive carcinomas. The cervical screening samples were analyzed before conization for the presence of HPV-DNA by PCR using the consensus primers PGMY09/11. HPV-DNA-positive samples were tested for the presence of HPV16, 18, 31 and 45 using type-specific primers for these HPV. RESULTS: HPV-DNA was detected in 67.5% of the studied women. HPV 16 (40%) was the most prevalent type in most ilesions, followed by HPV 31 (13.3%), 45 (13.3%), and 18 (4.1%). Multiple infections occurred in 15% of the cases and infections with other HPV types were detected in 14% of the sample. CONCLUSIONS: HPV 16 and 18 infections do not always occur as a single infection, and may be associated with other HPV types on different occasions.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2010;32(7):315-320
DOI 10.1590/S0100-72032010000700002
PURPOSE: to determine the prevalence of HPV 16, 18, 31 and 45 in cervical screening samples of women with cellular changes and/or colposcopy suggestive of persistent high grade or low grade lesion who were submitted to conization. METHODS: a total of 120 women were included in the study. Histological analysis of the cervical cones revealed 7 cases of cervicitis, 22 of CIN1, 31 of CIN2, 54 of CIN3, and 6 invasive carcinomas. The cervical screening samples were analyzed before conization for the presence of HPV-DNA by PCR using the consensus primers PGMY09/11. HPV-DNA-positive samples were tested for the presence of HPV16, 18, 31 and 45 using type-specific primers for these HPV. RESULTS: HPV-DNA was detected in 67.5% of the studied women. HPV 16 (40%) was the most prevalent type in most ilesions, followed by HPV 31 (13.3%), 45 (13.3%), and 18 (4.1%). Multiple infections occurred in 15% of the cases and infections with other HPV types were detected in 14% of the sample. CONCLUSIONS: HPV 16 and 18 infections do not always occur as a single infection, and may be associated with other HPV types on different occasions.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2009;31(4):189-195
DOI 10.1590/S0100-72032009000400006
PURPOSE: to identify species of lactobacillus isolated from the vaginal contents of healthy and asymptomatic women, determining the most prevalent species and characterizing them phenotypically. METHODS: lactobacillus have been isolated in selective milieu from samples of the vaginal contents of 135 women without complaints of vaginal secretion, and with negative laboratorial diagnosis of vaginal infection, followed up at an outpatient clinic. After being identified by multiplex PCR, the isolates have been submitted to RNAr 16S gene sequencing, when necessary. They have also been evaluated concerning the production of lactic acid, H2O2, bacteriocins and the ability to adhere to epithelial cells. RESULTS: eight-three lactobacillus strains were isolated and identified, L. crispatus (30.1%), L. jensenii (26.5%), L. gasseri (22.9%) e L. vaginalis (8.4%), being the prevalent species. Only 20 of those isolates did not present H2O2 production, in detectable amounts. From the 37 strains selected for the test of adhesion to the epithelial cells, 12 presented 50 to 69% of adhesion, 10 presented 70% or more, and the remaining, little or no adhesion at all. None of the tested strains produced bacteriocins. CONCLUSIONS: the lactobacillus species more prevalent in women without vulvovaginitis, isolated in selective culture milieu and identified by molecular methods were L. crispatus, L. jensenii and L. gasseri. Besides the fact of being more prevalent, these strains also presented better production of H2O2, and reached lower pH values in the culture milieu.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2009;31(4):189-195
DOI 10.1590/S0100-72032009000400006
PURPOSE: to identify species of lactobacillus isolated from the vaginal contents of healthy and asymptomatic women, determining the most prevalent species and characterizing them phenotypically. METHODS: lactobacillus have been isolated in selective milieu from samples of the vaginal contents of 135 women without complaints of vaginal secretion, and with negative laboratorial diagnosis of vaginal infection, followed up at an outpatient clinic. After being identified by multiplex PCR, the isolates have been submitted to RNAr 16S gene sequencing, when necessary. They have also been evaluated concerning the production of lactic acid, H2O2, bacteriocins and the ability to adhere to epithelial cells. RESULTS: eight-three lactobacillus strains were isolated and identified, L. crispatus (30.1%), L. jensenii (26.5%), L. gasseri (22.9%) e L. vaginalis (8.4%), being the prevalent species. Only 20 of those isolates did not present H2O2 production, in detectable amounts. From the 37 strains selected for the test of adhesion to the epithelial cells, 12 presented 50 to 69% of adhesion, 10 presented 70% or more, and the remaining, little or no adhesion at all. None of the tested strains produced bacteriocins. CONCLUSIONS: the lactobacillus species more prevalent in women without vulvovaginitis, isolated in selective culture milieu and identified by molecular methods were L. crispatus, L. jensenii and L. gasseri. Besides the fact of being more prevalent, these strains also presented better production of H2O2, and reached lower pH values in the culture milieu.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2008;30(9):437-444
DOI 10.1590/S0100-72032008000900003
PURPOSE: to verify the accuracy of uterine cervix cytology for HPV diagnosis, as compared to polymerase chain reaction (PCR) in samples of women with HIV. METHODS: 158 patients who had undergone a first collection of material from the uterine cervix with Ayre's spatula for PCR were included in the study. Then, another collection with Ayre's spatula and brush for oncotic cytology was performed. Only 109 slides were reviewed, as 49 of them had already been destructed for have being filed for over two years. RESULTS: the prevalence of HPV was 11% in the cytological exam and 69.7% in the PCR. Age varied from 20 to 61 years old, median 35 years. The HIV contagious route was heterosexual in 91.8% of the cases, and 79.1% of the patients had had from one to five sexual partners along their lives. The most frequent complaint was pelvic mass (5.1%), and 75.3% of the women had looked for the service for a routine medical appointment. The categorical variable comparison was done through contingency tables, using the χ2 test with Yates's correction to compare the ratios. The Fisher's test was used when one of the expected rates was lower than five. In the comparison of diagnostic tests, sensitivity, specificity and similarity ratios have been calculated. Among the 76 patients with HPV, detected by PCR, only 12 had the diagnosis confirmed by cytology (sensitivity=15.8%), which on the other hand did not present any false-positive results (specificity=100%). Concerning the HPV presence, the cytological prediction for positive results was 100% and 33.3% for negative, when both results were compared. Among the 12 patients with HPV positive cytology, four (33.3%) presented cervical intraepithelial neoplasia (OR=56; positive similarity ratio=positive infinity; negative similarity ratio=0.83). CONCLUSIONS: As the cytology specificity is quite high, it is possible to rely on the positive result, which means that a positive result will surely indicate the presence of HPV. The low sensitivity of cytology does not qualify it as a survey exam for HPV detection in this female group.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 2008;30(9):437-444
DOI 10.1590/S0100-72032008000900003
PURPOSE: to verify the accuracy of uterine cervix cytology for HPV diagnosis, as compared to polymerase chain reaction (PCR) in samples of women with HIV. METHODS: 158 patients who had undergone a first collection of material from the uterine cervix with Ayre's spatula for PCR were included in the study. Then, another collection with Ayre's spatula and brush for oncotic cytology was performed. Only 109 slides were reviewed, as 49 of them had already been destructed for have being filed for over two years. RESULTS: the prevalence of HPV was 11% in the cytological exam and 69.7% in the PCR. Age varied from 20 to 61 years old, median 35 years. The HIV contagious route was heterosexual in 91.8% of the cases, and 79.1% of the patients had had from one to five sexual partners along their lives. The most frequent complaint was pelvic mass (5.1%), and 75.3% of the women had looked for the service for a routine medical appointment. The categorical variable comparison was done through contingency tables, using the χ2 test with Yates's correction to compare the ratios. The Fisher's test was used when one of the expected rates was lower than five. In the comparison of diagnostic tests, sensitivity, specificity and similarity ratios have been calculated. Among the 76 patients with HPV, detected by PCR, only 12 had the diagnosis confirmed by cytology (sensitivity=15.8%), which on the other hand did not present any false-positive results (specificity=100%). Concerning the HPV presence, the cytological prediction for positive results was 100% and 33.3% for negative, when both results were compared. Among the 12 patients with HPV positive cytology, four (33.3%) presented cervical intraepithelial neoplasia (OR=56; positive similarity ratio=positive infinity; negative similarity ratio=0.83). CONCLUSIONS: As the cytology specificity is quite high, it is possible to rely on the positive result, which means that a positive result will surely indicate the presence of HPV. The low sensitivity of cytology does not qualify it as a survey exam for HPV detection in this female group.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 1999;21(3):127-131
DOI 10.1590/S0100-72031999000300002
Purpose: the frequency of point mutation at codon 12 of the K¾ras gene was determined in paraffin blocks of surgical specimens from patients who had ductal invasive breast cancer. Material and Methods: Fifty surgical specimens blocked in paraffin from patients with ductal invasive breast cancer, with histological degree II and III, were used. The polymerase chain reaction (PCR) was used for amplification of DNA fragments studied. The material cleavage was obtained with restriction fragment length polymorphisms (RFLP). The electrophoresis in agarose gel, with Ladder 123 (GIBCO-BRL) marker, was employed to verify if some mutation had occurred. The results were shown using ultraviolet beam and recorded by photos. Results: mutations at codon 12 of K-ras gene were found in five samples (10%) and all of them were polymorphic for this caracter. The five patients whose tumors expressed mutation were in the postmenopausal period. Four patientes had tumors of histological degree II and one, III.
Summary
Revista Brasileira de Ginecologia e Obstetrícia. 1999;21(3):127-131
DOI 10.1590/S0100-72031999000300002
Purpose: the frequency of point mutation at codon 12 of the K¾ras gene was determined in paraffin blocks of surgical specimens from patients who had ductal invasive breast cancer. Material and Methods: Fifty surgical specimens blocked in paraffin from patients with ductal invasive breast cancer, with histological degree II and III, were used. The polymerase chain reaction (PCR) was used for amplification of DNA fragments studied. The material cleavage was obtained with restriction fragment length polymorphisms (RFLP). The electrophoresis in agarose gel, with Ladder 123 (GIBCO-BRL) marker, was employed to verify if some mutation had occurred. The results were shown using ultraviolet beam and recorded by photos. Results: mutations at codon 12 of K-ras gene were found in five samples (10%) and all of them were polymorphic for this caracter. The five patients whose tumors expressed mutation were in the postmenopausal period. Four patientes had tumors of histological degree II and one, III.
