Effect of Genital Sampling Site on the Detection and Quantification of Ureaplasma Species with Quantitative Polymerase Chain Reaction during Pregnancy.

Objective. This study aimed to compare the qualitative and quantitative reproducibility of quantitative PCR (qPCR) for Ureaplasma species (Ureaplasma spp.) throughout pregnancy and according to the genital sampling site. Study Design. Between 5 and 14 weeks of gestation (T1), vaginal, fornix, and two cervical samples were taken. Sampling was repeated during the 2nd (T2) and 3rd (T3) trimester in randomly selected T1 positive and negative women. Qualitative and quantitative reproducibility were evaluated using, respectively, Cohen's kappa (κ) and interclass correlation coefficients (ICC) and repeated measures ANOVA on the log-transformed mean number of DNA copies for each sampling site. Results. During T1, 51/127 women were positive for U. parvum and 8 for U. urealyticum (4 patients for both). Sampling was repeated for 44/55 women at T2 and/or T3; 43 (97.7%) remained positive at the three timepoints. κ ranged between 0.83 and 0.95 and the ICC for cervical samples was 0.86. Conclusions. Colonization by Ureaplasma spp. seems to be very constant during pregnancy and vaginal samples have the highest detection rate.

Primary maternal cytomegalovirus infections: accuracy of fetal ultrasound for predicting sequelae in offspring.

BACKGROUND: Cytomegalovirus infection is the most common perinatal viral infection that can lead to severe long-term medical conditions. Antenatal identification of maternal cytomegalovirus infections with proven fetal transmission and potential postnatal clinical sequelae remains a major challenge in perinatology. There is a need to improve the prenatal counseling offered to patients and guide future clinical management decisions in cases of proven primary cytomegalovirus infection. OBJECTIVE: We sought to evaluate the accuracy of fetal ultrasound for predicting sequelae in fetuses infected with congenital cytomegalovirus after maternal primary infection. STUDY DESIGN: We conducted a prospective observational study from 1996 through 2012 in pregnant women with serological evidence of primary cytomegalovirus infection and proven vertical transmission to the fetus, based on viral load in the amniotic fluid. Fetal ultrasound was performed in all patients. Pregnancy termination was presented as an option for infected fetuses. Hearing and neurological clinical assessments were performed for all neonates with cytomegalovirus-positive urine samples. RESULTS: A total of 67 patients (69 fetuses) with proven vertical transmission were included in this study, including 64 singleton and 3 twin pregnancies. Eight fetuses were lost to follow-up. Of the remaining 61 fetuses, termination of the pregnancy was performed for 26, including 11 with fetal ultrasound anomalies. Autopsy provided histological evidence of fetal cytomegalovirus infection in all cases. In the 15 terminated fetuses without ultrasound anomalies, histological evidence of damage caused by fetal infection was detected in 13 cases. Among the 35 live-born infants, 12 had fetal ultrasound anomalies suggestive of congenital infection. Of these 12 infants, 6 had normal clinical evaluations, whereas 6 presented with either hearing and/or neurological anomalies, classified as severe in 4 cases. Among the 23 live-born infants with normal prenatal ultrasound, 5 developed hearing impairments and 1 showed mild neurological developmental delay. CONCLUSION: Fetal ultrasound anomalies were detected in 37.7% of pregnant women with primary cytomegalovirus infection acquired in early pregnancy and proven fetal infection, and were confirmed by autopsy or postnatal clinical evaluation in 73.9%. Autopsy or postnatal clinical evaluation also detected cytomegalovirus-related anomalies in 55% of infants with normal fetal ultrasound evaluations.

Anaerobic bacteraemia: a 10-year retrospective epidemiological survey.

In order to identify current trends in anaerobic bacteraemia, a 10-year retrospective study was performed in the University Hospital Brussel, Belgium. All clinically relevant bacteraemia detected from 2004 until 2013 were included. Medical records were reviewed in an attempt to define clinical parameters that might be associated with the occurrence of anaerobic bacteraemia. 437 of the isolated organisms causing anaerobic bacteraemia were thawed, subcultured and reanalyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). There were an average of 33 cases of anaerobic bacteraemia per year during 2004-2008 compared to an average of 27 cases per year during 2009-2013 (P = 0.017), corresponding to a decrease by 19% between the first and the latter period. Also, the total number of cases of anaerobic bacteraemia per 100,000 patient days decreased from 17.3 in the period from 2004 to 2008 to 13.7 in the period 2009 to 2013 (P = 0.023). Additionally, the mean incidence of anaerobic bacteraemia decreased during the study period (1.27/1000 patients in 2004 vs. 0.94/1000 patients in 2013; P = 0.008). In contrast, the proportion of isolated anaerobic bacteraemia compared to the number of all bacteraemia remained stable at 5%. Bacteroides spp. and Parabacteroides spp. accounted for 47.1% of the anaerobes, followed by 14.4% Clostridium spp., 12.6% non-spore-forming Gram-positive rods, 10.5% anaerobic cocci, 8.2% Prevotella spp. and other Gram-negative rods and 7.1% Fusobacterium spp. The lower gastrointestinal tract (47%) and wound infections (10%) were the two most frequent sources for bacteraemia, with the origin remaining unknown in 62 cases (21%). The overall mortality rate was 14%. Further studies focusing on the antimicrobial susceptibility and demographic background of patients are needed to further objectify the currently observed trends.

Association of abnormal vaginal flora and Ureaplasma species as risk factors for preterm birth: a cohort study.

OBJECTIVE: To find out whether the presence of Ureaplasma species (U. spp.) in combination with an alteration of the normal vaginal flora is an independent risk factor for preterm delivery. DESIGN: Prospective observational study. SETTING: Department of Obstetrics, University Hospital in Brussels. POPULATION: A total of 1,988 singleton pregnancies were included. METHODS: From each woman, a cervical culture for U. spp. was obtained and the vaginal flora evaluated at the first prenatal visit. The presence of known risk factors for preterm delivery was recorded. Preterm birth was defined as delivery < 37 weeks. RESULTS: There were 97 (4.9%) preterm births. In patients delivered before 37 weeks, abnormal vaginal flora was detected in 22.7% and U. spp. in 53.6% of these. The conditions were found together in 17.5%. In patients delivered at term, an abnormal vaginal flora was detected in 14.4% and U. spp. in 41.4% of these women, while they co-existed in 8.2%. Using a logistic regression analysis taking into account known risk factors for preterm birth and the microbiological parameters, preterm delivery was correlated with the presence of U. spp. (odds ratio (OR) 1.64; 95% confidence interval (CI) 1.08-2.48; p = 0.02) and abnormal vaginal flora in combination with U. spp. (OR 2.35; 95% CI 1.35-4.10; p = 0.003). No significant correlation between the presence of abnormal vaginal flora and preterm delivery (p = 0.09) was found. CONCLUSIONS: Preterm delivery was significantly correlated with the presence of U. spp. The risk for preterm delivery increased when U. spp. was associated with an abnormal vaginal flora.

Evaluation of different cytomegalovirus (CMV) DNA PCR protocols for analysis of dried blood spots from consecutive cases of neonates with congenital CMV infections.

Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (P = 0.036).

A 10-year prospective study of sensorineural hearing loss in children with congenital cytomegalovirus infection.

OBJECTIVE: To determine the incidence, characteristics, and evolution of sensorineural hearing loss (SNHL) in infants with a congenital cytomegalovirus infection (cCMV). STUDY DESIGN: In a prospective 10-year study, 14 021 unselected live-born infants were screened for cCMV by virus isolation in urine. Congenitally infected newborns were evaluated for SNHL during the first 5 years of life. RESULTS: A total of 74 of the 14 021 infants (0.53%) were congenitally infected; of these, 4 (5.4%) were symptomatic at birth. Hearing testing could be performed in 60 of the infants. SNHL was found in 21% of the asymptomatic and in 33% of symptomatic congenitally infected infants. Late-onset hearing loss was detected in 5%, progression in 11%, fluctuation in 16%, and improved hearing threshold in 18% of the infants with cCMV. SNHL was observed in 15% of infected infants born after a maternal primary infection, in 7% born after a maternal recurrent infection, and in 40% after a maternal infection of indeterminate timing. CONCLUSIONS: In our study population, 0.53% of the infants had cCMV infection, 22% of whom developed SNHL. Long-term follow up and repeated audiologic testing is needed, because progression, fluctuation, improvement, and late-onset hearing loss are important features of cCMV infection. The search for a neonatal screening program to detect all cCMV is worthwhile.

Detection of CMV DNA in the perilymph of a 6-year-old boy with congenital cytomegalovirus infection.

We present the case of a 6-year-old boy who received a cochlear implant for profound sensorineural hearing loss after being born with cytomegalovirus (CMV) infection. Even after 6 years, CMV DNA was still found in the perilymph of the cochlea. Our case shows that CMV DNA can be present in the cochlea years after congenital CMV infection, and it can explain why progressive and/or late-onset hearing loss occurs in these children.

Comparison of two methods for serotyping Ureaplasma urealyticum clinical isolates.

A newly developed enzyme linked immunosorbent assay (ELISA) method using monoclonal antibodies (MAbs) to the 14 serotypes of Ureaplasma urealyticum was compared to immunofluorescence assay (IFA) for serotyping U. urealyticum clinical isolates. Of the 102 vaginal isolates of U. urealyticum, five strains were lost and were excluded from analysis. Of the 97 strains analysed, a total of 86 (89%) strains were typeable by ELISA and a total of 89 (92%) strains were typeable by IFA. Eighty-six strains were typeable by both methods, three by IFA only and eight strains were not typeable neither by ELISA nor by IFA. Of the 86 strains typeable by both methods, complete concordance in serotyping results was found. The three strains not typeable by ELISA were typeable as serotype 4 by IFA. These three strains were reanalysed by ELISA after major modifications of the antigen preparation and were typeable as serotype 4. In conclusion, the ELISA was found suitable for serotyping clinical isolates. However, since the ELISA had a somewhat lower performance than IFA, strains not typeable by ELISA, should be retested by another technique such as IFA.

Hearing thresholds in children with a congenital CMV infection: a prospective study.

OBJECTIVE: Hearing thresholds in children with a congenital cytomegalovirus (cCMV) infection are not always stable. Children can develop late onset hearing loss, fluctuations, progression (worsening) and improvement of hearing loss. Knowledge about these characteristics is important to understand why long term follow up in these children is mandatory. METHODS: We prospectively follow a cohort of 154 children with cCMV infection, 68 of which met the inclusion criteria of at least 3 hearing evaluations over a period of at least 18 months in the absence of other risk factors for hearing loss. In those 68 children we evaluated the occurrence of unstable hearing thresholds: late onset hearing loss, fluctuations, progression and improvement of hearing loss. RESULTS: Unstable hearing thresholds were observed in 29.4% of children with cCMV infection of which 19.2% were found in the group of children with ultimately normal hearing and in 62.5% of children with sensorineural hearing loss (SNHL) (p=0.0027). Fluctuations occurred in 16.2%. Late onset hearing loss occurred in 4.3% of children with a normal hearing at birth. In children with SNHL, progression or worsening of hearing thresholds occurred in 27.3% and improvement of thresholds in 40.9%. Important changes in thresholds only occurred in 13.2% of all children and predominantly in children who finally develop SNHL. CONCLUSIONS: Unstable hearing thresholds are frequently found in children with cCMV infection and occur not only in children who develop hearing losses but also in children who have a normal hearing at the last visit. Important changes in hearing thresholds of > 30 dB are more frequently seen in children who ultimately will develop SNHL.

Modified real-time PCR for detecting, differentiating, and quantifying Ureaplasma urealyticum and Ureaplasma parvum.

We evaluated a previously described quantitative real-time PCR (qPCR) for quantifying and differentiating Ureaplasma parvum and U. urealyticum. Because of nonspecific reactions with Staphylococcus aureus DNA in the U. parvum PCR, we developed a modified qPCR and designed new primers. These oligonucleotides eradicated cross-reactions, indicating higher specificity. The detection limits of the qPCR were determined at 1 and 3 colony-forming units/ml for U. parvum and U. urealyticum, respectively. The quantification limits of the assay for both Ureaplasma species ranged from 2.10(6) to 2.10(1) copy numbers per PCR. A total of 300 patient samples obtained from the lower genital tract were tested with this newly designed qPCR assay and compared with culture results. Of the samples, 132 (44.0%) were culture positive, whereas 151 (50.3%) tested positive using qPCR. The U. parvum and U. urealyticum species were present in 79.5% and 12.6% of the qPCR-positive samples, respectively. Both species were found in 7.9% of those samples. Quantification of U. parvum and U. urealyticum in the samples ranged from less than 2.5 × 10(3) to 7.4 × 10(7) copies per specimen. In conclusion, the modified qPCR is a suitable method for rapid detection, differentiation, and quantification of U. parvum and U. urealyticum.

Hearing loss in children with congenital cytomegalovirus infection in relation to the maternal trimester in which the maternal primary infection occurred.

OBJECTIVES: The purpose of this work was to study the relation between maternal trimester of primary infection with cytomegalovirus and the occurrence of sensorineural hearing loss in the congenitally infected offspring. PATIENTS AND METHODS: Thirty-four consecutive live-born children diagnosed with a congenital cytomegalovirus infection after maternal primary cytomegalovirus infections were included in the study. Five were lost for follow-up, and 1 died. Of the remaining 28 congenitally infected children, an estimation of the maternal trimester in which cytomegalovirus primary infection occurred was performed. All of the children were investigated for potential sensorineural hearing loss. RESULTS: Five of the maternal infections occurred in the first trimester, 12 in the second trimester, and 11 in the third trimester of pregnancy. Sensorineural hearing loss was detected in 4 (80%) of the 5 congenitally infected children who were infected after a primary maternal infection in the first trimester of pregnancy and in 1 (8%) of the 12 children when the maternal infection occurred in the second trimester of pregnancy. No sensorineural hearing loss was detected after primary maternal infection occurring in the third trimester. Fluctuation and improvement of sensorineural hearing loss were seen regardless the trimester of pregnancy during which maternal primary infection occurred. Progression of sensorineural hearing loss occurred in 2 children born after a maternal primary infection of the first trimester. CONCLUSIONS: Hearing loss seemed more common in infants with congenital cytomegalovirus infection who were born to women who experienced a primary cytomegalovirus infection in the first trimester of pregnancy than when infection took place later in pregnancy.

A serologic strategy for detecting neonates at risk for congenital cytomegalovirus infection.

OBJECTIVES: To evaluate the feasibility of a serologic screening program in pregnant women to detect neonates at risk for a congenital cytomegalovirus infection. STUDY DESIGN: Unselected mother-infant pairs (n = 7140) were studied. In the mother, serologic screening consisted of the testing for cytomegalovirus antibodies at the first prenatal visit and at birth. In the neonate, cytomegalovirus urine culture was performed to diagnose congenital infection. RESULTS: Serologic screening showed evidence of past infection in 3850 women (53.9%); 192 (2.7%) women had both immunoglobulin (Ig)G and IgM antibodies when first tested during pregnancy. Seroconversion was detected in 44 seronegative women (1.4%). Forty-four congenital infections were diagnosed (0.62%): 8 in women with past infections, 22 in women who seroconverted, and 14 in women who initially had positive IgM antibodies. CONCLUSIONS: Screening at the first prenatal visit and at birth defines two major risk groups for congenital cytomegalovirus infection: women with seroconversion during pregnancy and women with IgM antibodies in their first prenatal serum sample (0.6% and 2.7%, respectively, of the pregnant population). In these selected babies (3.3% of the study group), cytomegalovirus urine culture should be performed. This type of screening allows the detection of 82% of all congenital cytomegalovirus infections.

European multicenter study of the LIAISON automated diagnostic system for determination of Toxoplasma gondii-specific immunoglobulin G (IgG) and IgM and the IgG avidity index.

The LIAISON system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles. The system allows fast and precise measurement of Toxoplasma-specific immunoglobulin G (IgG) and IgM antibody levels and measurement of the IgG avidity index even at low levels of Toxoplasma-specific IgG antibodies in a single step without manual interference. Seven European centers participated in a multicenter evaluation of the LIAISON system. The sensitivity and specificity of the LIAISON system compared to the Sabin-Feldman dye test were 99.3 and 96.8%, respectively. In a comparison of the LIAISON Toxoplasma-specific IgM assay with an immunosorbent agglutination assay, the LIAISON assay had a sensitivity of 96.7% and a specificity of 95.4%. The LIAISON IgG assay showed agreements of 91, 100, and 100% with the AXSYM IgG (Abbott), VIDAS IgG (bioMerieux), and Platelia IgG (Bio-Rad) assays, respectively. The LIAISON IgM assay showed agreements of 95% with the AXSYM IgM and Platelia IgM assays, 96% with the ISAGA IgM assay (bioMerieux), and 97% with the VIDAS IgM assay. The coefficient of correlation between the LIAISON system and the VIDAS Toxoplasma-specific IgG avidity index was 0.81. By use of the Toxoplasma-specific IgG avidity index assay with specific IgM-positive samples, the diagnosis of infection with Toxoplasma gondii in early pregnancy has been improved significantly. The LIAISON avidity assay is a valuable assay for the exclusion of recently acquired infection with T. gondii (less than 4 months) in pregnant women, and it decreases significantly the necessity for follow-up testing.

Evaluation of tRNA gene PCR for identification of mollicutes.

We evaluated the applicability of tRNA gene PCR in combination with fluorescent capillary electrophoresis with an ABI310 genetic analyzer (Applied Biosystems, Calif.) for the identification of different mollicute species. A total of 103 strains and DNA extracts of 30 different species belonging to the genera Acholeplasma, Mycoplasma, and Ureaplasma were studied. Reproducible peak profiles were generated for all samples, except for one M. genitalium isolate, the three M. gallisepticum isolates, and 8 of the 24 Ureaplasma cultures, where no amplification could be obtained. Clustering revealed numerous discrepancies compared to the identifications that had been previously obtained by means of biochemical and serological tests. Final identification was obtained by 16S rRNA gene amplification followed by sequence analysis and/or restriction digestion. This confirmed the identification obtained by tRNA gene PCR in all cases. Seven samples yielded an unexpected tRNA gene PCR profile. Sequence analysis of the 16S rRNA genes showed that six of these samples were mixed and that one had a unique sequence that did not match any of the published sequences, pointing to the existence of a not-yet-described species. In conclusion, we found tRNA gene PCR to be a rapid and discriminatory method to correctly identify a large collection of different species of the class of Mollicutes and to recognize not-yet-described groups.

Prevention of toxoplasmosis during pregnancy--an epidemiologic survey over 22 consecutive years.

BACKGROUND: Prevention of congenital toxoplasmosis is most often based on the results of a serological screening program in pregnant women followed by prenatal and postnatal treatment of women and their newborns when infection is already established during pregnancy or on cord blood (secondary prevention). Little effort has been made to study primary prevention of toxoplasmosis during pregnancy. OBJECTIVE: To assess the possibilities of two different programs aimed at preventing the acquisition of toxoplasmosis during pregnancy. METHODS: During the first study period (1979-1982) the natural incidence of toxoplasmosis in pregnancy was studied in 2986 pregnant women. In the second study period (1983-1990) the incidence of toxoplasmosis was studied in 8300 women. During this period, seronegative women received a written list of recommendations on how to avoid a toxoplasma infection during pregnancy. In the third study period (1991-2001) the incidence of toxoplasmosis was studied in 16,541 women. During this period, the prevention campaign consisted of a leaflet explaining a) toxoplasmosis as a disease and b) what measures should be taken to avoid toxoplasmosis during pregnancy. The third part of the campaign involved a reiteration of these recommendations during antenatal classes held around mid-gestation. The impact of the two prevention programs was studied by measuring the seroconversion rate in seronegative women. RESULTS: Twenty of 1403 seronegative women in the first period (1.43%), 19 of 3605 women in the second period (0.53%) and 8 of 8492 in the third period (0.09%) seroconverted during pregnancy. The first prevention campaign reduced the seroconversion rate by 63% (p<0.05 OR 2.729 95% CI 1.452-5.084). The second prevention program resulted in a reduction rate of 92% compared to the seroconversion rate in the first period (p<0.0001 OR 15.34 95% CI 6.741-34.89). CONCLUSION: Promotion of simple measures is very effective in the prevention of toxoplasmosis during pregnancy. Primary prevention should not only be based on education about preventive measures given by physicians, but these guidelines should be reiterated during antenatal classes and leaflets distributed containing written recommendations on the nature of the disease and its avoidance.