Incidence of congenital CMV in children at a hearing rehabilitation center.

OBJECTIVE: To evaluate the incidence of children with congenital CMV (cCMV) infection in a hearing rehabilitation center. METHODOLOGY: This was a retrospective review of 309 children followed in a rehabilitation center for mild to total sensorineural hearing loss (SNHL). Seventy-five children had dried blood spots that we retrieved and retrospectively analyzed for the presence of CMV DNA by real time PCR. The children were born in Belgium after January 1996. The etiology of the SNHL was investigated for each child. RESULTS: The CMV DNA was detected in the dried blood spots for 8 of the 75 children tested (10.6%) by real time PCR. In three children, an alternative etiology of SNHL was suspected before the cCMV infection was diagnosed. CONCLUSIONS: The incidence of children infected with cCMV in a hearing rehabilitation center is high (10.6%). The detection of CMV DNA in dried blood spots is useful and improves the etiological diagnosis of SNHL.

Galactomannan enzymatic immunoassay cross-reactivity caused by Prototheca species.

We report a reactive Aspergillus galactomannan enzymatic immunoassay against the serum of a patient with invasive Prototheca zopfii infection. Analysis of the supernatants of suspensions of P. zopfii and other Prototheca isolates revealed positive results as well. These data suggest cross-reactivity with the serum Aspergillus galactomannan assay in invasive protothecosis.

Production of recombinant antigens of Ureaplasma parvum serotypes 3 and 6 for development of a serological assay.

Recombinant antigens of Ureaplasma parvum serotypes 3 and 6 were produced in order to develop a serological assay for Ureaplasma antibody detection. The genes of the multiple banded antigen (MBA) were amplified by PCR and cloned in a pTrcHis TOPO plasmid. Purified recombinant proteins were evaluated in Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies and human sera. Our approach was successful in the production of the recombinant MBAs (rMBAs) for serotypes 3 and 6. The antigens tested positive with serotype-specific monoclonal antibodies in Western blotting and in ELISA. Prominent reactions were detected with the rMBAs and their homologous monoclonal antibodies. Strong cross-reactions were visible in ELISA between rMBA 3 and the monoclonal antibodies from the other U. parvum serotypes. A weak cross-reaction was seen with rMBA 3 and the monoclonal antibody from serotype 4. rMBA 6 showed cross-reaction only with the monoclonal antibody from U. parvum serotype 1. Fifty-one percent of the sera obtained from culture-positive women reacted with one or both rMBAs. Only three (15%) of the sera from culture-negative women reacted with the rMBA. The positive reactions were observed only with rMBA 6. These preliminary tests showed the potential usefulness of the rMBAs produced for detecting an antibody response against Ureaplasma antigens.

Multicenter evaluation of strategies for serodiagnosis of primary infection with Toxoplasma gondii.

The diagnostic performance of single-serum assays for toxoplasma-specific immunoglobulin (Ig)M. IgA. IgG, and IgE antibodies and of different combinations of such antibody assays in 20 European reference centers was assessed. A panel of 276 sera, of which 73 came from patients who seroconverted within 3 months (acute infection), 49 from patients who had seroconverted 3-12 months earlier (convalescence), and 154 from subjects who had two IgG-positive samples obtained 12 months apart (past infection), was tested with 20 toxoplasma-antibody assays and 195 combinations. In general, every assay with high diagnostic sensitivity showed low diagnostic specificity, i.e. no assay performed alone could reliably distinguish acute from past infection. Furthermore, no single assay (or combination) could separate convalescence from the other stages of toxoplasma infection. However, excellent diagnostic performances were reached by sequential use of highly sensitive IgM assays and methods examining IgG avidity or stage specificity. IgA or IgM assays were less suitable for confirmation of toxoplasma-IgM positivity. This study documents the strength of test combinations in assessing the stage of toxoplasma infection.

Development of an enzyme-linked immunosorbent assay for serotyping ureaplasma urealyticum strains using monoclonal antibodies.

Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains of U. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.

Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula.

We describe an outbreak of necrotizing enterocolitis (NEC) that occurred in the neonatal intensive care unit of our hospital. A total of 12 neonates developed NEC in June-July 1998. For two of them, twin brothers, the NEC turned out to be fatal. Enterobacter sakazakii, a known contaminant of powdered milk formula, was isolated from a stomach aspirate, anal swab, and/or blood sample for 6 of the 12 neonates. A review of feeding procedures revealed that 10 of the 12 patients were fed orally with the same brand of powdered milk formula. E. sakazakii was isolated from the implicated prepared formula milk as well as from several unopened cans of a single batch. Molecular typing by arbitrarily primed PCR (AP-PCR) confirmed, although partially, strain similarity between milk and patient isolates. No further cases of NEC were observed after the use of the contaminated milk formula was stopped. With this outbreak we show that intrinsic microbiological contamination of powdered milk formula can be a possible contributive factor in the development of NEC, a condition encountered almost exclusively in formula-fed premature infants. The use of sterilized liquid milk formula in neonatal care could prevent problems with intrinsic and extrinsic contamination of powdered milk formula.

Prevention of congenital toxoplasmosis.

In utero infection with Toxoplasma gondii may result in congenital defects such as hydrocephalus, chorioretinitis and mental retardation; these defects may be present at birth or may develop later in life. Prevention of this disease can be achieved in different ways. The most effective measure is to prevent the acquisition of the disease during pregnancy by avoiding risk factors for Toxoplasma gondii infection. Health education may decrease the incidence of toxoplasmosis during pregnancy by 60%. A second preventive measure is based on serologic screening during pregnancy to identify infected women. Treatment during pregnancy results in a significant reduction in the incidence of sequelae including severe handicaps. A third possible intervention is treating infected neonates. Antibiotic treatment of infected children has a beneficial effect on the development of sequelae and the sooner therapy is started after birth, the better the outcome. This overview presents the potential benefits and harms of these different options available for the prevention of congenital toxoplasmosis.

A rare manifestation of Kingella kingae infection.

Kingella kingae is an aerobic gram-negative coccobacillus that has been associated predominantly with bone and joint infection but also with septicemia and endocarditis. Until now, only four cases of proven K. kingae meningitis have been reported. We describe a case of a K. kingae meningitis in a male adolescent who presented with a history of fever of unclear origin.

Development of monoclonal antibodies against Ureaplasma urealyticum serotypes and their use for serotyping clinical isolates.

Monoclonal antibodies (MAbs) against Ureaplasma urealyticum serotype 2, 5, 7, 8, 10, 11, 12, and 13 reference strains were developed. The reactivities of these MAbs with the 14 serotype reference strains was verified by colony immunofluorescence assay and Western blot assay. MAbs against serotypes 2, 7, 10, 11, and 12 were serotype specific, whereas MAbs against serotypes 5, 8, and 13 showed cross-reactivity. All MAbs against serotype 5 were cross-reactive with serotype 2, and one showed, in addition, cross-reactivity to serotypes 9 and 10. Mutual cross-reactivities were observed between MAbs against serotypes 8 and 13. The usefulness of the MAbs for the serotyping of U. urealyticum strains was evaluated by serotyping 21 selected clinical isolates. A complete set of MAbs (the newly developed MAbs and the previously described MAbs against serotypes 1, 3, 4, 6, 9, and 14) as well as a complete set of polyclonal antibodies (PAbs), PAbs 1 to 14, were used. MAbs were able to identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more valuable tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity.

Diagnosis of congenital toxoplasmosis in the neonatal period: A multicenter evaluation.

OBJECTIVE: To evaluate different laboratory tests used to diagnose congenital toxoplasmosis in the neonatal period. STUDY DESIGN: A retrospective multicenter study of 294 pregnant women who experienced seroconversion for Toxoplasma gondii and subsequently delivered live-born infants. Fetal infection was assessed via specific IgM and IgA antibodies (cord and neonatal blood) and detection of T gondii in placenta and cord blood by mouse inoculation. RESULTS: Ninety-three (32%) of the 294 infants were congenitally infected. The sensitivity of IgA in cord blood and in neonatal blood was 64% and 66%; the sensitivity of IgM was 41% and 42%, respectively. Mouse inoculation of the placenta and cord blood had sensitivities of 45% and 16%. Positive results of the serologic tests in congenitally infected children correlated significantly with the gestational age at the time of maternal infection but was not significantly influenced by the administration of specific antiparasitic treatment during pregnancy. CONCLUSION: Specific T gondii IgA antibody is a more sensitive test than IgM for detecting congenital toxoplasmosis in the neonatal period. The overall specificity is better for serologic tests performed on neonatal blood than for those on cord blood. Neonatal screening with IgM or IgA antibodies will not detect the majority of children with congenital toxoplasmosis when the maternal infection occurred before the 20th week of pregnancy.

Prenatal diagnosis of congenital toxoplasmosis: a multicenter evaluation of different diagnostic parameters.

OBJECTIVE: Our purpose was to evaluate different methods of diagnosing congenital toxoplasmosis prenatally by amniocentesis and cordocentesis. STUDY DESIGN: In a retrospective multicenter study, we investigated consecutive women who had seroconversion for Toxoplasma gondii during pregnancy and who underwent either amniocentesis or cordocentesis or both to obtain a prenatal diagnosis of fetal toxoplasmosis. Data were obtained from 122 patients recruited in 6 different European Toxoplasma reference centers. Infants born to these mothers were followed up until 1 year of age to confirm or exclude congenital toxoplasmosis. Sensitivity, specificity, positive predictive value, and negative predictive value were measured for the following parameters: (1) detection of the parasite in amniotic fluid by mouse inoculation, (2) detection of the parasite in amniotic fluid by in vitro cell culture, (3) detection of Toxoplasma deoxyribonucleic acid in amniotic fluid by a polymerase chain reaction assay, (4) detection of the parasite in fetal blood by mouse inoculation, (5) detection of specific immunoglobulin M antibodies in fetal blood, and (6) detection of specific immunoglobulin A antibodies in fetal blood. RESULTS: The polymerase chain reaction test performed on amniotic fluid had the highest level of sensitivity (81%) and also a high level of specificity (96%). The combination of the polymerase chain reaction test and mouse inoculation of amniotic fluid increased sensitivity to 91%. The sensitivity of immunoglobulins M and A in fetal blood was 47% and 38%, respectively. In congenitally infected fetuses a negative correlation was observed between positive serologic parameters and gestational age at the time of maternal infection and at prenatal diagnosis. CONCLUSION: Congenital toxoplasmosis is best predicted by prenatal examination with the combination of T gondii polymerase chain reaction and mouse inoculation of amniotic fluid. The role of cordocentesis in the diagnosis of congenital toxoplasmosis is limited.

Neonatal screening for congenital cytomegalovirus infections.

We evaluated a screening program for the detection of congenital cytomegalovirus in 3075 unselected pregnant women. From each live-born child urine for CMV culture was collected within 7 days after birth. Each fetus expelled after a spontaneous second trimester abortion and each stillborn infant were also evaluated for a possible congenital CMV infection. For each congenital infection stored maternal sera were analysed to determine whether maternal infection was primary or recurrent. Fifteen out of the 3075 pregnancies studied resulted in a congenitally infected infant (0.49%). Nine maternal CMV infections were primary infections; five were recurrent infections, and in one case the type of infection could not be determined. Three congenital infections resulted in severe sequelae, leading to the termination of pregnancy in two instances and to neonatal death in one case. One of these severe fetal infections was due to a recurrent maternal infection. Follow-up of the other 12 neonates demonstrated hearing disorders in two children. One was born after a primary maternal infection and one after a recurrent maternal infection. We conclude that congenital CMV infections occurs in 0.49% of all pregnancies in the population studied. Twenty percent of the congenitally infected infants present severe sequelae at birth or during pregnancy, and an additional 17% have audiological deficits at 1 year of age. Severe sequelae may occur after both primary and recurrent maternal CMV infection.

Treatment of toxoplasmosis during pregnancy: a multicenter study of impact on fetal transmission and children's sequelae at age 1 year.

OBJECTIVE: Toxoplasmosis during pregnancy can cause fetal infection, with unpredictable sequelae in later life. We measured the effects of prenatal antibiotic therapy on the fetomaternal transmission of Toxoplasma gondii and on the appearance of sequelae in the congenitally infected child at age 1 year. STUDY DESIGN: In a multicenter study we investigated consecutive women with Toxoplasma seroconversion during pregnancy. Data were obtained from 144 women recruited in 5 different Toxoplasma reference centers. Through multivariate analysis we assessed the association between transmission and appearance of sequelae as a function of the following parameters: estimated gestational age at infection, administration of antibiotic therapy, duration of antibiotic therapy, and time lapse between infection and the start of antibiotic therapy. RESULTS: Sixty-four of the 144 women (44%) gave birth to a congenitally infected infant. Multivariate analysis showed that transmission was predicted neither by whether antibiotics had been administered nor by the time lapse between infection and the start of antibiotic therapy, but only by the gestational age at which maternal infection occurred (P <.0001). Sequelae were found in 19 children (13%), 9 of whom (6%) had severe sequelae. Administration of antibiotics was predictive of the absence of sequelae (P =.026, odds ratio 0.30, 95% confidence interval 0.104-0.863), in particular the absence of severe sequelae (P =.007, odds ratio 0.14, 95% confidence interval 0.036-0.584). The sooner antibiotics were given after the infection, the less frequently sequelae were seen (P =. 021). CONCLUSION: Prenatal antibiotic therapy after toxoplasmosis during pregnancy had no impact on the fetomaternal transmission rate but reduced the rate of sequelae among the infected infants. The early start of treatment resulted in a significant reduction in the number of severely affected infants.

A second European collaborative study on polymerase chain reaction for Toxoplasma gondii, involving 15 teams.

In order to investigate the accuracy and practicability of the polymerase chain reaction (PCR) in the antenatal diagnosis of congenital toxoplasmosis, a collaborative study involving 15 European laboratories was performed under the auspices of the Biomed 2 Programme of the European Community. Each team received 12 aliquots (four negative, eight positive) of 'artificial samples' made of amniotic fluid spiked with tachyzoites of the RH strain of Toxoplasma gondii. Each team performed its own PCR protocol (all were different). Nine of the 15 laboratories were able to detect a single parasite, but two of the 15 found all samples negative. Four of the 15 laboratories found one or more control samples to be falsely positive. This study highlights the lack of homogeneity between PCR protocols and performance and underlines the need for an external quality assurance scheme which could provide 'reference' samples that could be used by any laboratory wanting to establish and maintain an accurate diagnostic test based on PCR.

Discordant prenatal diagnosis of congenital toxoplasmosis in a dizygotic pregnancy.

Prenatal diagnosis of toxoplasmosis gondii in twin pregnancies has been described twice. In both cases they were accomplished by prenatal blood sampling of the foetuses. We report the first prenatal diagnosis with a discordant result in a dizygotic pregnancy. One of the foetuses died in utero and the other was born unaffected at term.

Development of a monoclonal antibody to a Ureaplasma urealyticum serotype 9 antigen.

We produced a monoclonal antibody (MAb) to Ureaplasma urealyticum Vancouver, the serotype 9 standard strain. By immunoblotting, this MAb showed a single, 85-kDa band with the homologous serotype and a minor, 100-kDa band with serotype 2 but did not react with any other serotype standard strain. Clinical isolates of U. urealyticum were tested with this MAb and with two sets of polyclonal antisera against the 14 serotype standard strains. The use of MAb 9-2H9 correctly identified certain serotype 9 strains but did not react with wild-type strains lacking the serotype 9 determinant.

Markers of infection and their relationship to preterm delivery.

In this study we evaluated different markers of infection and their relationship to preterm delivery. Forty-four consecutive women with singleton pregnancies in uncomplicated preterm labor were investigated. C-reactive protein (CRP) in peripheral maternal blood, amniotic fluid cytokines, amniotic fluid leukocyte count, and amniotic fluid culture were performed in all patients. Thirty-six patients responded to standard tocolytic therapy and delivered after 34 weeks' gestation. In eight patients treatment failed and they delivered before 34 weeks' gestation. Two of these eight patients had a positive amniotic fluid culture for Ureaplasma urealyticum. The positive culture was accompanied by an elevated neutrophil count in the amniotic fluid. Elevated amniotic fluid levels of tumor necrosis factor (TNF) (more than 23 pg/mL), interleukin-6 (IL-6) (more than 2292 pg/mL) and interleukin-8 (more than 164 pg/mL) correlated with early preterm delivery. CRP levels in serum had a low sensitivity (38%) but a high specificity (94%) in predicting preterm delivery. This study indicates that preterm labor can be initiated by infection. Markers of infection obtained by amniocentesis have a better sensitivity and positive predictive value than noninvasive markers. Elevated IL-6 (more than 2292 pg/mL) seems to be the best predictor for preterm delivery, with a sensitivity of 75% and a specificity of 97%.

Identification of serotype 1-, 3-, and 6-specific antigens of Ureaplasma urealyticum by using monoclonal antibodies.

Little is known about the antigens responsible for serotype specificity in Ureaplasma urealyticum. We produced monoclonal antibodies to U. urealyticum serotypes 1, 3, and 6, the serotypes most commonly found in pregnant women, and analyzed serotype-specific antigens for the three serotypes. Clinical isolates belonging to serotype 1, 3, or 6 were tested in immunoblots with these monoclonal antibodies. The immunoblot patterns of these isolates were, in most cases, different from each other as well as from those of the reference strains, indicating a high rate of antigenic variation among U. urealyticum strains.