Prenatal diagnosis of congenital cytomegalovirus infection in 115 cases: a 5 years' single center experience.

OBJECTIVE: The objective of this study is to investigate the diagnostic value of invasive prenatal diagnosis (PD) of congenital cytomegalovirus (CMV) infection from amniotic fluid (AF) and fetal blood (FB). METHODS: A retrospective study was conducted on 115 pregnancies with CMV primary infection. A total of 111 AF and 106 FB samples were investigated for various virological and non-virological markers. Detailed ultrasound examinations were performed at time of PD. RESULTS: Overall sensitivity of CMV PCR in FB (75.6%; 95%CI 60-87) and AF (72.7%; 95%CI 57-85) was comparable. In women with amniocentesis >8 weeks between seroconversion and PD, we did not observe significant differences between amniocentesis performed ≥17 + 0 (sensitivity 90.9%; 95%CI 71-99) and ≥20 + 0 gestational weeks (sensitivity 90.0%; 95%CI 68-99). Virological markers in FB were higher in symptomatic compared with asymptomatic fetuses (p < 0.05). No significant differences were observed for non-virological markers. However, platelet counts <120 × 10e9/L and beta-2 microglobulin values >14 mg/L were more frequently found in fetuses with severe ultrasound abnormalities compared with fetuses with no or mild abnormalities (p < 0.001). CONCLUSION: Optimal timing of amniocentesis in women with primary infection in early gestation should be reevaluated in a prospective study. Analysis of FB markers may be beneficial in the individual management of pregnant women with confirmed congenital CMV infection. © 2017 John Wiley & Sons, Ltd.

Detection of high risk HPV and Chlamydia trachomatis in vaginal and cervical samples collected with flocked nylon and wrapped rayon dual swabs transported in dry tubes.

A dual collection device containing flocked and wrapped rayon swabs was used to collect vaginal and cervical samples from 494 women. The swabs were separated into individual tubes and sent to the laboratory in a dry state, where they were hydrated and tested for high risk HPV DNA [Digene-Qiagen hybrid capture 2] and Chlamydia trachomatis using in-house real-time PCR. The flocked swabs identified more high risk HPV and C. trachomatis infections from both sampling sites.

Cyclooxygenase-2 overexpression abrogates the antiproliferative effects of TGF-beta.

The influence of cyclooxygenase-2 (COX-2) overexpression on the development of tumours has been well documented. The underlying mechanism however has still not been completely elucidated. An escape of proliferating cells from the regulatory influence of TGF-beta for example in the intestine has been discussed as well as a preponderance or prolongation of growth factor stimulation. The experiments presented here demonstrated that COX-2 transfection of a TGF-beta-sensitive cell line abrogates the growth inhibitory effects of TGF-beta. However, analysis of the TGF-beta/Smad-signalling pathway clearly revealed that COX-2 overexpression did not interfere with that. Neither TGF-receptor expression nor Smad phosphorylation and signal transfer into the nucleus were influenced by COX-2 overexpression. In addition, a TGF-beta reporter assay revealed no difference between controls and COX-2-transfected cells. Thus, the proliferation inhibiting effects must have been well compensated by growth-inducing stimuli. Indications for this came from experiments showing an induction of TGF-alpha expression and secretion with a higher and prolonged stimulation of the ERK 1/2 (p42/44) pathway in COX-2 transfectants. This effect could have been triggered by direct prostaglandin receptor stimulation or changes in intracellular lipid mediators. An increase in PPAR signalling as proven by a reporter assay is indication for the latter. Therefore, inhibiting both COX-2 as well as the PPAR and TGF/EGF pathway could be effective in the inhibition of adenoma or even carcinoma development in the intestine.

Cationic lipid complexed camptothecin (EndoTAG-2) improves antitumoral efficacy by tumor vascular targeting.

Neo-vascular targeting by cationic colloidal carriers enables to realize an innovative approach for tumor therapy. EndoTag-2 is a novel vascular targeting agent, comprising the mammalian topoisomerase I inhibitor camptothecin in its carboxylate form complexed to cationic lipid (cationic lipid complexed camptothecin). Here we studied tumor vascular targeting properties, antitumoral effects and mode of action of EndoTag-2. Tumor vascular targeting properties of fluorescently labelled EndoTag-2 were investigated by in vivo microscopy using A-MEL-3 tumors grown in the dorsal skinfold chamber preparation and by fluorescence histology of s.c. LLC-1 carcinomas. Therapeutic effects have been investigated in the s.c. LLC-1 carcinoma model and the L3.6pl human pancreatic cancer model implanted orthotopically in athymic nude mice. Antivascular effects have been studied by histological investigation of tumor microvessel density and non invasive investigation of tumor blood flow by dynamic contrast enhanced MRI imaging (DCE-MRI). EndoTag-2 selectively targeted tumor microvessels as confirmed by quantitative fluorescence microscopy. Compared to controls EndoTag-2 revealed remarkable antitumoral efficiency in s.c. LLC-1 carcinomas implanted in C57/Bl6 mice. Growth and metastasis of orthotopic L3.6pl human pancreatic tumors was significantly inhibited by EndoTag-2 treatment. Quantitative analysis of tumor microvessel density revealed significant reduction of microvessel density in lewis lung carcinomas up to 50%. DCE-MRI confirmed significant reduction of intratumoral vascular volume as well as tumor perfusion upon EndoTag-2 treatment. In conclusion this study shows that cationic lipid complexed camptothecin (EndoTag-2) is a markedly active antitumor agent based on an innovative vascular targeting approach.

Current epidemiological aspects of human parvovirus B19 infection during pregnancy and childhood in the western part of Germany.

This investigation was undertaken to provide detailed information on the epidemiology of human parvovirus B19 (B19) infection during pregnancy and childhood in the western part of Germany. Between 1997 and 2004, 40,517 sera from pregnant women aged 17-45 years and 6060 sera from children and young adults were tested for B19 IgG and IgM in our laboratory. In pregnant women, both the history of a 'specific' (OR 7.7, 95% CI 5.2-11.4) and a 'non-specific' rash (OR 3.3, 95% CI 1.5-7.1) was predictive for B19 IgM positivity. The B19 IgG prevalence was 69.2% (4097/5924) in a subgroup of asymptomatic pregnant women screened for B19 antibodies. In children, the age-specific IgG-positivity rate increased from 12.2% (66/541) at 2 years of age to 71.9% (396/551) in those older than 10 years. In conclusion, the prevalence of B19 IgG in pregnant women from the western part of Germany is higher then previously reported. Contact with children aged 3-10 years is a major risk factor for exposure to B19. Pregnant women with the history of a 'non-specific' rash should also be evaluated for acute B19 infection.

Prenatal ultrasound diagnosis, follow-up, and outcome of congenital varicella syndrome.

OBJECTIVES: To report on a case of fetal varicella infection following the diagnosis of maternal infection at 16 weeks of gestation. METHODS: Diagnosis was based on serology testing and prenatal ultrasound, confirmed by DNA detection in amniotic fluid (Lightcycler-PCR). Serial ultrasound examinations were performed. RESULTS: Sonographic anomalies included borderline ventriculomegaly, intracerebral, intrahepatic and myocardial calcifications, limb deformities, articular effusions, and intrauterine growth retardation (confirmed postpartally). The newborn showed a severe encephalopathy and could not be stabilized sufficiently. The child died 23 days after birth. CONCLUSION: The outcome of an affected fetus may be very serious and prenatal ultrasound is a helpful tool to recognize the severity of the infection.

Different expression and activity of the alpha1 and alpha4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny.

Two catalytic isoforms of the Na,K-ATPase, alpha1 and alpha4, are present in testis. While alpha1 is ubiquitously expressed in tissues, alpha4 predominates in male germ cells. Each isoform has distinct enzymatic properties and appears to play specific roles. To gain insight into the relevance of the Na,K-ATPase alpha isoforms in male germ cell biology, we have studied the expression and activity of alpha1 and alpha4 during spermatogenesis and epididymal maturation. This was explored in rat testes at different ages, in isolated spermatogenic cells and in spermatozoa from the caput and caudal regions of the epididymis. Our results show that alpha1 and alpha4 undergo differential regulation during development. Whereas alpha1 exhibits only modest changes, alpha4 increases with gamete differentiation. The most drastic changes for alpha4 take place in spermatocytes at the mRNA level, and with the transition of round spermatids into spermatozoa for expression and activity of the protein. No further changes are detected during transit of spermatozoa through the epididymis. In addition, the cellular distribution of alpha4 is modified with development, being diffusely expressed at the plasma membrane and intracellular compartments of immature cells, finally to localize to the midregion of the spermatozoon flagellum. In contrast, the alpha1 isoform is evenly present along the plasma membrane of the developing and mature gametes. In conclusion, the Na,K-ATPase alpha1 and alpha4 isoforms are functional in diploid, meiotic and haploid male germ cells, alpha4 being significantly upregulated during spermatogenesis. These results support the importance of alpha4 in male gamete differentiation and function.

Sensitivity of a new commercial enzyme-linked immunospot assay (T SPOT-TB) for diagnosis of tuberculosis in clinical practice.

Diagnosis of active and latent tuberculosis (TB) remains a challenge; however, over the last few years, a new approach based on detecting Mycobacterium tuberculosis-specific T cells has shown much promise. In particular, there is substantial published evidence showing that the detection of ESAT-6- and CFP-10-specific T cells using the ex vivo enzyme-linked immunospot technique is a marked improvement over the existing tuberculin skin test. This technique, which detects gamma interferon-producing T cells, is now available as the commercial assay T SPOT-TB (Oxford Immunotec, Oxford, UK). In the present study, the usefulness of the T SPOT-TB test for diagnosis of TB in "real-world" clinical practice was investigated. Ninety patients of a southern German referral centre for TB with confirmed or suspected TB were randomly selected for this study. The results of the T SPOT-TB test were compared with the results of conventional diagnostic tools. The T SPOT-TB test detected 70 of 72 patients with pulmonary or extrapulmonary TB, indicating a sensitivity of 97.2% (95% confidence interval, 90.3-99.7). For 45 of these patients, tuberculin skin test (TST) results were also available. Only 40 (89%) of these 45 patients were positive in the TST compared to all 45 (100%) in the T SPOT-TB test (p=0.056). Among 12 of 90 patients for whom active TB disease was ruled out, the T SPOT-TB test was negative for 11 (92%), allowing the rapid exclusion of TB in patients suspected to have active TB disease. The T SPOT-TB test is a sensitive assay for detection of TB and represents a useful addition to the diagnostic algorithm available for detecting TB in low-incidence settings.

Pre- and periconceptional primary cytomegalovirus infection: risk of vertical transmission and congenital disease.

OBJECTIVE: To estimate the risk of congenital cytomegalovirus infection and disease following primary maternal infection around the time of conception compared with the risk during later stages of pregnancy. DESIGN: Cohort study between 1990 and 2003. SETTING: Germany. PARTICIPANTS: One hundred and sixty-six pregnant women with serologically confirmed primary cytomegalovirus infection and known outcome. METHODS: Timing of primary cytomegalovirus infection by analysing the kinetics of cytomegalovirus-specific IgG and IgM antibodies, the IgG avidity index and neutralising antibodies. MAIN OUTCOME MEASURE: Onset of maternal primary infection in relation to congenital infection and disease. RESULTS: Preconceptional (between eight and two weeks before onset of the last menstrual period) was determined in three women and did not lead to congenital infection. Periconceptional infection (between one week before and five weeks after last menstrual period) occurred in 20 women with congenital infection in nine cases (45%). Timing was less precise (between eight weeks before and five weeks after last menstrual period) in an additional 10 women, three cases of which resulted in congenital infection. Of the 12 pregnancies in which congenital infection occurred, seven were terminated, six before the 12th week of gestation (WG 12) and one at WG 19 due to fetal hyperechogenic bowel. One of the five infected live-born infants delivered to a mother with periconceptional infection showed dystrophy and mild microcephaly at birth, but had a rather normal development at two years of age. Primary infections occurring between WG 6-20 and WG 20-38 resulted in transmission rates of 30% (27/89) and 58% (18/31), respectively. CONCLUSIONS: Counselling of women with periconceptional primary cytomegalovirus infection should be adjusted to offer prenatal diagnosis and high-level ultrasound controls due to the considerable risk for fetal infection and uncertainty of clinical outcome and disease.

[Frequency of spontaneous abortion and premature birth after acute mumps infection in pregnancy]. / Abort- und Fruhgeburtenrate nach akuter Mumpsinfektionin der Schwangerschaft.

OBJECTIVE: Since mumps infection is endemic, the occurrence of acute mumps infection during pregnancy is rare. As a result, data on the possible negative consequences of acute mumps infection on pregnancy outcome are limited. PATIENTS AND METHODS: The clinical diagnosis of acute mumps infection was serologically confirmed in 79 pregnant women between January 1985 and December 2002. Data on pregnancy outcome were obtained from the gynaecologist or obstetrician. Cord blood from 26 of the 57 live-born infants was investigated for mumps-specific IgM and IgG antibodies by enzyme-linked immunoassay. RESULTS: Sixty-two patients were prospectively followed up with respect to pregnancy outcome. Two of the 62 pregnancies were electively terminated. The overall rate of fetal loss was 6.6% (4/60). Only 2 cases of spontaneous abortion occurred during the first trimester. However, all 4 spontaneous abortions occurred in women who had contracted mumps infection during the first trimester. The time interval between mumps infection and fetal loss varied widely. The median gestational age at delivery was 40 weeks. Only 2 of the 57 live-born infants (3.5%) were delivered before completion of the 37th week of pregnancy. Mumps-specific IgM anti-bodies were not detected in any of the 26 cord blood samples tested. CONCLUSION: The lack of mumps-specific antibodies in the cord blood samples tested suggests that prenatal mumps infection did not occur. The frequency of premature birth and spontaneous abortion following mumps infection in pregnancy does not appear to be increased over normal levels.

Gene profiling--chances and challenges.

Microarray analysis has been emerged as a tool to characterize the overall reaction of cells in culture or tissue to different stimuli e.g. stressful events by analysing bulk RNA present at a particular time point. It has supplemented or even replaced more traditional methods like cDNA-bank sequencing or conventional differential display. The commercial availability of several different precoated arrays and the ease of handling has supported the broad distribution of this new technique. The basic protocol involves the hybridization of complementary strands of labelled DNA or RNA from cells/tissue with representations of known genes spotted onto a solid support (nylon, glass). Labelling can be radioactive (p32/33), by a hapten group (biotin, digoxigenin, aminoallyl) or by fluorescent (Cy3, Cy5 etc.) nucleotides. Detection is performed by autoradiography, chemiluminescence or fluorescence scanning. There are different setups of arrays available: either known genes/gene-groups (apoptosis, cytokines etc.) are spotted as PCR fragments, plasmids or synthetic oligonucleotides or representations of the known genome are directly synthesized as short sequence tags of 20-70 oligonucleotides on glass chips. The latter allow the identification of newly expressed genes whereas the former deal with known genes. Ideally, the intensity of the signal can be correlated with the relative expression of a known gene and allows the comparison with a standard. Problems arise from the quality of the sample material, the standardization of the protocols and the data management. Nevertheless, gene profiling by cDNA-arrays will definitely be integrated into routine screening programs.

Can we continue research in splenectomized dogs? Mycoplasma haemocanis: old problem--new insight.

We report the appearance of a Mycoplasma haemocanis infection in laboratory dogs, which has been reported previously, yet, never before in Europe. Outbreak of the disease was triggered by a splenectomy intended to prepare the dogs for a hemorrhagic shock study. The clinical course of the dogs was dramatic including anorexia and hemolytic anemia. Treatment included allogeneic transfusion, prednisone, and oxytetracycline. Systematic follow-up (n = 12, blood smears, antibody testing and specific polymerase chain reaction) gives clear evidence that persistent eradication of M. haemocanis is unlikely. We, therefore, had to abandon the intended shock study. In the absence of effective surveillance and screening for M. haemocanis, the question arises whether it is prudent to continue shock research in splenectomized dogs.

[Congenital varicella syndrome - is it infectious?]. / Kongenitales Varizellensyndrom - besteht eine Infektionsgefahr für die Umgebung?

A 30-year-old gravida 2 suffered from chickenpox in the 13th week of gestation. In the 38th week of gestation she delivered by caesarean section a baby with typical congenital varicella syndrome (CVS). In the serum of the newborn IgG antibodies but no IgM or IgA antibodies were found by VZV ELISA test (Enzygnost Behring). On the 2nd, 5th, 8th and 11th days of life, VZV DNA was detectable in the cerebrospinal fluid by PCR in elevated copy numbers as well as in the fluid of skin lesions on the 3rd day of life in higher copy numbers, on the 11th day of life it was still detectable in low copy numbers. In the EDTA blood samples taken on the 5th day of life VZV DNA was detectable in low copy numbers. The positive VZV DNA detection in skin lesions led to the conclusion that newborns with CVS and skin lesions should be considered as infectious until the time of crusting and isolated in nursery care.

Evaluation of a commercial IgG/IgM Western blot assay for early postnatal diagnosis of congenital toxoplasmosis.

The aim of this study was to evaluate a commercial Western blot IgG/IgM assay for use in the early serological diagnosis of congenital toxoplasmosis. This assay compares the immunological profile of mother and infant and allows differentiation between passive transmitted maternal antibodies and newly synthesized antibodies of the infant within the first 3 months of life. Over a 6-year period (1995-2001), the sera from 169 mothers and their 175 offspring (6 had twins) were examined for specific anti- Toxoplasma gondii IgG, IgM and IgA antibodies with an enzyme-linked immunosorbent assay or an immunosorbent agglutination assay. All mothers had primary Toxoplasma infection during pregnancy. Serological and clinical follow-up of the infants during the first year of life confirmed 36 cases of congenital toxoplasmosis. In 139 cases, infection could be ruled out. Three hundred fifty-one paired samples from 175 mother-child pairs were tested retrospectively for IgG and IgM patterns by Toxoplasma Western blot IgG/IgM (LDBIO Diagnostics, France). The results of conventional serological analysis (immunosorbent agglutination assay or enzyme-linked immunosorbent assay) to detect IgM or IgA were compared with the results of the Toxoplasma Western blot IgG/IgM on samples obtained within the first 3 months of life. The performance of the combination of the two methods was also assessed. At birth, the sensitivity values of conventional serological analysis and the Toxoplasma Western blot were 52% and 67%, with specificity values being 99% and 96%, respectively. Combination of the Western blot and conventional serological analysis increased the sensitivity at birth to 78% and within the first 3 months of life to 85%. Overall, the combination of both methods detected 94% of congenital infections. Therefore, this commercial Western blot represents a useful tool for early postnatal diagnosis of congenital toxoplasmosis.

Studies on the immunogenicity of hCEA in a transgenic mouse model.

BACKGROUND AND AIMS: Immunization protocols in mice have shown that the tumor-associated antigen hCEA could be a target for active immunization; however, human CEA is foreign to mice. Success may depend in part on a simple anti-xenoresponse. Using hCEA-transfected syngeneic tumor cells in hCEA-transgenic mice should bypass this problem and allow testing for new vaccination strategies. MATERIALS AND METHODS: We established a hCEA transgenic model of the haplotype H2(d), which may differ from other haplotypes in cytokine production and in effectiveness of antigen presentation, and tested two vaccination protocols in wild-type and transgenic mice. RESULTS: Syngeneic wild-type mice built up an immune response with high antibody titers; only 65% of animals developed solid tumors after tumor challenge. In contrast, hCEA-transgenic mice developed no antibody response and accepted the tumor in more than 90% of cases, thus demonstrating the role of human CEA as a foreign antigen. Accordingly, active immunization using tumor lysate or lymphocytes loaded with hCEA resulted in a CTL response and tumor-rejection in up to 80% of wild-type mice. hCEA-transgenic mice could be induced with both immunization protocols to build up a CTL response, although the number of CTL were much lower and the cytotoxic response weaker than in wild-type mice. In vivo hCEA-transgenic mice rejected hCEA-positive tumors only after immunization with the tumor lysate in about 60% whereas there was no rejection of tumors after immunization with the human hCEA-loaded autologous lymphocytes. CONCLUSION: The findings clearly show the importance of transgenic models when testing the effects of immunization towards human tumor associated antigens such as hCEA because results differ in wild-type and transgenic mice.

[Diagnostic and therapy of a severe fetal parvovirus-B19-infection with persistence of viral DNA in the mothers blood but inconspicuous serological tests. Case report]. / Diagnostik und Therapie einer schweren fetalen Parvovirus-B19-Infektion bei mütterlicher Viruspersistenz, aber unauffälliger Serologie. Fallbericht.

In the 24th week of gestation we diagnosed a severe hydrops fetalis in a 25 year old VI gravid III para, who had contact to parvovirus B19 in the 14th week of gestation. Because of the severe anaemia of the fetus and the massively increased bilirubinoides in the amniotic fluid we decided at the same day to apply the first of four intrauterine transfusions. The serological patterns of maternal blood with highly positive parvovirus-B19-IgG and negative IgM suggested that an infection had occurred. Parvoviral DNA was found in maternal and fetal blood confirming the diagnosis of an acute intrauterine parvovirus-B19 infection. No viral DNA was detected in fetal ascites. IgM in fetal blood was negative. By means of four transfusions, the pregnancy could be prolonged until the 32 + 5th week of gestation while the ascites was declining. When rupture of membranes occurred, a cesarean section had to be performed due to contractions and presentation of the feet. The newborn's blood count exhibited a thrombocytopenia with normal haemoglobin and haematocrit. Five days after delivery, a blood exchange had to be done because of a hyperbilirubinaemia. After seven weeks, the child could be dismissed from hospital in good general status, with decreasing ascites, normal liver function and normal neurological status. The blood of the newborn was tested to be positive for IgG, while IgM-antibodies and parvovirus-B19-DNA were negative. The diagnosis of a parvovirus-B19 infection of a fetus with severe hydrops and anaemia could be verified by a positive proof for DNA in maternal blood, with negative IgM and highly positive parvovirus-B19-IgG and on the other hand highly positive viral DNA in fetal blood and in the amniotic fluid. 10 weeks after contact to parvovirus B19, i. e. in the 24th week of gestation, positive IgG- and negative IgM-antibodies were found in the mother's blood, whereas fetal complications were noticed. These data demonstrate that following an acute parvovirus-B19-infection of the mother IgM-antibodies can be proofed for 6 - 8 (- 10) weeks. On the other side parvovirus-B19-DNA in the mothers blood is detectable by means of PCR for 8 - 10 weeks and in some cases even more than 15 weeks.

Recurrent cytomegalovirus infection during pregnancy: ultrasonographic diagnosis and fetal outcome.

Fetal infection as a consequence of recurrent disease is uncommon. We present a case of recurrent cytomegalovirus infection in the second trimester of pregnancy. Fetal infection was detected through severely abnormal findings on ultrasound examination and verified by detecting cytomegalovirus DNA in the amniotic fluid and cytomegalovirus-specific immunoglobulin M antibodies in the fetal blood and associated pancytopenia. Because of the severity of the infection, a fatal outcome was predicted. A Cesarean section was performed at 33+5 weeks of gestation; the child died shortly after birth.