Establishing correlates of maternal-fetal cytomegalovirus transmission-one step closer through predictive modeling.

BACKGROUND: Determinants of maternal-fetal cytomegalovirus (CMV) transmission and factors influencing the severity of congenital CMV (cCMV) infection are not well understood. METHODS: We conducted a descriptive, multi-center study in pregnant women ≥18 years old with primary CMV infection and their newborns (NCT01251744) to explore maternal immune responses to CMV and determine potential immunologic/virologic correlates of cCMV following primary infection during pregnancy. We developed alternative approaches looking into univariate/multivariate factors associated with cCMV, including a participant clustering/stratification approach and an interpretable predictive model-based approach using trained decision trees for risk prediction (post-hoc analyses). RESULTS: Pregnant women were grouped in three distinct clusters with similar baseline characteristics, particularly gestational age at diagnosis. We observed a trend for higher viral loads in urine and saliva samples from mothers of infants with cCMV versus without cCMV. When using a trained predictive-model approach that accounts for interaction effects between variables, anti-pentamer IgG antibody concentration and viral load in saliva were identified as biomarkers jointly associated with the risk of maternal-fetal CMV transmission. CONCLUSION: We identified biomarkers of CMV maternal-fetal transmission. After validation in larger studies, our findings will guide the management of primary infection during pregnancy and the development of vaccines against cCMV.

The human cytomegalovirus (CMV) is common and usually causes no symptoms in healthy individuals. However, CMV infections can be life-threatening in individuals with improperly functioning or immature immune systems, such as fetuses. Women can become infected with CMV for the first time (primary infection) during pregnancy. If CMV is transmitted from mother to fetus before the second trimester, the infant can suffer from severe disorders such as hearing loss and delayed development. We aimed to identify characteristics of pregnant women with a primary CMV infection that may increase the likelihood of transmitting CMV to the fetus. We considered demographical, clinical, and behavioral characteristics, as well as immune responses and the quantity of virus detected in the women's blood, urine, saliva, and vaginal mucus. Because we could not identify one single characteristic that could predict a high risk of CMV transmission, we developed new data analysis models to study how they can be combined. We found that antibodies targeting a pentameric antigen of the virus envelope and the presence of virus in saliva can together predict the risk of CMV transmission from mother to fetus. Our results can help improve the care of CMV-infected pregnant women and the design of CMV vaccines.

Incidence of Cytomegalovirus Primary and Secondary Infection in Adolescent Girls: Results From a Prospective Study.

Developing a vaccine to prevent congenital cytomegalovirus (CMV) infection and newborn disability requires an understanding of infection incidence. In a prospective cohort study of 363 adolescent girls (NCT01691820), CMV serostatus, primary infection, and secondary infection were determined in blood and urine samples collected at enrollment and every 4 months for 3 years. Baseline CMV seroprevalence was 58%. Primary infection occurred in 14.8% of seronegative girls. Among seropositive girls, 5.9% had ≥4-fold increase in anti-CMV antibody, and 23.9% shed CMV DNA in urine. Our findings provide insights on infection epidemiology and highlight the need for more standardized markers of secondary infection.

Cytomegalovirus (CMV) can be passed from a woman to her unborn baby during pregnancy, which can result in disabilities in the baby. This can happen after a first infection with the virus during pregnancy, after a subsequent infection with a different strain ("reinfection"), or after "reactivation", which means that a virus present from a previous infection becomes active again. Vaccinating adolescent girls against CMV may be a future strategy to help prevent CMV infection during pregnancy. To provide information to design trials evaluating a CMV vaccine, it is important to know how common primary/secondary CMV infection is in adolescent girls and if this can be measured with available tools. We followed adolescent girls living in Finland, Mexico or the United States for three years. At study start, 58% of these girls showed evidence of previous CMV infection. During the three-year follow-up, a first CMV infection occurred in 15% of girls, and reinfection or reactivation in 6% to 24% of girls (depending on the method used). The obtained estimates of CMV infection rates in adolescent girls provide valuable information for future studies to evaluate CMV vaccines, but standardized markers for secondary infection are needed.

A human papilloma virus testing algorithm comprising a combination of the L1 broad-spectrum SPF10 PCR assay and a novel E6 high-risk multiplex type-specific genotyping PCR assay.

Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection and genotyping systems. To improve HPV detection by PCR, the broad-spectrum L1-based SPF10 PCR DNA enzyme immunoassay (DEIA) LiPA system and a novel E6-based multiplex type-specific system (MPTS123) that uses Luminex xMAP technology were combined into a new testing algorithm. To evaluate this algorithm, cervical swabs (n = 860) and cervical biopsy specimens (n = 355) were tested, with a focus on HPV types detected by the MPTS123 assay (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6, and 11). Among the HPV-positive samples, identifications of individual HPV genotypes were compared. When all MPTS123 targeted genotypes were considered together, good overall agreement was found (κ = 0.801, 95% confidence interval [CI], 0.784 to 0.818) with identification by SPF10 LiPA, but significantly more genotypes (P < 0.0001) were identified by the MPTS123 PCR Luminex assay, especially for HPV types 16, 35, 39, 45, 58, and 59. An alternative type-specific assay was evaluated that is based on detection of a limited number of HPV genotypes by type-specific PCR and a reverse hybridization assay (MPTS12 RHA). This assay showed results similar to those of the expanded MPTS123 Luminex assay. These results confirm the fact that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample. Therefore, a testing algorithm combining the broad-spectrum PCR and a range of type-specific PCRs can offer a highly accurate method for the analysis of HPV infections and diminish the rate of false-negative results and may be particularly useful for epidemiological and vaccine studies.

Quantification of Bifidobacterium spp. and Lactobacillus spp. in rat fecal samples by real-time PCR.

The microbiota of the rat intestinal tract constitutes a complex ecosystem of microorganisms. We have developed a real-time quantitative PCR assay based on genus-specific 16S rDNA primers and 3' minor groove binder (MGB) probes for accurate detection and quantification of a wide range of Bifidobacterium spp. (30 species) and Lactobocillus spp. (15 species) in rat fecal samples. Real-time PCR detection of serially diluted DNA isolated from reference strains of Bifidobacterium longum and Lactobacillus acidophilus was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. The method proved applicable to the detection of Bifidobacterium spp. and Lactobacillus spp. at concentrations down to 10 CFU per PCR, corresponding to 5 x 10(4) CFU/g feces. The inter-extract reproducibility was high, with a coefficient of variation ranging from 0.24% to 1.07% for the Bifidobacterium assay and from 0.05% to 1.28% for the Lactobacillus assay. We conclude that real-time PCR is a very sensitive and precise technique for extensive quantitative evaluation of gut Bifidobacterium spp. and Lactobacillus spp. Thus, the approach used here to detect and quantify bacteria with group-specific primers should contribute to further studies of the composition and dynamics of the rat intestinal microbiota.

Estimation of bioreactor efficiency through structured hydrodynamic modeling case study of a Pichia pastoris fed-batch process.

In this article, two theories are unified to investigate the effect of hydrodynamics on a specific bioprocess: the network-of-zones (NOZ) hydrodynamic structured modeling approach (developed by several researchers but applied to only a few bioprocesses) and the effectiveness factor eta approach. Two process scales were investigated (20 and 500 L), and for each, hydrodynamics were quantified using an NOZ validated by homogeneity time measurements. Several impeller combinations inducing quite different hydrodynamics were tested at the 20-L scale. After this step, effectiveness factors were determined for each fermentation run. To achieve this, a perfectly mixed microbial kinetic model was evaluated by using simple Monod kinetics with a fed-batch mass balance. This methodology permitted determination of the effectiveness factor with more accuracy because of the relation with the perfect case deduced from the Monod kinetics. It appeared that for the small scale, eta decreased until reaching a value of approx 0.7 (30% from the ideal case) for the three impeller systems investigated. However, stirring systems that include hydrofoils seemed to maintain higher effectiveness factors during the course of the fermentation. This effect can be attributed to oxygen transfer performance or to homogenization efficiency exhibited by the hydrofoils. To distinguish the oxygen transfer from the homogenization component of the effectiveness factor, these phenomena were analyzed separately. After determining the evolution of etaO2 linked to oxygen transfer for each of the fermentation runs, the NOZ model was employed to quantify substrate gradient appearance. After this step, another effectiveness factor, etamix, related to mixing was defined. Consequently, it is possible to distinguish the relative importance of the mixing effect and oxygen transfer on a given bioprocess. The results have highlighted an important scale effect on the bioprocess that can be analyzed using the NOZ model.

Isolation and biomass production of a Saccharomyces cerevisiae strain binding copper and zinc ions.

Copper and zinc are essential trace elements participating in many physiological functions, notably immunity and protection against oxidative stress. Yeasts and the yeast Saccharomyces cerevisiae, in particular, possess in their genome tandem repeats of the CUP1 gene coding for a protein (a metallothionein) capable of capturing and binding toxic elements such as copper ions. The number of copies of this gene in a cell determines its physiological level of resistance to these ions. This paper describes the selection, characterization, and production of a new copper-resistant yeast strain that can bind large quantities of copper and zinc. This approach should lead to increasing the bioavailability of these trace elements and hence to reducing their emission into the environment.

Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris.

This is the first report of an insect esterase efficiently expressed in the methylotrophic yeast Pichia pastoris (so far insect esterases have been produced only in the baculovirus system). Having isolated a Tribolium castaneum carboxylesterase cDNA (TCE), we were initially unable to express it in Escherichia coli or P. pastoris despite significant transcription levels. As codon usage bias is different in T. castaneum and P. pastoris, we assumed this was a possible explanation for the translational barrier observed in yeast. Accordingly, we designed and constructed by recursive PCR a synthetic TCE gene (synTCE) optimized for heterologous expression in P. pastoris, i.e., a gene in which certain TCE codons are replaced with synonymous codons 'preferred' in P. pastoris. When the altered gene was placed under the control of either the P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase (AOX1) promoter and introduced on an expression vector into P. pastoris, its product was produced intracellularly. We also successfully explored the possibility of obtaining a secreted product: P. pastoris cells expressing an in-frame fusion of synTCE with the alpha-factor secretion signal under the control of the GAP promoter were found to secrete the recombinant esterase into the external medium (to a concentration of 7 mg/L). In addition to this demonstration of TCE production in yeast, our results suggest that the GAP promoter could advantageously replace the AOX1 promoter as a driver of synTCE expression. TCE specific activity was approximately 5 U/mg when p-nitrophenyl acetate was used as substrate.

Generation of the Brucella melitensis ORFeome version 1.1.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.