Toxocara seropositivity, atopy and asthma: a study in Cuban schoolchildren.

INTRODUCTION: Evidence suggests that human toxocariasis (HT) could stimulate the onset of allergic diseases such as asthma. More specifically, in subjects having a hypothetical 'atopic genotype', HT could boost preexistent allergy symptoms. We tested the latter hypothesis in Cuba, a country where both asthma and HT are prevalent. MATERIAL AND METHODS: In a group of Cuban school-aged children (n = 958), we investigated the association of Toxocara seropositivity and atopic status with asthma. Toxocara seropositivity was diagnosed with ELISA and atopy by allergen skin prick test. Both physician-diagnosed asthma and current wheeze, as determined by International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire, were considered. Associations were assessed using multivariable logistic regression analyses, with either 'physician-diagnosed asthma' or 'current wheeze' as outcome variable. RESULTS: 40.1% of the children were Toxocara seropositive. Prevalences were 21.7% for current wheeze and 32.7% for physician-diagnosed asthma. The odds of having asthma were almost two times higher in atopic children, but only reached borderline significance (OR=1.90, CI 95%: 0.95-3.80 for physician-diagnosed asthma and OR=1.94, CI 95%: 0.98-3.85 for current wheeze). Toxocara seropositivity and physician-diagnosed asthma were associated (OR=1.51, CI 95%: 1.01-2.26). Moreover, in children without antibodies to Toxocara, being atopic was significantly associated with having physician-diagnosed asthma (OR=2.53, CI 95%: 1.63-3.90), while this association was not present in Toxocara positives (OR=1.38, CI 95%: 0.82-2.37). CONCLUSION: Our data confirm previous observations of higher Toxocara seropositivity rates in asthmatic children. Toxocara seropositivity appeared to abrogate the apparent association between atopy and asthma in Cuban children. Although this observation was limited to physician-diagnosed asthma, it challenges the hypothesis that HT stimulates the onset of allergic diseases such as asthma in atopic individuals.

Frequency of antibodies to Toxocara in Cuban schoolchildren.

OBJECTIVE: The aim of the study was to determine the frequency of antibodies to Toxocara in Cuban schoolchildren. METHODS: The frequency of antibodies to Toxocara canis was assessed with a commercial enzyme-linked immunosorbent assays kit in school-aged children from two municipalities of Cuba. Univariate analysis and a multivariable logistic regression analysis adjusted for age, sex, municipality and co-infection with helminth and/or protozoa were conducted. RESULTS: The percentage of children with antibodies to Toxocara was 38.8% (392/1011; 95% CI = 36.8-42.8). Antibody positivity was significantly associated with gender and co-infections with intestinal parasites, but not with age or municipality. CONCLUSION: Cuban children are highly exposed to the Toxocara parasite, corresponding well with reported environmental contamination with Toxocara parasite eggs and T. canis prevalences in dogs in Cuba. Relevant policy makers and the Cuban population need to be better informed about this preventable infection.

Modelling Yersinia enterocolitica inactivation in coculture experiments with Lactobacillus sakei as based on pH and lactic acid profiles.

In food processing and preservation technology, models describing microbial proliferation in food products are a helpful tool to predict the microbial food safety and shelf life. In general, the available models consider microorganisms in pure culture. Thus, microbial interactions are ignored, which may lead to a discrepancy between model predictions and the actual microbial evolution, particularly for fermented and minimally processed food products in which a background flora is often present. In this study, the lactic acid mediated negative microbial interaction between the lactic acid bacterium Lactobacillus sakei and the psychrotrophic food pathogen Yersinia enterocolitica was examined. A model describing the lactic acid induced inhibition (i.e., early induction of the stationary phase) of the pathogen [Vereecken, K.M., Devlieghere, F., Bockstaele, A., Debevere, J., Van Impe, J.F., 2003. A model for lactic acid induced inhibition of Yersinia enterocolitica in mono- and coculture with Lactobacillus sakei. Food Microbiology 20, 701-713.] was extended to describe the subsequent inactivation (i.e., decrease of the cell concentration to values below the detection limit). In the development of a suitable model structure to describe the inactivation process, critical points in the variation of the specific evolution rate mu [1/h] with the dynamic (time-varying) pH and undissociated lactic acid profiles were taken into account. Thus, biological knowledge, namely, both pH and undissociated lactic acid have an influence on the microbial evolution, was incorporated. The extended model was carefully validated on new data. As a result, the newly developed model is able to accurately predict the growth, inhibition and subsequent inactivation of Y. enterocolitica in coculture as based on the dynamic pH and lactic acid profiles of the medium.

Mother-to-child transmission of different HIV-1 subtypes among ARV Naïve infected pregnant women in Nigeria.

The rate of mother-to-child transmission (MTCT) of HIV as well as the implications of the circulating multiple subtypes to MTCT in Nigeria are not known. This study was therefore undertaken to determine the differential rates of MTCT of HIV-1 subtypes detected among infected pregnant women before ARV intervention therapy became available in Nigeria. Twenty of the HIV-positive women who signed the informed consent form during pregnancy brought their babies for follow-up testing at age 18-24 months. Plasma samples from both mother and baby were tested for HIV antibody at the Department of Virology, UCH, Ibadan, Nigeria. All positive samples (plasma and peripheral blood mononuclear cells-PBMCs) were shipped to the Institute of Tropical Medicine, Antwerp, Belgium, where the subtype of the infecting virus was determined using the HMA technique. Overall, a mother-to-child HIV transmission rate of 45% was found in this cohort. Specifically, 36.4%, 66.7% and 100% of the women infected with HIV-1 CRF02 (IbNg), G and B, respectively, transmitted the virus to their babies. As far as it can be ascertained, this is the first report on the rate of MTCT of HIV in Nigeria. The findings reported in this paper will form a useful reference for assessment of currently available therapeutic intervention of MTCT in the country.

Towards a novel class of predictive microbial growth models.

Food safety and quality are influenced by the presence (and possible proliferation) of pathogenic and spoilage microorganisms during the life cycle of the product (i.e., from the raw ingredients at the start of the production process until the moment of consumption). In order to simulate and predict microbial evolution in foods, mathematical models are developed in the field of predictive microbiology. In general, microbial growth is a self-limiting process, principally due to either (i) the exhaustion of one of the essential nutrients, and/or (ii) the accumulation of toxic products that inhibit growth. Nowadays, most mathematical models used in predictive microbiology do not explicitly incorporate this basic microbial knowledge. In this paper, a novel class of microbial growth models is proposed. In contrast with the currently used logistic type models, e.g., the model of Baranyi and Roberts [Baranyi, J., Roberts, T.A., 1994. A dynamic approach to predicting bacterial growth in food. International Journal of Food Microbiology 23, 277-294], the novel model class explicitly incorporates nutrient exhaustion and/or metabolic waste product effects. As such, this novel model prototype constitutes an elementary building block to be extended in a natural way towards, e.g., microbial interactions in co-cultures (mediated by metabolic products) and microbial growth in structured foods (influenced by, e.g., local substrate concentrations). While under certain conditions the mathematical equivalence with classical logistic type models is clear and results in equal fitting capacities and parameter estimation quality (see Poschet et al. [Poschet, F., Vereecken, K.M., Geeraerd, A.H., Nicolai, B.M., Van Impe, J.F., 2004. Analysis of a novel class of predictive microbial growth models and application to co-culture growth. International Journal of Food Microbiology, this issue] for a more elaborated analysis in this respect), the biological interpretability and extendability represent the main added value.

Analysis of a novel class of predictive microbial growth models and application to coculture growth.

In this paper, a novel class of microbial growth models is analysed. In contrast with the currently used logistic type models (e.g., the model of Baranyi and Roberts [Baranyi, J., Roberts, T.A., 1994. A dynamic approach to predicting bacterial growth in food. International Journal of Food Microbiology 23, 277-294]), the novel model class, presented in Van Impe et al. (Van Impe, J.F., Poschet, F., Geeraerd, A.H., Vereecken, K.M., 2004. Towards a novel class of predictive microbial growth models. International Journal of Food Microbiology, this issue), explicitly incorporates nutrient exhaustion and/or metabolic waste product effects inducing stationary phase behaviour. As such, these novel model types can be extended in a natural way towards microbial interactions in cocultures and microbial growth in structured foods. Two illustrative case studies of the novel model types are thoroughly analysed and compared to the widely used model of Baranyi and Roberts. In a first case study, the stationary phase is assumed to be solely resulting from toxic product inhibition and is described as a function of the pH-evolution. In the second case study, substrate exhaustion is the sole cause of the stationary phase. Finally, a more complex case study of a so-called P-model is presented, dealing with a coculture inhibition of Listeria innocua mediated by lactic acid production of Lactococcus lactis.

Interpatient genetic variability of HIV-1 group O.

OBJECTIVE: To analyse the genetic and phylogenetic characteristics of HIV-1 group O viruses. MATERIALS AND METHODS: The env gene, encoding the gp160 glycoprotein, and a partial p24-encoding gag gene fragment of a Cameroonian (CA9) and a Gabonese (VI686) HIV-1 group O virus, isolated from cultured peripheral blood mononuclear cells of symptomatic patients, were sequenced, aligned with other representatives of group O for which the same region has been documented, and genetically and phylogenetically analysed. RESULTS: Phylogenetic analysis of the env gene (gp160) revealed that CA9, VI686, ANT70, and four Ha strains formed a separate cluster, which was supported by 100% of all bootstrap trees. In addition, these seven isolates were part of the same clade in the p24 phylogeny. VAU and MVP5180 may represent two other subtypes. CONCLUSION: We have characterized two group O viruses, originating from Cameroon and Gabon, which show a close evolutionary relationship to ANT70 and four Ha strains based on the entire env gene, suggestive of a first group O subgroup, tentatively named the HIV-1 group O env ANT70 clade or subtype.

Study of HIV type 1 gag/env variability in The Gambia, using a multiplex DNA polymerase chain reaction.

A multiplex DNA PCR assay was developed for the simultaneous first-round amplification of HIV-1 gag and env fragments for the heteroduplex mobility assay (HMA). This assay was compared with the conventional amplification assay, using DNA extracted from PBMC samples from 30 HIV-1-seropositive individuals from The Gambia, who were enrolled between 1992 and 1997. From 27 of 30 (90%) samples both gag and env HMA fragments were amplified simultaneously. In one sample only the gag HMA fragment could be amplified by multiplex DNA PCR, and in two samples amplification was negative for both gag and env HMA in multiplex as well as the mono-DNA PCR. Of the 28 Gambian isolates subtyped by gag/env HMA or by sequencing and phylogenetic analysis, the majority (19 of 28; 68%) were intersubtype recombinant. Fifteen of 28 (53%) samples were circulating recombinant form (CRF) CRF02.AG variants. Two isolates clustering with the previously documented Gambian isolate GM4 (previously described as an env GC recombinant) are classified as gag A/env J recombinants.

The neutralization relationship of HIV type 1, HIV type 2, and SIVcpz is reflected in the genetic diversity that distinguishes them.

Neutralizing antibody (NA) patterns in the sera of individuals naturally infected with human immunodeficiency virus (HIV) type 1, HIV-2, and the simian immunodeficiency virus (SIVcpz) to their homologous and heterologous isolates were determined in a peripheral blood mononuclear cell-based neutralization assay. We examined the role of the V3 loop of HIV-1 and SIVcpz in neutralization and the cross-reactivities among them. Cross-neutralization by sera of humans and chimpanzees naturally infected, respectively, with HIV-1 and SIVcpz isolates was more extensive than the infrequent and low-titer cross-neutralizations observed between HIV-1 and HIV-2. Neutralization of 9 of the 16 HIV-1 isolates by 9 of 10 HIV-2 and all 3 SIVcpz antibody-positive sera were weak and sporadic (titer, 1:10-1:160). Twelve of 15 HIV-1 sera neutralized the 2 SIVcpz isolates with titers of 1:10-1:320 but only sporadically neutralized the 6 HIV-2 isolates (titers: 1:10-1:20). The majority of HIV-1 and SIVcpz sera bound to the V3 peptides although their binding capacity did not readily reflect their neutralizing capacity. The HIV-2 sera did not or only weakly bound to the V3 peptides. These results suggest that HIV-1 and SIVcpz share some structural and functional similarities that set them apart from HIV-2.

Development of a one-tube multiplex reverse transcriptase-polymerase chain reaction assay for the simultaneous amplification of HIV type 1 group M gag and env heteroduplex mobility assay fragments.

The emergence of intersubtype recombinant HIV-1 isolates has made it imperative to analyze different regions of HIV-1 genomes. For this purpose a one-tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplification of gag and env heteroduplex mobility assay (HMA) fragments from different HIV-1 group M isolates, was developed, starting with plasma samples. The multiplex RT-PCR assay is sensitive: 115 of 136 (84.5%) samples were positive for both gag and env, positive amplification of the gag fragment was observed in 130 of 136 (95.6%) samples, while for the env fragment 119 of 136 (87.5%) tested positive. The multiplex RT-PCR in combination with gag and env HMA makes large-scale HIV-1 subtyping fast, simple, and more economical.

Intrapatient variability of HIV type 1 group O ANT70 during a 10-year follow-up.

HIV-1 ANT70 is the first HIV-1 group O virus isolate obtained from a 25-year-old Cameroonian woman, who seroconverted in March 1987. This individual has remained asymptomatic and clinically healthy (clinical stage WHO 1, CDC II) even though she did not receive any antiretroviral therapy for HIV-1 before 97 months post-seroconversion. CD4+ T cell counts declined steadily to 200/microl at 70 months postseroconversion. The HIV-1 ANT70 nucleotide and amino acid sequence diversity of the V3C3-encoding env fragment within this individual was followed over a 10-year period. RT-PCR, cloning, sequencing, and genetic analyses were performed on eight plasma follow-up samples. Extensive increasing intra- and intersample variation was observed. This is the first long-term (>10 years) follow-up of the genetic variability of an HIV-1 group O-infected individual. As the course of the disease in the HIV-1 ANT70-infected woman was similar in many aspects to that of group M-infected individuals, it remains to be elucidated whether the changes observed in the V3 loop are critical for disease progression.

Differential diagnosis of HIV type 1 group O and M infection by polymerase chain reaction and PstI restriction analysis of the pol gene fragment.

HIV-1 group O serological screening or confirmation strategies so far have not proved 100% sensitive and specific, indicating a lack of antibody reactivity or cross-reactivity with group O antigens. Therefore, genetic analysis currently represents the only method by which confirm presumed HIV-1 group O or group O/M infections. We have optimized the sensitivity (100%) and specificity (100%) of an HIV-1 group O/M-specific PCR of a pol gene fragment. In addition, we report on a highly sensitive (97.2%) and specific (100%) method for differentiation between HIV-1 group O and group M viruses, using PCR and PstI enzyme restriction fragment analysis of a pol fragment. Compared with sequencing, these methods are fast, inexpensive, and simple.

Reactivity and amplification efficiency of the NASBA HIV-1 RNA amplification system with regard to different HIV-1 subtypes.

In view of the genetic diversity of the human immunodeficiency virus Type 1, we assessed the sensitivity and quantification efficiency of the HIV-1 RNA NASBA amplification system with respect to different HIV-1 subtypes and recombinants. Twenty cell culture supernatants representing 17 HIV-1 group M and 3 group O strains were tested, and NASBA RNA loads were compared with results obtained with a RT-PCR based HIV-1 RNA quantitation method, with p24-antigen concentrations and with the infective dose. The current HIV-1 RNA NASBA seemed suitable to quantitate representatives of different HIV-1 M subtypes. Differences between NASBA and RT-PCR loads were observed for certain HIV-1 M strains. Significantly lower RT-PCR loads were measured for most gag A, gag B and gag F strains, whereas NASBA detected lower copy numbers in 1 gag H strain and 1 gag H/env G recombinant. NASBA was not able to quantify 1 HIV-1 group M recombinant. Some of these differences could be explained by the presence and position of mismatches with primers. HIV-1 group O strains were not detectable by both RNA amplification methods. A firm correlation was not observed between the measured RNA loads and either the p24-antigen concentration or the infective dose.

Design and evaluation of an in-house HIV-1 (group M and O), SIVmnd and SIVcpz antigen capture assay.

An enzyme-linked immuno-sorbent assay (ELISA) for the detection of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVcpz/SIVmnd) antigens was designed using immunoreagents from naturally infected individuals, and compared to the commercially available Vironostika HIV-1 Antigen Microelisa System (Organon Teknika). The in-house assay proved to be specific for HIV-1 isolates belonging to group M (A-H) and group O and for SIVcpz and SIVmnd isolates, but was less sensitive than the Vironostika HIV-1 Antigen Microelisa System, except for SIVmnd. For the strains belonging to HIV-2, SIVmac and SIVagm, the in-house assay could not detect antigen to an appreciable degree. This study shows that a considerably less expensive but sufficiently accurate HIV-1 antigen capture assay can be developed to monitor HIV-1 (group M and O), SIVcpv and SIVmnd antigen in the supernatants of virus cultures.

Effect of chemicals on the microbial evolution in foods.

In contrast with most chemical hazardous compounds, the concentration of food pathogens changes during processing, storage, and meal preparation, making it difficult to estimate the number of microorganisms or the concentration of their toxins at the moment of ingestion by the consumer. These changes are attributed to microbial proliferation, survival, and/or inactivation and must be considered when exposure to a microbial hazard is assessed. The number of microorganisms can also change as a result of physical removal, mixing of food ingredients, partitioning of a food product, or cross-contamination (M. J. Nauta. 2002. Int. J. Food Microbiol. 73:297-304). Predictive microbiology, i.e., relating these microbial evolutionary patterns to environmental conditions, can therefore be considered a useful tool for microbial risk assessment, especially in the exposure assessment step. During the early development of the field (late 1980s and early 1990s), almost all research was focused on the modeling of microbial growth over time and the influence of temperature on this growth. Later, modeling of the influence of other intrinsic and extrinsic parameters garnered attention. Recently, more attention has been given to modeling of the effects of chemicals on microbial inactivation and survival. This article is an overview of different applied strategies for modeling the effect of chemical compounds on microbial populations. Various approaches for modeling chemical growth inhibition, the growth-no growth interface, and microbial inactivation by chemicals are reviewed.

Effects of gelatin concentration on the growth parameters of Listeria innocua in a model gel system.

In this work, the growth of Listeria innocua was studied responding to the addition of different concentrations of gelatin (see text) model gel system in a modi_ed Brain Heart Infusion medium at 12 C and an initial pH of 6.2. The global number of viable cells as a function of incubation time and the corresponding pH, lactic acid concentration and glucose concentration were measured. Each set of data was fitted with the growth model of Baranyi and Roberts (1994) to estimate the maximum specific growth rate and the maximum cell concentration. Gelatin had a significant e_ect on the growth rate of Listeria innocua, which reduced as the gelatin concentration increased. A tail was observed after a certain concentration of gelatin indicating that there exists a maximum concentration beyond which no further reduction could be observed. There was, however, within the gelatin concentration range studied, no appreciable effect on the maximum cell concentration. A distinct morphological change of colonies was also observed with increasing gelatin concentration.

Predicting inhibition and inactivation of Yersinia enterocolitica through lactic acid production by Lactobacillus sakei.

In food technology, there is a need for models taking into account the interactions between microorganisms, in order to correctly predict the safety and shelf life of food products. When leaving these interactions out of consideration, a discrepancy between the model prediction and the actual microbial evolution may occur for certain types of food products. In this study, a model describing the inhibition of the pathogenic Yersinia enterocolitica in mono- and coculture with Lactobacillus sakei was extended to describe also the subsequent inactivation of Y. enterocolitica. During the development of a suitable model structure to describe the inactivation process, biological knowledge about this process was incorporated. The extended model was able to predict evolution of Y. enterocolitica in coculture as well as in monoculture.