vRNA structured population model for Hepatitis C Virus dynamics.

Improvements in the understanding of the Hepatitis C Virus (HCV) life-cycle have led to the identification of targets and the development of drugs affecting the intracellular reproduction of the virus. These advancements have presented new modeling challenges as the classic models have focused on describing the macroscopic viral kinetics only. Our primary objective is to apply the existing theory of Physiologically Structured Population (PSP) modeling to describe dynamics of viral RNA (vRNA) in infected hepatocytes of patients receiving treatment with Direct-acting Antiviral Agents (DAA). Using vRNA as a physiological structure this work expands on previous structured population models allowing exploration of micro- and macroscopic implications of such treatments. The PSP model provides a description of vRNA distribution in the infected cells at steady state and its time evolution following treatment. The long term behavior of the model predicts viral load time courses in plasma and permits to quantify conditions for the virus eradication. Finally, we demonstrate that PSP models can account for additional structures, which are essential for the viral replication process with potentially far reaching implications in our understanding of HCV infections and treatment options.

Strategies to increase the stability of intermediate moisture foods towards Zygosaccharomyces rouxii: the effect of temperature, ethanol, pH and water activity, with or without the influence of organic acids.

Intermediate moisture foods (IMF) are in general microbiologically stable products. However, due to health concerns consumer demands are increasingly forcing producers to lower the fat, sugar and preservatives content, which impede the stability of the IMF products. One of the strategies to counteract these problems is the storage of IMF products at lower temperatures. Thorough knowledge on growth/no growth boundaries of Zygosaccharomyces rouxii in IMF products, also at different storage temperatures is an important tool for ensuring microbiologically stability. In this study, growth/no growth models for Z. rouxii, developed by Vermeulen et al. (2012) were further extended by incorporating the factor temperature. Three different data sets were build: (i) without organic acids, (ii) with acetic acid (10,000 ppm on product basis) and (iii) with sorbic acid (1500 ppm on product basis). For each of these data sets three different growth/no growth models were developed after 30, 60 and 90 days. The results show that the influence of temperature is only significant in the lower temperature range (8-15 °C). Also, the effect of pH is negligible (pH 5.0-6.2) unless organic acids are present. More specific, acetic acid had only an additive effect to ethanol and aw at low pH, whereas sorbic acid had also an additive effect at the higher pH values. For incubation periods longer than 30 days the growth/no growth boundary remained stable but enlarged gradually between day 60 and 90, except for the lower temperature range (<12 °C) where the boundary shifts to more stringent environmental conditions.

Newborn hearing screening and cochlear implantation: impact on spoken language development.

OBJECTIVES: This study retrospectively evaluates the effect of newborn hearing screening on age at diagnosis, age at cochlear implantation and spoken language development in severely hearing-impaired children. METHODS: Age at diagnosis, age at cochlear implantation and language development were evaluated in a group of early screened (n = 149) and a group of late screened (n = 139) severely hearing-impaired children. Language outcomes were quantified as language quotients (LQs) on the Reynell Developmental Language Scales and Schlichting Expressive Language Test at 1,2, and 3 years after cochlear implantation. RESULTS: Early screened children were significantly younger than late screened children at the time of hearing loss diagnosis and cochlear implantation. Furthermore, early screening was associated with better receptive and expressive spoken language skills after cochlear implantation. CONCLUSION: The results of this retrospective study indicate that the newborn hearing screening program in Flanders and The Netherlands resulted in earlier intervention in deaf children, which beneficially influenced spoken language development.

Poikilocytosis of Angora goats is associated with erythrocyte density and reticulocytosis.

Angora goats in South Africa experience several syndromes that result in notable morbidity and mortality in juveniles and adults, but not kids. Insight into their causes is hampered by the lack of normal reference values for this breed, and the present study therefore aimed to characterise (1) differences in the haematology of healthy kids at birth and weaning, and (2) the haematology of apparently healthy yearlings. Selected variables were measured by blood smear analysis, and complete blood counts were performed using an ADVIA 2120i. Variables at 1, 11, and 20 weeks of age were compared using the Friedman test and associations between variables of yearlings were determined by correlation analysis. In kids, red blood cell count, mean corpuscular haemoglobin concentration (MCHC), and poikilocytosis increased over time, while mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) decreased. Yearlings displayed a lower MCHC, and higher haemoglobin distribution width than previously reported for goats, and these were positively correlated with poikilocytosis, as were reticulocyte counts. White cell counts of yearlings exceeded normal values previously reported for goats, with some individuals displaying remarkably high mature neutrophil counts. Changes in haemoglobin variant expression or cation and water fluxes are possible explanations for the findings in kids, while in yearlings, the associations between MCHC, HDW, poikilocytosis, and reticulocytosis suggest alterations in red cell hydration in adulthood that are associated with increased red cell turnover. These findings may prove informative in the further investigation of various clinical syndromes in this population.

Screening of different stress factors and development of growth/no growth models for Zygosaccharomyces rouxii in modified Sabouraud medium, mimicking intermediate moisture foods (IMF).

The microbial stability of intermediate moisture foods (IMF) is linked with the possible growth of osmophilic yeast and xerophilic moulds. As most of these products have a long shelf life the assessment of the microbial stability is often an important hurdle in product innovation. In this study a screening of several Zygosaccharomyces rouxii strains towards individual stress factors was performed and growth/no growth models were developed, incorporating a(w), pH, acetic acid and ethanol concentrations. These stress factors are important for sweet IMF such as chocolate fillings, ganache, marzipan, etc. A comparison was made between a logistic regression model with and without the incorporation of time as an explanatory variable. Next to the model development, a screening of the effect of chemical preservatives (sorbate and benzoate) was performed, in combination with relevant stress factors within the experimental design of the model. The results of the study showed that the influence of the investigated environmental stress factors on the growth/no growth boundary of Z. rouxii is the most significant in the first 30-40 days of incubation. Incorporating time as an explanatory variable in the model had the advantage that the growth/no growth boundary could be predicted at each time between 0 and 60 days of incubation at 22 °C. However, the growth/no growth boundary enlarged significantly leading to a less accurate prediction on the growth probability of Z. rouxii. The developed models can be a useful tool for product developers of sweet IMF. Screening with chemical preservatives revealed that benzoic acid was much less active towards Z. rouxii than sorbic acid or a mixture of both acids.

On the critical evaluation of growth/no growth assessment of Zygosaccharomyces bailii with optical density measurements: liquid versus structured media.

Growth/no growth (G/NG) studies that include the effect of medium structure have typically been performed for (pathogenic) bacteria and on the basis of gelatin/agar as a gelling agent. In this study, the growth potential of the spoilage yeast Zygosaccharomyces bailii was investigated in two model systems that resemble the macroscopic physicochemical and rheological properties of acidic sauces. In a Carbopol model system, the effect of pH (3.5-4.5), glycerol concentration (17-32%), acetic acid concentration (1.5-2.0%) and medium structure (3 levels) was investigated. In xanthan gum, the behavior of the yeast was studied at different levels of pH (3.5-4.5), NaCl concentration (0.5-13.5%), acetic acid concentration (0-2.0%) and medium structure (2 levels). Rheologically, viscoelastic moduli failed to discriminate between different forms of microbial growth, whereas yield stress data appeared to provide a better indication. In general, G/NG results revealed an unexpected increase of growth probability as a function of medium structure, both at 22 and 30 °C. Whether this behavior is the result of an underlying growth-promoting mechanism could not be explained from a macroscopic point of view (e.g., macrorheology, a(w)), but may be more related to the local microscopic properties of the gels. In a second part of this study, the potential use and information content of optical density measurements for G/NG data collection in structured media were critically evaluated and confronted with their practical relevance to the food industry.

Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmission-blocking antibodies in the mosquito.

Plasmodium falciparum gametocytes contain specific antigens, some of which (Mr 230,000, 48,000, 45,000) are expressed on the surface of the newly emerged macrogamete. A different antigen (Mr 25,000) surrounds the surface of the ookinete and, although present to some extent in the developing gametocyte, is synthesized in high quantities by the macrogamete/zygote and expressed progressively on the transforming zygote surface. These antigens are targets of transmission blocking antibodies that are effective at two distinct points after gametogenesis: fertilization of the macrogamete and ookinete to oocyst development. The antigens involved in the fertilization blockade are the Mr 48 and 45 proteins, which are expressed on the macrogamete surface. The Mr 230 K coprecipitating protein probably plays no part in transmission block. mAb directed against the Mr 25 K ookinete surface protein blocked transmission without inhibiting ookinete formation, indicating that this protein has an important role in the transformation of ookinete into oocyst. A combination of mAb recognizing different epitopes on the same protein molecule acted synergistically in inhibiting oocyst formation. Using a mixture of two blocking mAb reacting against the Mr 48/45 and 25 K proteins, respectively, an additive blocking effect could be demonstrated.

Appropriateness of computed tomography and magnetic resonance imaging scans in a rural regional hospital in South Africa: A 6-year follow-up study.

BACKGROUND: Requests for computed tomography (CT) and magnetic resonance imaging (MRI) scans by doctors with different levels of experience have cost and risk implications globally. Evidence-based appropriateness criteria guide doctors to the suitable use of radiology imaging. There are few studies regarding appropriateness of CT requests in the South African (SA) context. Previous research in the Garden Route district of Western Cape Province, SA, evaluated the appropriateness of scans. OBJECTIVES: To review the appropriateness of CT and MRI scans done in a 6-year follow-up study at one facility. METHODS: This was a retrospective descriptive study. All CT and MRI scans performed during October 2018 at George Hospital were classified according to American College of Radiology guidelines as: usually appropriate (UA), might be appropriate (MBA), or not appropriate (NA). Stratified analysis allowed simple statistics and some comparison with the previous study. RESULTS: A total of 515 CT and MRI scans were included, of which 81.4% were UA, 7.4% MBA and 11.2% NA. Most scans were requested by medical officers (n=255), followed by consultants (n=126) and junior doctors (n=70). Medical officers made the majority of inappropriate requests. Second-year interns requested the lowest number of inappropriate scans, with registrars not requesting any inappropriate scans. Most of the inappropriate scans were requested after hours. Thirty-seven of the 123 (30.1%) after-hours scans were inappropriately requested compared with 21 of 392 (5.4%) scans during normal working hours, which were inappropriate. CONCLUSIONS: Although the majority of scans were being ordered appropriately, pre-authorisation by experienced physicians and incorporation of guidelines would make requests more complete and possibly more appropriate, especially after hours.

Design of an experimental viscoelastic food model system for studying Zygosaccharomyces bailii spoilage in acidic sauces.

Within the field of predictive microbiology, the number of studies that quantify the effect of food structure on microbial behavior is very limited. This is mainly due to impracticalities related to the use of a nonliquid growth medium. In this study, an experimental food model system for studying yeast spoilage in acid sauces was developed by selecting a suitable thickening/gelling agent. In a first step, a variety of thickening/gelling agents was screened, with respect to the main physicochemical (pH, water activity, and acetic acid and sugar concentrations) and rheological (weak gel viscoelastic behavior and presence of a yield stress) characteristics of acid sauces. Second, the rheological behavior of the selected thickening/gelling agent, Carbopol 980, was extensively studied within the following range of conditions: pH 4.0 to 5.0, acetic acid concentration of 0 to 1.0% (vol/vol), glycerol concentration of 0 to 15% (wt/vol), and Carbopol concentration of 1.0 to 1.5% (wt/vol). Finally, the applicability of the model system was illustrated by performing growth experiments in microtiter plates for Zygosaccharomyces bailii at 0, 0.5, 1.0, and 1.5% (wt/vol) Carbopol, 5% (wt/vol) glycerol, 0% (vol/vol) acetic acid, and pH 5.0. A shift from planktonic growth to growth in colonies was observed when the Carbopol concentration increased from 0.5 to 1.0%. The applicability of the model system was illustrated by estimating mu(max) at 0.5% Carbopol from absorbance detection times.

Chromosome-sized DNA molecules of Plasmodium falciparum.

At least seven chromosome-sized DNA molecules (750 to 2000 kilobases in length and one fraction of undetermined molecular weight) from cultured clones and isolates of Plasmodium falciparum have been separated by pulsed-field gradient gel electrophoresis. Whereas asexual blood stages and sexual stages of the same line have identical molecular karyotypes, the length of chromosome-sized DNA molecules among different geographical isolates and several clones derived from a single patient is different. These length alterations of chromosomes are the result of DNA rearrangements that must occur unrelated to sexual differentiation.

A peculiar stimulatory effect of acetic and lactic acid on growth and fermentative metabolism of Zygosaccharomyces bailii.

Stimulatory or protective effects of organic acids at low concentrations, e.g. acetic and lactic acid, on microorganisms have previously been reported. Especially in case of Zygosaccharomyces bailii, a peculiar growth stimulation by these two acids has recently been noticed. In order to elucidate this interesting phenomenon, growth and fermentative metabolism of Z. bailii was investigated in media with low pH (pH 4.0), high sugar (15% (w/v)) and different acetic and lactic acid concentrations. At both experimental temperatures (7 and 30 degrees C), a growth stimulation in the presence of 2.5% (v/v) lactic acid was observed. Furthermore at 7 degrees C, the yeast exhibited another unusual behaviour as it grew much faster in media containing 1.25% (v/v) acetic acid than in the control (without any acid). Production of fermentative metabolites was also increased together with the enhanced growth at both temperatures. These possible stimulatory effects of acetic and lactic acid should be taken into consideration when the acids are used at low doses for food preservative purpose. Presence of the acids may stimulate Z. bailii growth and fermentative metabolism, particularly at refrigeration temperature, consequently resulting in an earlier spoilage.

Patterns of renal disease: A 30-year renal biopsy study at Chris Hani Baragwanath Academic Hospital, Soweto, Johannesburg, South Africa.

BACKGROUND: Data on renal pathology from sub-Saharan Africa are limited. OBJECTIVES: To report on biopsy-confirmed renal pathology over 30 years in Soweto, South Africa (SA). METHODS: Retrospective analysis was conducted of 1 848 adult native renal biopsies performed at Chris Hani Baragwanath Academic Hospital between 1 January 1982 and 31 December 2011. RESULTS: There was an even gender distribution, and 96.4% of patients were of black ethnicity. The mean (standard deviation) age of patients was 33.5 (12.6) years. The main clinical indications for renal biopsy were nephrotic syndrome (47.7%), acute kidney injury (19.8%) and asymptomatic urine abnormalities (8.1%). Secondary glomerular diseases (SGNs) (49.3%) occurred more frequently than primary glomerular diseases (PGNs) (39.7%). SGNs increased during the study period, while PGNs decreased (p<0.001). The most frequent PGNs were focal segmental glomerulosclerosis (FSGS) (29.6%), membranous glomerulonephritis (25.7%) and membranoproliferative glomerulonephritis (18.1%). Lupus nephritis (LN) (31.0%) was the most frequent SGN, followed by HIV-associated nephropathy (HIVAN) (13.3%) and malignant hypertension (12.5%). HIV-positive biopsies constituted 19.7% of all biopsies, and the proportion increased over time. In HIV-positive patients, the most common diagnoses were HIVAN (32.7%), HIV immune complex disease (11.8%) and FSGS (11.3%). CONCLUSIONS: This study contributes to our knowledge of renal pathology in SA and shows some data that differ from studies in other regions. The increase in SGNs probably reflects the influence of HIV and LN in the study population.

Modelling the unexpected effect of acetic and lactic acid in combination with pH and aw on the growth/no growth interface of Zygosaccharomyces bailii.

Microbial spoilage of shelf-stable acidified sauces is predominantly caused by lactic acid bacteria and yeasts. A specific spoilage yeast in these products is Zygosaccharomyces bailii, as this fructophilic, osmotolerant, and weak acid resistant yeast is difficult to control. A growth/no growth model was developed describing the influence of (i) pH in a range from pH 3.0 to pH 5.0 (5 levels), (ii) acetic acid in a range from 0 to 3.5% (w/v), and (iii) lactic acid in a range from 0 to 3.0% (w/v). aw was fixed at a level of 0.95 which is representative for acidified sauces with high sugar content. Modified Sabouraud medium was inoculated at +/- 10(4) CFU/ml, incubated at 30 degrees C and growth was assessed by optical density measurements. All combinations of environmental conditions were tested in at least twelve replicates, yielding precise values for the probability of growth. Results showed that replacing acetic acid by lactic acid, which has a milder taste, may imply some risks on food spoilage because, under some conditions, stimulation of growth by lactic acid was observed. This stimulation had also consequences on the model development: (i) only ordinary logistic regression models were able to describe this phenomenon due to their flexible behaviour, (ii) it was necessary to split up the data set into two subsets to have the best description of the obtained data. Two different ordinary logistic regression models were fitted on these data sets taking either the total acid concentration as one of the explanatory variables or differentiating between the undissociated and dissociated acid concentrations. The obtained models were compared with the CIMSCEE code [CIMSCEE, 1992. Code for the production of microbiologically safe and stable emulsified and non-emulsified sauces containing acetic acid. Comité des Industries des Mayonnaise et Sauces Condimentaires, de la Communauté Economique Européenne, Brussels, Belgium], a formula which is nowadays often used by the food industry to predict the stability of acidified products based on the undissociated acetic acid, NaCl and sugars concentration. Comparing this formula and the newly developed models showed that the CIMSCEE code made a slight underestimation of the growth probability. Advantages of the newly developed models are the description of the gradual transition zone between growth and no growth and the incorporation of the effect of lactic acid, alone or in combination with acetic acid.

Heat resistance of Bacillus cereus emetic toxin, cereulide.

AIMS: The study describes the effects of heating temperature and exposure time on the thermal stability of cereulide under different conditions (pH, presence/absence of oil phase and cereulide concentration). METHODS AND RESULTS: Cereulide heat inactivation was investigated at 100, 121 and 150 degrees C under different alkaline pH values (8.6-10.6) and in the presence of oil phase (0.6-1.4%). Three different cereulide concentrations (0.5, 5 and 6 microg ml(-1)) were used. Cereulide detection was performed with computer-aided semen analyzer and with HPLC-MS. Highly alkaline pH was needed to achieve inactivation. At lower cereulide concentrations less drastic conditions were needed. Removal of alkaline buffer after the heat treatment resulted in the recovery of toxic activity. CONCLUSIONS: Heat stability of cereulide has been proved to be remarkable, even at highly alkaline pH values, at all temperatures tested. The loss of activity appeared to be reversible. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the inability of any heat treatment used in the food industry to inactivate cereulide. Food safety has to rely on prevention and cold chain maintenance. Cleaning practices also need to be adapted as cereulide may remain in its active form upon sterilization of used material.

Speech perception in congenitally, pre-lingually and post-lingually deaf children expressed in an equivalent hearing loss value.

OBJECTIVES: To investigate the speech perception performance of children with a cochlear implant (CI) after 3 and 4 years of follow-up and to study the influence of age at implantation, duration of deafness and communication mode on the variability in speech perception performance. STUDY DESIGN: A broad battery of speech perception tests was administered to 67 children with a CI. The results were reduced into one measure: the 'equivalent hearing loss (EHL)'. This outcome measure refers to the performance of a reference group of severely and profoundly hearing impaired children with conventional hearing aids. PARTICIPANTS: The population comprised 35 congenitally, 17 pre-lingually and 15 post-lingually deaf children implanted between 1989 and 1999. The population was homogeneous with respect to cognition, residual hearing and support at home as a result of conservative inclusion criteria. RESULTS: During the first 2 years after implantation, post-lingually deaf children showed the fastest rate of improvement in speech perception. After 3 years of implant use, the early implanted pre-lingually deaf children and congenitally deaf children implanted under the age of 6 years caught up with the post-lingually deaf children. Pre-lingually deaf children implanted after a relatively long-duration of deafness tended to show poorer performance than those with a shorter duration. Performance of congenitally deaf children implanted after the age of 6 years was poorer and progress was slower. In the congenitally deaf children, 36% of the variability in performance was explained by duration of deafness, whereas in the children with pre- and post-lingually acquired deafness, communication mode explained 69% of the variance. CONCLUSIONS: All children derived benefit from their CI for speech perception tasks, but performance varied greatly. Several children reached EHL levels around 70 dB; their speech perception was equal to that of a child with conventional hearing aids who has 70 dB HL. After early implantation, the levels of performance that were eventually achieved differed no more than 10 dB, irrespective of whether the onset of deafness was pre-lingual or postlingual. In congenitally deaf children, duration of deafness played a major role in speech perception performance, whereas in the children with pre-lingually and post-lingually acquired deafness together, mode of communication was a major factor.

Solid fat influences sorbic acid partitioning and enhances the preservation effect on C. guilliermondii in biphasic food model systems.

Preservation of emulsions relies on factors including pH, temperature, structure and the application of carboxylic acid preservatives, such as sorbic and benzoic acid. Organic acid preservatives tend to migrate to the lipid phase of emulsions. Taking into account the fact that organic acid in the aqueous phase is solely responsible for antimicrobial activity, this partitioning behavior is considered as a loss of preservative. The influence of microstructure properties on sorbic acid distribution and preservation effect was investigated in model food systems comprising of aqueous phase, and liquid oil and solid fat as lipid phase, which represent major constituents of water-oil emulsions. The aqueous phase of the food model systems was comprised of Yeast Nitrogen Broth (YNB) in phosphate buffer and buffered at pH 4.5, 5.5, 6.5. Sorbic acid (100 mg/L) in the form of potassium sorbate was added to the aqueous phase. Candida guilliermondii (2 log CFU/mL) was inoculated in the aqueous phase to resemble industrial post-contamination CFU levels. Growth parameters, generation time (GT) and lag phase (λ) of C. guilliermondii in the aqueous phase of the food model systems were quantified during 1 month at 7 °C and maximum 80 h at 22 °C. HPLC analyses were performed to evaluate total sorbic acid content in each food model system. Sorbic acid inhibited growth of C. guilliermondii in YNB + SA at pH 4.5 at 7 °C and 22 °C. The presence of liquid oil caused partitioning of sorbic acid into the lipid phase in YNB + SA|oil at pH 4.5 at 7 °C and 22 °C, reducing its inhibitory effect. Adding solid fat into the model food systems significantly prolonged lag phase duration in the YNB + SA|oil + fat system at pH 4.5 at 22 °C and growth was inhibited at 7 °C. HPLC analysis showed a final aqueous sorbic acid concentration of 34 mg/L and 44 mg/L in YNB + SA|oil system at pH 4.5 at 7 °C and 22 °C after 1 month and 168 h, respectively. In YNB + SA|oil + fat system at pH 4.5, 87 mg/L of aqueous sorbic acid was measured after 1 month at 7 °C and 168 h at 22 °C, indicating that the presence of solid fat retards sorbic acid partitioning. Results show that structural components such as liquid oil and solid fat have an effect on the aqueous sorbic acid concentration and its preservation effect. The presence of solid fat reduces the tendency of sorbic acid to partition into the lipid phase, which is reflected in the inhibitory effect of sorbic acid on C. guilliermondii.

Growth/no growth models describing the influence of pH, lactic and acetic acid on lactic acid bacteria developed to determine the stability of acidified sauces.

Growth/no growth models were developed for two spoilage bacteria typical for acidified sauces, L. plantarum and L. fructivorans. Influencing factors embedded in the model are also those typically encountered in these acidified sauces. The pH was varied between 3.0 and 5.0 (5 levels), and the acetic and lactic acid concentration ranged from 0 to 3% (6 levels). Modified MRS broth was inoculated at a high inoculation level (10(6) CFU/ml), incubated at 30 degrees C and growth was assessed by optical density measurements. All combinations of environmental conditions were tested in twelvefold yielding precise values for the probability of growth. Data were modelled by means of ordinary logistic regression. A comparison was made between a model containing the total acid concentrations as explanatory variables, on the one hand, and a model differentiating between the dissociated and undissociated concentrations, on the other hand. Results showed that (i) L. plantarum and L. fructivorans behave differently, resulting in a clearly distinct growth/no growth interface, (ii) there was no great difference between the established models with different explanatory variables, (iii) in some cases, growth/no growth boundaries at very low probabilities (which are more practical in industry) show illogical behaviour. The results of this study were also compared with the CIMSCEE code, which is often used by food producers to determine the stability of their acidified food products.

Influence of pH, water activity and acetic acid concentration on Listeria monocytogenes at 7 degrees C: data collection for the development of a growth/no growth model.

Growth/no growth models can be used to determine the chance that microorganisms will grow in specific environmental conditions. As a consequence, these models are of interest in the assessment of the safety of foods which can be contaminated with food pathogens. In this paper, growth/no growth data for Listeria monocytogenes (in a monoculture and in a mixed strain culture) are presented. The data were gathered at 7 degrees C in Nutrient Broth with different combinations of environmental factors pH (5.0-6.0, six levels), water activity (0.960-0.990, six levels) and acetic acid concentration (0-0.8% (w/w), five levels). This combination of environmental factors for the development of a growth/no growth model was based on the characteristics of sauces and mayonnaise based salads. The strains used were chosen from screening experiments in which the pH, water activity and acetic acid resistance of 26 L. monocytogenes strains (LFMFP culture collection) was determined at 30 degrees C in Brain Heart Infusion broth. The screening showed that most L. monocytogenes strains were not able to grow at a(w)<0.930, pH<4.3 or a total acetic acid concentration >0.4% (w/w). Among these strains, the ones chosen were the most resistant to one of these factors in the hope that, if the resulting model predicted no growth at certain conditions for those more resistant strains, then these predictions would also be valid for the less resistant strains. A mixed strain culture was also examined to combine the strains that were most resistant to one of the factors. A full factorial design with the selected strains was tested. The experiments were performed in microtiter plates and the growth was followed by optical density measurements at 380 nm. The plates were inoculated with 6 log CFU/ml and twenty replicates were made for each treatment combination. These data were used (1) to determine the growth/no growth boundary and (2) to estimate the influence of the environmental conditions on the time to detection. From the monoculture and mixed strain data, the growth boundary of L. monocytogenes is shown not to be a straight cut-off but a rather narrow transition zone. The experiments also showed that in the studied region, a(w) did not have a pronounced influence on the position of the growth/no growth boundary while a low concentration of acetic acid (0.2% (w/w)) and a pH decrease from 6.0 to 5.8 was sufficient to significantly reduce the possibility of growth. The determination of the time to detection showed a significant increase at the combinations of environmental conditions near the 'no growth zone'. For example, at 0.2% (w/w) acetic acid, there was an increase from +/-10 days to 30 days by lowering pH from 5.8 to 5.6 at a(w) values of 0.985 and 0.979, while at pH 5.4 less than 50% growth occurred for all a(w) values.

Exploring the performance of logistic regression model types on growth/no growth data of Listeria monocytogenes.

Several model types have already been developed to describe the boundary between growth and no growth conditions. In this article two types were thoroughly studied and compared, namely (i) the ordinary (linear) logistic regression model, i.e., with a polynomial on the right-hand side of the model equation (type I) and (ii) the (nonlinear) logistic regression model derived from a square root-type kinetic model (type II). The examination was carried out on the basis of the data described in Vermeulen et al. [Vermeulen, A., Gysemans, K.P.M., Bernaerts, K., Geeraerd, A.H., Van Impe, J.F., Debevere, J., Devlieghere, F., 2006-this issue. Influence of pH, water activity and acetic acid concentration on Listeria monocytogenes at 7 degrees C: data collection for the development of a growth/no growth model. International Journal of Food Microbiology. .]. These data sets consist of growth/no growth data for Listeria monocytogenes as a function of water activity (0.960-0.990), pH (5.0-6.0) and acetic acid percentage (0-0.8% (w/w)), both for a monoculture and a mixed strain culture. Numerous replicates, namely twenty, were performed at closely spaced conditions. In this way detailed information was obtained about the position of the interface and the transition zone between growth and no growth. The main questions investigated were (i) which model type performs best on the monoculture and the mixed strain data, (ii) are there differences between the growth/no growth interfaces of monocultures and mixed strain cultures, (iii) which parameter estimation approach works best for the type II models, and (iv) how sensitive is the performance of these models to the values of their nonlinear-appearing parameters. The results showed that both type I and II models performed well on the monoculture data with respect to goodness-of-fit and predictive power. The type I models were, however, more sensitive to anomalous data points. The situation was different for the mixed strain culture. In that case, the type II models could not describe the curvature in the growth/no growth interface which was reversed to the typical curvatures found for monocultures. This unusual curvature may originate from the fact that (i) an interface of a mixed strain culture can result from the superposition of the interfaces of the individual strains, or that (ii) only a narrow range of the growth/no growth interface was studied (the local trend can be different from the trend over a wider range). It was also observed that the best type II models were obtained with the flexible nonlinear logistic regression, although reasonably good models were obtained with the less flexible linear logistic regression with the nonlinear-appearing parameters fixed at experimentally determined values. Finally, it was found that for some of the nonlinear-appearing parameters, deviations from their experimentally determined values did not influence the model fit. This was probably caused by the fact that only a limited part of the growth/no growth interface was studied.