The use of 16s rDNA restriction fragment length polymorphism analysis for the identification of non-fermenting gram negative bacilli in a cystic fibrosis microbiology referral service

The utility of 16s rDNA restriction fragment length polymorphism (RFLP) analysis for the partial genomovar differentiation of Burkholderia cepacia complex bacterium is well documented. We compared the 16s rDNA RFLP signatures for a number of non-fermenting gram negative bacilli (NF GNB) LMG control strains and clinical isolates pertaining to the genera Burkholderia, Pseudomonas, Achromobacter (Alcaligenes), Ralstonia, Stenotrophomonas and Pandoraea. A collection of 24 control strain (LMG) and 25 clinical isolates were included in the study. Using conventional PCR, a 1.2 kbp 16s rDNA fragment was generated for each organism. Following restriction digestion and electrophoresis, each clinical isolate RFLP signature was compared to those of the control strain panel. Nineteen different RFLP signatures were detected from the 28 control strains included in the study. TwentyoneyTwenty- five of the clinical isolates could be classified by RFLP analysis into a single genus and species when compared to the patterns produced by the control strain panel. Four clinical B. pseudomallei isolates produced RFLP signatures which were indistinguishable from B. cepacia genomovars I, III and VIII. The identity of these four isolates were confirmed using B. pseudomallei specific PCR. 16s rDNA RFLP analysis can be a useful identification strategy when applied to NF GNB, particularly for those which exhibit colistin sulfate resistance. The use of this molecular based methodology has proved very useful in the setting of a CF referral laboratory particularly when utilised in conjunction with B. cepacia complex and genomovar specific PCR techniques. Species specific PCR or sequence analysis should be considered for selected isolates; especially where discrepancies between epidemiology, phenotypic and genotypic characteristics occur.

Differences in lung function, growth parameters and frequency of hospital admissions between adolescents with cystic fibrosis-related diabetes and controls

As survival of patients with CF increases,glucose intolerance and cystic fibrosisrelated diabetes (CFRD),ar e increasingly recognised common complications. CFRD may be preceded by a pre-diabetic state. Using markers identified as being associated with CFRD may improve targeted screening. Aim: To identify features consistently predicting CFRD in paediatric patients. Patients diagnosed with CFRD between January 1997–January 2002 were compared with age and sex matched controls. Clinical,micr obiological, and hospitalisation data was collected at time of CFRD diagnosis,and at six monthly intervals for 3 yr prior to diagnosis. Eight patients with CFRD were identified,mean age 13.7 yr (S.D. 3.49) at time of diagnosis. Control patients underwent OGTT to ensure normal glucose tolerance. Patients with CFRD had a lower FEV1 up to 12 months prior to diagnosis however, this was only significant at diagnosis. There was no difference in weight and height z scores between the 2 groups; however,the decrease in weight and height z scores in the CFRD group over 3 yr prior to diagnosis was significant. Mean number of days in hospital and admissions per patient significantly increased in the CFRD group,6 months prior to diagnosis. No other significant differences were observed between the 2 groups. Conclusions: This study has shown a difference in lung function,gr owth parameters and frequency of hospital admissions between patients with CFRD and controls. These differences may be utilised as tools for targeted screening in the paediatricyadolescent population. Further larger scale studies are required to improve guidelines for targeted screening in this population.

Gender differences in CF phenotype associated with low bone mineral density

Adolescents and adults with CF have lower bone mineral density (BMD) than normal, but its relationship with phenotype is not well understood. Point FEV1% predicted (FEV) and rate of change of FEV are biased estimates of disease severity, because progressively older subjects represent a selected survivor population, with females at greater risk of death than males. To investigate the relationship between BMD and phenotype we used an index (predicted age at death) derived from Bayesian estimates of slope and intercept of FEV, age at last measurement and survival status. Predictive equations for the index were derived from 97 subjects (78 survivors) from the RCH CF clinic, and applied to a group of 102 comparable subjects who had BMD measured, classified as having‘mild’ ()75th), ‘moderate’ (25– 75th), or ‘severe’ (-25th centile) phenotype. Total body (TB) and lumbar spine (LS) BMD z-scores (Z) were compared, adjustingfor gender effects, using 2-way ANOVA. Annual mean change in FEV segregated, as expected, according to phenotype, ‘severe’ (ns25), ‘moderate’ (ns51) and ‘mild’ (ns25) y3.01(y3.73 to y2.30)%, y0.85(y1.36 to y0.35)%, 2.70(1.92 to 3.46)%, respectively, with no gender difference. LS and TB BMDZ were different in each phenotype (P-s 0.002), LS BMDZ for ‘severe’, ‘moderate’ and ‘mild’ y1.63(CI: y2.07 to y 1.19), y0.86(CI: y1.17 to y0.55), y0.06(CI: y0.54 to 0.41). Males had lower LS BMDZ than females overall (y1.22 (CI: y1.54 to y0.91) vs. y0.48(CI: y 0.84 to y0.12) Ps0.002). In the ‘severe’ group, males had lower TB BMDZ and LS BMDZ (PF0.002). Low BMD is associated with ‘moderate’ and ‘severe’ phenotypes, with relative preservation in females in the ‘severe’ group. Female biology (reproductive fitness) might promote resistance to bone resorption at a critical level of BMD loss.

Mortality from asthma in the state of S. Paulo, Brazil (1970-1992)

Mortality from asthma has shown important variations over time in several countries. In Brazil, a mortality study performed in the 60s, covering the cities of S.Paulo and Ribeirão Preto, and other ten cities showed that S.Paulo presented the lowest death rate from asthma among of them all. It was decided to study the time trends of deaths from asthma and from the whole set of respiratory diseases from 1970 to 1992, in the population aged 15-34 yrs. old in the State of S.Paulo, as well as to compare them with those of other countries. Asthma mortality rates during the 23 years of observation since 1975, showed an oscillatory declining pattern with a peak of deaths in the initial years. The linearization of the curve allows the calculation of Pearson's correlation coefficient that was significantly negative, suggesting a decline in the mortality over this period, mainly in the 5-9 yrs. old and 30-34 yrs. old strata. The segmentation of data between the period of ICD-9, 1970 to 1978, and of ICD-9, 1979 and subsequent years, shows that there is stability within each period, in all age-groups, except for that of 5-9 yr. olds between 1970-1978. Comparing the rates of the population aged 15-34 yrs. old for the State of S. Paulo, Brazil, with trends observed in 14 other countries, an intermediate pattern for the first triennial period (1970-1972) as well as for the subsequent triennial periods, emerges. A prevalence study of asthma, a follow up program meant for using emergency rooms and a surveillance of deaths due to all respiratory diseases and specifically to asthma are strongly recommended.

Assessing morbidity in the paediatric community

INTRODUCTION: Morbidity information is easily available from medical records but its scope is limited to the population attended by the health services. Information on the prevalence of diseases requires community surveys, which are not always feasible. These two sources of information represent two alternative assessments of disease occurrence, namely demand morbidity and perceived morbidity. The present study was conceived so as to elicit a potential relationship between them so that the former could be used in the absence of the latter. METHODS: A community of 13,365 families on the outskirts of S. Paulo, Brazil, was studied during the period from 15/Nov/1994 to 15/Jan/1995. Data regarding children less than 5 years old were collected from a household survey and from the 2 basic health units in the area. Prevalence of diseases was ascertained from perceived morbidity and compared to estimates computed from demand morbidity. RESULTS: Data analysis distinguished 2 age groups, infants less than 1 year old and children 1 to less than 5. The most important groups of diseases were respiratory diseases, diarrhoea, skin problems and infectious & parasitical diseases. Basic health units presented a better coverage for infants. Though disease frequencies were not different within or outside these units, a better coverage was found for diarrhoea and infectious & parasitical diseases in the infant group, and for diarrhoea in the older age group. Equivalence between the two types of morbidity was found to be limited to the infant group and concerned only the best covered diseases. The odds of a disease being seen at the health service should be of at least 4:10 to ensure this equivalence. CONCLUSION: It was concluded that, provided that health service coverage is good, demand morbidity can be taken as a reliable estimate of community morbidity.

Occupational exposure to Aspergillus by swine and poultry farm workers in Portugal

Aspergillus is among a growing list of allergens that aggravate asthmatic responses. Significant pulmonary pathology is associated with Aspergillus-induced allergic and asthmatic lung disease. Environments with high levels of exposure to fungi are found in animal production facilities such as for swine and poultry, and farmers working with these are at increased risk for occupational respiratory diseases. Seven Portuguese poultry and seven swine farms were analyzed in order to estimate the prevalence, amount, and distribution of Aspergillus species, as well as to determine the presence of clinical symptoms associated with asthma and other allergy diseases in these highly contaminated settings. From the collected fungal isolates (699), an average incidence of 22% Aspergillus was detected in poultry farms, while the prevalence at swine farms was 14%. The most frequently isolated Aspergillus species were A. versicolor, A. flavus, and A. fumigatus. In poultry farms, A. flavus presented the highest level of airborne spores (>2000 CFU/m3), whereas in swine farms the highest was A. versicolor, with an incidence fourfold greater higher than the other mentioned species. Eighty workers in these settings were analyzed, ranging in age from 17 to 93 yr. The potentially hazardous exposure of poultry workers to mold allergens using sensitization markers was evaluated. Although no significant positive association was found between fungal contamination and sensitization to fungal antigens, a high incidence of respiratory symptoms in professionals without asthma was observed, namely, wheezing associated with dyspnea (23.8%) and dyspnea after strenuous activities (12.3%), suggesting underdiagnosed respiratory disturbances. Further, 32.5% of all exposed workers noted an improvement of respiratory ability during resting and holidays. From all the analyzed workers, seven were previously diagnosed with asthma and four reported the first attack after the age of 40 yr, which may be associated with their occupational exposure. Some of the fungi, namely, the Aspergillus species detected in this study, are known to induce hypersensitivity reactions in humans. This study confirmed the presence and distribution of Aspergillus in Portuguese poultry and swine farms, suggesting a possible occupational health problem and raising the need for preventive and protective measures to apply to avoid exposure in both occupational settings.

Exposure to particles in Portuguese swine production

Several studies have shown that human exposures to airbome dust and microorganisms, such as bacteria and fungi, can cause respiratory diseases. Agricultural workers have been found to be at high risk of exposures to airborne particles. From a human health perspective dust exposure in pig farming is the most important risk because of the large number of workers needed in pig production and the increasing number of working hours inside enclosed buildings. In the pig buildings, particulate matters like dust play a role in not only deteriorating indoor air quality but also can cause an adverse health effect on workers. Generally, dust is recognized to adsorb and transport odorous compounds and biological agents. The aim of this study was to determine particles contamination in 7 swine farms located in Lisbon district, Portugal.

Fungal contamination: comparation between Portuguese poultries and swines

Animal confinement tends to increase the overall microbial load in the production environment caused by high amounts of feed and organic residuals (manure and wastewater) present in those environments. The number of animais and the handling and management required to work in these settings also contribute to enhance that microbial ioad. Animal housing typically exposes workers to substantial concentrations of bioaerosols, such as fungi and their metabolites. Therefore, agricultural workers, and especially pig and poultry farmers, are at increased risk of occupational respiratory diseases. Exposure to bioaerosols in poultries and swines may vary depending upon the stage of the animals' growth, density, manure management procedures, litter type and used floor coverage, among others. Gathering temporal information about the quantity and the composition of fungal load is necessary to better understand the relationship between these factors and adverse health symptoms of workers. This study aimed to characterize and compare fungal contamination between these two different settings.