A retrospective assessment of the completeness and timeliness of meningococcal disease notifications in the Republic of Ireland over a 16-year period, 1999-2015.

OBJECTIVES: To assess how invasive meningococcal disease (IMD) records held by the Irish Meningitis & Sepsis Reference Laboratory (IMSRL) compare to records of IMD notifications reported on the national integrated electronic Computerised Infectious Disease Reporting (CIDR) system. STUDY DESIGN: We assessed the completeness, data quality and timeliness of IMD notifications and reference laboratory records for the period between 01 July 1999 and 30 June 2015 by identifying discrepant and/or missing data items in a matched case data set and by measuring the timeliness of case reporting. METHODS: We matched anonymised cases notified to CIDR to records based at the IMSRL using birth, reporting and onset dates with gender and laboratory parameters of meningococcal strain characteristics and method of confirmation. Completeness, data quality and the timeliness of notifications were assessed by a stratified sensitivity-based technique and by calculating the average difference between IMSRL and CIDR reporting dates. RESULTS: CIDR recorded a total of 3163 notifications, of which 2759 (87.2%) were matched to IMSRL records. Completeness of IMD case classification as confirmed was estimated to be >99%. Examining the levels of discrepant or missing data in both matched CIDR and IMSRL records as a measure of data quality, recording of demographic items and meningococcal group showed least differences, recording of laboratory case confirmation method and meningococcal strain characteristics were less well recorded, with detail on clinical presentation/diagnosis least well recorded. Overall average annual difference between CIDR and IMSRL recording dates was 3.2 days (95% confidence interval 2.6-3.8). CONCLUSIONS: A high quality of IMD surveillance in Ireland was demonstrated, but scope for improvements in timeliness and capture of enhanced surveillance data regarding date of onset and strain-specific characteristics were identified.

Update on World Health Organization HIV drug resistance prevention and assessment strategy: 2004-2011.

The HIV drug resistance (HIVDR) prevention and assessment strategy, developed by the World Health Organization (WHO) in partnership with HIVResNet, includes monitoring of HIVDR early warning indicators, surveys to assess acquired and transmitted HIVDR, and development of an accredited HIVDR genotyping laboratory network to support survey implementation in resource-limited settings. As of June 2011, 52 countries had implemented at least 1 element of the strategy, and 27 laboratories had been accredited. As access to antiretrovirals expands under the WHO/Joint United Nations Programme on HIV/AIDS Treatment 2.0 initiative, it is essential to strengthen HIVDR surveillance efforts in the face of increasing concern about HIVDR emergence and transmission.

Monitoring of early warning indicators for HIV drug resistance in antiretroviral therapy clinics in Zimbabwe.

Monitoring human immunodeficiency virus drug resistance (HIVDR) early warning indicators (EWIs) can help national antiretroviral treatment (ART) programs to identify clinic factors associated with HIVDR emergence and provide evidence to support national program and clinic-level adjustments, if necessary. World Health Organization-recommended HIVDR EWIs were monitored in Zimbabwe using routinely available data at selected ART clinics between 2007 and 2009. As Zimbabwe's national ART coverage increases, improved ART information systems are required to strengthen routine national ART monitoring and evaluation and facilitate scale-up of HIVDR EWI monitoring. Attention should be paid to minimizing loss to follow-up, supporting adherence, and ensuring clinic-level drug supply continuity.

Effect of liposomal composition on photoactivated liposome fusion.

Bennett and O'Brien [(1995) Biochemistry 34, 3102] showed that the ultraviolet light exposure of two-component large unilamellar liposomes (LUV) composed of a 3:1 molar mixture of dioleoylphosphatidylethanolamine (DOPE) and 1,2-bis[10-(2'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphatidyl- choline (bis-SorbPC) facilitated liposome fusion. The rate and extent of liposome fusion was dependent on the extent of photopolymerization, the temperature, and the pH. Examination of the temperature dependence of fusion of photolyzed and unphotolyzed liposomes demonstrated that an enhancement of the rate of fusion occurred in the temperature range associated with the initial appearance of precursors to the inverted cubic (QII) phase [Barry et al. (1992) Biochemistry 31, 10114]. Here, the effect of the molar lipid ratio of the DOPE/bis-SorbPC liposomes on the temperature for the onset of fusion, i.e. the critical fusion temperature, was characterized by changing the relative amounts of unreactive polymorphic lipid and reactive lamellar lipid. In each case, photopolymerization of bis-SorbPC lowered the critical fusion temperature by ca. 15-20 degrees C. The photoreaction of the bis-SorbPC-containing LUV yields cross-linked poly-SorbPC, enhancing the lateral separation of the DOPE and the polylipid and causing isothermal induction of liposome fusion by lowering the temperature for the onset of fusion. Evidence is presented to support the hypothesis that the critical temperature for fusion of two LUV populations depends on the molar ratio of the monomeric lipids in heterodimers of the two LUV. This analysis indicates that the photopolymerization of appropriately designed LUV can decrease the critical fusion temperature from above to below 37 degrees C.

Molecular genetic approaches to identification, epidemiology and taxonomy of non-albicans Candida species.

The reported incidence of fungal infections associated with non-albicans species from the Candida genus is increasing. Most of these infections occur in immunocompromised patients, particularly those infected with HIV. The role of molecular genetic techniques alongside the existing techniques for the identification and typing of these organisms is discussed. Species-specific genomic DNA fragments cloned from C. tropicalis and C. krusei have been developed for identification and strain typing. Analysis of tRNA profiles has been shown to be effective for the identification of C. glabrata, C. guilliermondii, C. parapsilosis and C. tropicalis. A PCR method employing primers complimentary to large ribosomal subunit genes and the lanosterol-alpha-demethylase gene has been applied for several species, including C. glabrata, C. krusei and C. tropicalis. Strain typing by comparison of genomic DNA fingerprints has been demonstrated for C. tropicalis and C. krusei following hybridisation analysis with species-specific probes. Synthetic oligonucleotide probes--which do not have to be species-specific and which can detect minor polymorphisms--have also been used for strain typing of isolates of several non-albicans species. Random amplification of polymorphic DNA (RAPD) has also been used for analysis of C. glabrata, C. lusitaniae and C. tropicalis isolates. The potential for the application of these and other techniques to Candida spp. taxonomy--and the example of a recently discovered novel species, C. dubliniensis--is discussed.

Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals.

Atypical oral Candida isolates were recovered from 60 HIV-infected and three HIV-negative individuals. These organisms were germ-tube-positive and produced abundant chlamydospores which were frequently arranged in triplets or in contiguous pairs. They belonged to C. albicans serotype A and had atypical carbohydrate assimilation profiles. Fingerprinting the genomic DNA of a selection of these organisms with the C. albicans-specific probe 27A and five separate oligonucleotides, homologous to eukaryotic microsatellite repeat sequences, demonstrated that they had a very distinct genomic organization compared to C. albicans and C. stellatoidea. This was further established by random amplified polymorphic DNA (RAPD) and karyotype analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from nine atypical isolates and the corresponding sequences determined from C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that they atypical organisms formed a homogeneous cluster (100% similarity) that was significantly different from the other Candida species analysed, but was most closely related to C. albicans and C. stellatoidea. These genetic data combined with the phenotypic characteristics of these atypical organisms strongly suggest that they constitute a novel species within the genus Candida for which the name Candida dubliniensis is proposed.

Photoactivated enhancement of liposome fusion.

The photopolymerization of two-component large unilamellar liposomes (LUV) composed of 3:1 dioleoylphosphatidylethanolamine (DOPE) and either 1,2-bis[10-(2'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphatidylc holine (bis-SorbPC) or 1-palmitoyl-2-[10-(2'-hexadienoyloxy)decanoyl]-sn- glycero-3-phosphatidylcholine (mono-SorbPC) facilitated liposome fusion. Fusion was characterized by fluorescent assays for lipid mixing, aqueous contents mixing, and aqueous contents leakage. The rate and extent of the liposome fusion was dependent on the extent of photopolymerization, temperature, and the fusion initiation conditions, including the pH and the presence of Mg2+ ions. Examination of the temperature dependence of fusion for unpolymerized and polymerized liposomes showed that an enhancement of the rate of fusion occurred in the temperature range delta TI, which previous NMR studies have identified as the initial appearance of precursors to the formation of the inverted cubic phase [Barry, J. A., et al. (1992) Biochemistry 31, 10114]. The phase behavior and fusion characteristics of the DOPE/bis-SorbPC (3:1) membranes provide unequivocal evidence that liposome fusion is mediated via intermediates associated with the lamellar to QII phase transition rather than the HII phase. Photopolymerization of SorbPC-containing liposomes forms poly-SorbPC, which enhances the lateral separation of the liposome components. The formation of enriched domains of polymorphic lipids, e.g., DOPE, causes isothermal induction of fusion by lowering the critical fusion temperature of the membranes.

Oral Candida in HIV infection and AIDS: new perspectives/new approaches.

Oral candidosis has become an increasingly important problem in HIV-infected individuals. At present, the small body of published literature on the characterization of the Candida strains and species found in HIV+ patients is full of confusion and contradictions. Some of these difficulties are the result of the methodological shortcomings of a number of the techniques that have been used. Examples of the problems that may be encountered on primary isolation and subculture are described and the drawbacks associated with the systems used to date for phenotyping Candida are quoted. While molecular characterization techniques would appear to offer a reliable and objective alternative, they too have their strengths and weaknesses. An attempt is made to summarize the progress that has been made recently in the detection and identification of Candida albicans and also the non-albicans species from HIV-infected individuals. What emerges is that the commensal Candida species that inhabit the oral cavities of HIV+ patients are subjected to a number of significant pressures that probably promote the selection of organisms with unusual phenotypes and genotypes. These Candida are more difficult to characterize and behave differently compared to their counterparts in HIV- individuals. It is clear that uncovering the factors that are important for the selection of treatment regimens and will be predictive of outcome will not be easy. Candida organisms are neither as benign nor as simple as once thought.

Reduced azole susceptibility of oral isolates of Candida albicans from HIV-positive patients and a derivative exhibiting colony morphology variation.

Approximately 50% (15/28) of a selection of oral isolates of Candida albicans from separate individuals infected with the human immunodeficiency virus (HIV) exhibited low susceptibility to ketoconazole as determined by hyphal elongation assessment. Nine of these isolates exhibited colony morphology variation or switching at 37 degrees C, of which six expressed low ketoconazole susceptibility. To determine whether colony morphology variation could give rise to derivatives with reduced azole susceptibility, several high-frequency switching variants of three HIV-patient isolates were recovered and assessed. All but one of the variants expressed similar azole susceptibility profiles to their respective parental strains. However, the C. albicans derivative 132ACR expressed significantly reduced susceptibility to ketoconazole in comparison to its parental strain 132A. In whole cells, on the basis of total growth the switched derivative 132ACR was markedly less susceptible than its parental isolate 132A to ketoconazole at 10 microM. A much smaller difference was observed with fluconazole at 10 microM, with the switched derivative 132ACR exhibiting a threefold lower susceptibility compared with the parental isolate 132A. The incorporation of [14C]acetate in control and azole-treated cells of both organisms was higher for the parental strain. When cell lysates of strain 132A and its derivative 132ACR were incubated with [14C]mevalonic acid and ketoconazole, the IC50 for 14C-label incorporation into C-4 demethyl sterols was fivefold higher for lysates of the switched derivative 132ACR compared with those of the parental strain 132A. With fluconazole the IC50 value for the derivative 132ACR was 25-fold higher than for strain 132A. The 14-sterol demethylase of the switched derivative 132ACR was possibly less sensitive to azole inhibition than that of the enzyme of strain 132A. These studies indicated that colony morphology variation in vitro can generate derivatives with stable, reduced azole susceptibility without prior exposure to azoles.

Infants born at risk: consequences for maternal post-partum adjustment.

The role of infant risk and social support as predictors of post-partum adjustment was investigated. Fifty-three mothers whose infants reflected the range of neonatal conditions were interviewed six weeks after discharge of their infant from hospital. They were assessed on measures of emotional distress, depressive symptoms, social support and perceptions of, and concerns about, their infant and themselves. The results indicated that mothers of higher risk infants reported higher levels of emotional distress and depressive symptomatology, more concerns about themselves and their baby, more difficulty in expressing affection towards their baby and greater dissatisfaction with their social support. Using multiple regression techniques, depressive symptoms were predicted by neonatal risk and dissatisfaction with social support from family and friends, while emotional distress was predicted by neonatal risk and dissatisfaction with social support from the infant's father. The study underlines the need to place more emphasis on infant variables as factors in maternal post-partum adjustment.

Exact versus asymptotic analysis for a matched case-control study.

We compare exact and asymptotic methods for variable selection in matched case-control studies. Data from a study of melanoma among the employees of the Lawrence Livermore National Laboratory illustrate the comparisons. Relative to large sample methods, the exact method almost always yielded larger p-values. The differences in p-values became more pronounced with inclusion of more variables in the logistic model. Thus, when the sample size is not large, and there are many covariates under study, use of the exact method tends to select more parsimonious models and avoids overfit of the data.

Dermatitis from plastic tote boxes impregnated with an antistatic agent.

An outbreak of dermatitis occurred among employees of a microelectronics firm. In a cross-sectional epidemiologic investigation, we found that dermatitis of the hands or arms had occurred among 14 of 29 (48.3%) employees of the incoming inspection department where plastic tote boxes recently purchased from one manufacturer had been used, compared to only one case among 17 (5.9%) employees in another department which had not used these boxes. Affected workers could detect an oily film on the surfaces of these new boxes, but not on older ones. We identified the oily film to be a surface accumulation of bis-hydroxyethyl-tallow amine (BHETA), an antistatic agent with which the tote boxes had been impregnated. Subsequent toxicologic investigation established that BHETA could provoke both follicular and nonfollicular irritant dermatitis, and was also a potential skin sensitizer. Antistatic agents should be considered as potential causes of dermatitis among employees who handle electrical parts transported in plastic boxes, particularly when affected employees can detect an oily film on the box surfaces.

The electronic and magnetic properties of rubredoxin: a low-temperature magnetic circular dichroism study.

Oxidized rubredoxin from Clostridium pasteurianum has been investigated by magnetic circular dichroism (MCD) spectroscopy over the temperature range 1.5 to 150 K and at magnetic fields between 0 and 4.5 tesla. The results show that studies of the temperature and field dependence of MCD transitions afford insight into the polarization of electronic transitions for ground states with large g-value anisotropy, in addition to estimates of ground-state g values and zero-field splitting parameters. In agreement with the assignment made by Eaton and Lovenberg (Eaton, W.A. and Lovenberg, W. (1973) in Iron-Sulfur Proteins, Vol. II (Lovenberg, W., ed.), pp. 131-162, Academic Press, New York), the ultraviolet-visible spectrum of oxidized rubredoxin is assigned to two S----Fe(III) charge transfer transitions (both 6A1----6T2 under tetrahedral symmetry), each spanning a range of 650-430 nm and 430-330 nm, respectively. The observed splitting in each of these transitions is attributed to a predominant axial distortion in the excited state resulting in effective D2d symmetry.

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus.

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.

Impaired left ventricular function in acute myocardial infarction assessed by Doppler measurement of ascending aortic blood velocity and maximum acceleration.

The Doppler-derived ejection variables systolic velocity integral, maximum acceleration and heart rate were recorded in 92 patients with acute myocardial infarction (AMI) and 73 age-matched normal subjects. Systolic velocity integral was validated as an index of stroke volume against a thermodilution technique in acutely ill patients. Patients with AMI were separated into clinically defined Forrester subsets and into survivors and nonsurvivors of the acute infarction period. Systolic velocity integral correlates significantly with stroke volume determined by thermodilution (r = 0.07) in patients with aortic root areas within the normal range. Patients had a 37% lower maximum acceleration (p less than or equal to 0.001), a 48% lower systolic velocity integral (p less than or equal to 0.001) and a 13% higher heart rate than the age-matched normal subjects (p less than or equal to 0.01). Systolic velocity integral and maximum acceleration both showed a systematic significant decrease through the Forrester subsets (p less than or equal to 0.01, p less than or equal to 0.001, respectively), and were also significantly different between the survivor and nonsurvivor groups (p less than or equal to 0.05, p less than or equal to 0.01, respectively.) Thus, noninvasive measurement of ascending aortic blood velocity and acceleration allows rapid assessment of left ventricular function and provides indexes closely related to the patients' clinical status and subsequent risk of mortality, indicating the potential of the Doppler technique in the prognosis and subsequent management of patients with myocardial infarction.

Magnetic circular dichroism studies of succinate dehydrogenase. Evidence for [2Fe-2S], [3Fe-xS], and [4Fe-4S] centers in reconstitutively active enzyme.

Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.

The iron-sulfur cluster composition of Escherichia coli nitrate reductase.

Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition. The results indicate approximately one 3Fe and three or four [4Fe-4S]2+,1+ centers/molecule of isolated enzyme. The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and [4Fe-4S] centers to be electronically analogous to those in bacterial ferredoxins. The form and spin quantitation of the EPR spectra from [4Fe-4S]1+ centers in the reduced enzyme were found to vary with the conditions of reduction. For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers. In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions. The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center. However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.

The DNA-based human karyotype.

Image cytometry and computer analysis are used to determine the relative DNA content and the DNA-based centromeric index of the 24 chromosomes of the human karyotype. A two-step procedure is used. Chromosomes of cells in metaphase first are stained with quinacrine and identified visually by their fluorescent Q-band patterns. They then are stained for DNA using gallocyanin-chrome alum. The chromosome images are scanned and recorded as digital values of optical density by an CYDAC image cytometric microscope system, CYDAC. The digital images are processed by computer to measure for each chromosome the relative DNA stain contents of the whole chromosome and of the p and q arms and the DNA-based centromeric index. About ten cells are analyzed for each of the donors, who are phenotypically normal men and women. The chromosome measurements are pooled by chromosome type for each donor and are compared among donors. The means of the chromosome measurements give the DNA-based human karyotype. Analysis of the DNA-based data shows that some chromosomes or portions of chromosomes vary significantly among donors. These variants do not correlate with detectable morphologic polymorphisms, such as Q- or C-band variants; thus they represent new and otherwise undetectable chromosome polymorphisms whose genetic basis and clinical significance are yet to be determined.

Quantitative methods of measuring the sensitivity of the mouse sperm morphology assay.

In this study murine sperm were subjected to graded doses of X irradiation (0 to 120 rad) to determine whether quantitative measurements made on enlarged photographs of the sperm heads are related to radiation dose. We found that the Mahalanobis distance statistic, when used to measure distance in a multivariate space from a control group of measurements, could be used to classify sperm as normal or abnormal. The percent classified as abnormal by this method was found to be linearly related to dose. The results suggest that sensitivity of the murine sperm assay can be improved by selecting an optimal set of measurements. This improvement can reduce the doubling dose from approximately 70 rad to 10 to 15 rad while keeping the percentage of abnormal sperm in control mice at 3%, equal to the current visual method.