Benefit of Sequential Audit Cycles in Improving Management of Vitamin D Deficiency in the HIV Infected Paediatric Population

Aims Lifelong HIV infection has an unknown impact on bone health in children. In view of this, we aimed to improve management of vitamin D deficiency. Methods Three audits over 8 years (2009-2017) were performed with interventions introduced intermittently in an effort to improve vitamin D deficiency. The interventions included education, a change in vitamin D dose and brand to increase compliance and a shift to nursing led management. Results The most striking result was the eradication of patients with deficient vitamin D levels (<25nmol/L) in 2017. In 2009 and 2015, 15% and 9% were deficient. In the earlier two studies, only 15% had 'sufficient' (>50nmol) vitamin D levels. This increased to 71% in 2017. 10% of patients had levels greater than >120nmol/L, increasing risk of vitamin D toxicity. 67% of patients with insufficient vit D (25-50nmol/L) were prescribed a stat high dose vitamin D (120,000 IU) to help avoid adherence issues. Conclusions Sequential audits along with a shift to nurse led management were the most likely reasons for sustained improvement. Similar projects in all medical departments could improve clinical outcomes.

Eleven new microsatellite loci for the tiger rattlesnake (Crotalus tigris).

Eleven microsatellite loci were isolated from an enriched genomic library from the tiger rattlesnake Crotalus tigris. Average observed heterozygosities in two populations were 0.456 and 0.427, respectively, and mean number of alleles were 7.54 (range 2-14) and 4.72 (range 2-13) respectively. No evidence of linkage disequilibrium was found across pairs of loci. The markers will be used in a long-term study examining the potential effects of urbanization on population dynamics and connectivity of this species in the mountain ranges surrounding Tucson, Arizona.

DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises.

DNA Surveillance is a Web-based application that assists in the identification of the species and population of unknown specimens by aligning user-submitted DNA sequences with a validated and curated data set of reference sequences. Phylogenetic analyses are performed and results are returned in tree and table format summarizing the evolutionary distances between the query and reference sequences. DNA Surveillance is implemented with mitochondrial DNA (mtDNA) control region sequences representing the majority of recognized cetacean species. Extensions of the system to include other gene loci and taxa are planned. The service, including instructions and sample data, is available at http://www.dna-surveillance.auckland.ac.nz.

Adherence issues in children and adolescents receiving highly active antiretroviral therapy.

The introduction of highly active antiretroviral therapies (HAART) for the treatment of paediatric HIV infection poses additional adherence challenges for children and families living with HIV A preliminary survey of 18 parents of children receiving HAART in Australia showed that although parents report high level of child adherence to HAART, specific features of the medication regimen, such as taste and number of medications made administration of HAART extremely difficult.Moreover, interaction between the treatment regimen and the day-to-day lives of families increases the adherence challenge. While some agreement exists in relation to the concerns families have about negative aspects of HAART; the diversity of issues suggests the need for ongoing and individualized support and information to families.

Some chemical properties of monochlorogallane: decomposition to gallium(I) trichlorogallate(III), Ga(+)[GaCl3H](-), and other reactions.

Thermal decomposition of monochlorogallane, [H2GaCl]n, at ambient temperatures releases H2 and results in the formation of gallium(I) species, including the new compound Ga[GaHCl3], which has been characterized crystallographically at 100 K (monoclinic P2(1)/n, a = 5.730(1), b = 6.787(1), c = 14.508(1) A, beta = 97.902(5) degrees ) and by its Raman spectrum. The gallane suffers symmetrical cleavage of the Ga(mu-Cl)2Ga bridge in its reaction with NMe3 but unsymmetrical cleavage, giving [H2Ga(NH3)2](+)Cl(-), in its reaction with NH3. Ethene inserts into the Ga-H bonds to form first [Et(H)GaCl]2 and then [Et2GaCl]2.

Improvement in aerosol delivery with helium-oxygen mixtures during mechanical ventilation.

In mechanically ventilated patients with airway obstruction, helium-oxygen (He-O2) mixtures reduce airway resistance and improve ventilation, but their influence on aerosol delivery is unknown. Accordingly, we determined the effect of various He-O2 mixtures on albuterol delivery from metered-dose inhalers (MDIs) and jet nebulizers in an in vitro model of mechanical ventilation. Albuterol delivery from a MDI was increased when the ventilator circuit contained 80% helium and 20% oxygen (He-O2 80/20) versus O2: 46.7 +/- 3.3 versus 30.2 +/- 1.3 (SE)% of the nominal dose (p < 0.001)-the difference was mainly due to decreased drug deposition in the spacer chamber, mean 39.2% and 55.2%, respectively (p < 0.001). Nebulizer efficiency at a flow rate of 6 L/min was five times lower with He-O2 80/20 than O2, and the amount of nebulized drug was inversely correlated with gas density (r = 0.94, p < 0.0001). When the nebulizer was operated with O2, greater albuterol delivery was achieved when the ventilator circuit contained He-O2 rather than O2. In summary, He-O2 mixtures in the circuit increased aerosol delivery for both MDIs and nebulizers in the mechanically ventilated model by as much as 50%. In conclusion, at appropriate flow rates and concentrations, He-O2 in the ventilator circuit may improve aerosol delivery in mechanically ventilated patients with severe airway obstruction.

The prevalence of osteopenia in pediatric renal allograft recipients varies with the method of analysis.

BACKGROUND: Pediatric renal allograft recipients often suffer from osteopenia and the potential for increased fractures. Although modern densitometers are widely available, their use in children is complicated by lack of optimal interpretive criteria. METHODS: We reviewed dual energy X-ray absorptiometry (DEXA) studies in 33 patients with functional renal allografts 4.4 +/- 3.6 years after transplantation. We interpreted our data using three previously described methods of assigning bone mineral density (BMD) Z scores. RESULTS: BMD was directly related to age, height, weight, body surface area, and pubertal status (p < 0.001). Using gender-mixed reference data matched by chronological age, the mean BMD Z score was -0.9 +/- 1.3 vs. 0.4 +/- 1.4 when matched by height-age (p < 0.001). Height-age adjustment particularly increased the BMD Z score of pubertal adolescents. In a subset of 22 patients, gender-matched reference data led to different results from the gender-mixed reference population (mean BMD Z score 0.0 +/- 1.7 vs. -0.8 +/- 1.4, p < 0.001). CONCLUSIONS: The perceived prevalence of osteopenia among pediatric kidney transplant recipients differs using analysis based on chronological age, height-age, or gender-matched reference data. Further studies are necessary to determine the clinical significance of measured bone density in this population.

Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR.

The <> assay (PE Applied Biosystems) combines PCR with concomitant release of fluorogenic nucleotides for immediate product detection by fluorometry. Yersinia pestis, the etiological agent of bubonic plague, expresses species-specific genes known to be located on two unique plasmids (9.6-kb pPCP and 100.9-kb pMT). Pesticin (pst) is a unique gene located on pPCP which encodes for a bacteriocin. Using fluorogenic probe coupled PCR as few as three copies of pst targets were detected from total Y. pestis genomic DNA. The pst probe used in this report was positive only for pesticinogenic isolates and did not show complementarity with Yersiniae nor with other bacteria targeted in this study suggesting, that the pst probe is very specific for Y. pestis. Under optimal conditions of Mg(2+)concentration and thermal cycle number, addition of extraneous DNA to respective assay mixtures had no effect on detection.

Preferential pulmonary retention of (S)-albuterol after inhalation of racemic albuterol.

The (R)-enantiomer of racemic albuterol produces bronchodilation, whereas the (S)-enantiomer may increase airway reactivity. After oral or intravenous administration of racemic albuterol, the (R)- enantiomer is metabolized several times faster than the (S)-enantiomer; however, enantiomer disposition after inhaling racemic albuterol with a metered-dose inhaler (MDI) is not known. Accordingly, 10 healthy subjects inhaled racemic albuterol with a MDI alone and with a MDI and holding chamber. We measured plasma levels of unchanged (R)- and (S)-albuterol before and up to 4 h after inhalation of racemic albuterol, and determined the unchanged R/S ratio in urine before and at 0.5, 4, 8, and 24 h later. The disposition of albuterol's enantiomers with a MDI and holding chamber was similar to that with a MDI alone. The area under the curve (AUC) of the plasma levels over time was significantly lower for the (S)- than for the (R)-enantiomer-395.5 +/- 141.0 (SE) versus 882.7 +/- 126.4 ng. ml(-)(1). min (p < 0.05)-indicating preferential retention of (S)-albuterol in the lung. The R/S ratio in urine at 0. 5 h after albuterol was > 1, reflecting the higher plasma level of the (R)-enantiomer. In conclusion, preferential retention of the (S)- compared with the (R)-enantiomer in the lung could lead to accumulation of the (S)-enantiomer after long-term use of racemic albuterol.

Detection of Yersinia pestis using branched DNA.

In contrast to target amplification methods, e.g. polymerase chain reaction, the branched DNA (bDNA) signal amplification method quantitates target nucleic acid at physiological levels, involving a series of hybridization reactions without thermal cycling. In this report, we describe a modification of the bDNA assay in which a <> preamplifier oligonucleotide (206 mer) is used in concert with ELISA and light addressable potentiometric sensor (LAPS) formats to detect the plasminogen activator (pla) gene of Yersinia pestis, the etiological agent of plague. Pla is encoded by a 9.6-kb plasmid pPCP, which is essential for virulence. The detection limit of the bDNA-ELISA and LAPS assays is less than 10 000 and 1000 molecules of Y. pestis plasmid DNA, respectively.

A comparative study of PCR product detection and quantitation by electro-chemiluminescence and fluorescence.

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)3(2+))-end-labelled primers. In this way, biotin for capture and Ru(bpy)3(2+) for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)3(2+)-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.

Sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor.

Extremely sensitive detection of various biotoxoids and bacterial spores using the commercial ORIGEN analyzer was achieved by capture on antibody-conjugated micron sized magnetic beads (MBs) followed by binding of ruthenium (II) trisbipyridal chelate (Ru(bpy)2+3-labelled reporter antibodies. Immunomagnetically captured target materials were collected on a magnet. Electrochemiluminescence (ECL) was evoked from the Ru(bpy)3(2+)-tagged reporter antibodies by application of an electrical potential. Femtogram sensitivity levels were obtained for all biotoxoids tested including botulinus A, cholera beta subunit, ricin and staphylococcal enterotoxoid B by this immunomagnetic (IM)-ECL approach. An IM-ECL assay for Bacillus anthracis spores yielded a detection limit of at least 100 spores. The ECL signal was a function of analyte quantity over several orders of magnitude, but the immunological 'hook' effect at high antigen loads made quantitation impossible over a broader range. All assays were performed with a maximum combined incubation and assay time of approximately 40 min. This work demonstrates the extreme sensitivity of the IM-ECL approach for soluble and particulate antigens.

Infectious vaginitis. Selecting therapy and preventing recurrence.

Vaginal candidiasis, trichomoniasis, and bacterial vaginosis can be difficult to manage because of predisposing factors and frequent recurrence. Thus, in addition to drug therapy, management should include attempts to minimize factors that contribute to recurrence. Both topical and oral agents are available for vaginal candidiasis and bacterial vaginosis, and both routes of administration appear equally safe and effective. Topical therapy may be used for these conditions in pregnant women. Only oral therapy is optimal for trichomoniasis. However, during pregnancy, the infection may be managed with topical clotrimazole. Currently, treatment of a woman's sexual partner is recommended in the management of trichomoniasis and is optional in cases of bacterial vaginosis. Sexual activity may also contribute to the recurrence of vaginal candidiasis; however, more studies are needed to evaluate the impact on recurrence rates of treating the male partner.

Therapeutic drug monitoring in ambulatory care.

There is a growing emphasis on developing ambulatory care services in an attempt to minimize healthcare costs through preventive medicine and outpatient therapeutic management. This creates an environment that can greatly benefit from a pharmacist-managed ambulatory TDM service. The implementation of a pharmacist-managed TDM service can improve patient care, contain the cost of healthcare, and enhance the education of pharmacy/medical students and physicians. The opportunity exists for further development of clinical pharmacy services to positively influence patient care in the ambulatory care setting. This development, however, may be hindered by inadequate reimbursement for services and lack of documentation to support the beneficial impact of clinical pharmacy services on patient outcome. Methods of documenting clinical pharmacists' interventions and outcome must be developed in order to obtain reimbursement for these services.

Chemical and behavioral ecology of foraging in prairie rattlesnakes (Crotalus viridis viridis).

Free-ranging prairie rattlesnakes (Crotalus viridis viridis) exhibit lengthy vernal migrations upon emergence from winter hibernation. A series of laboratory experiments was designed to test hypotheses regarding the function and causation of vernal movements. Rattlesnakes obtained from Wyoming and Colorado populations were used. First, we hypothesized that the function of vernal movements is to locate small mammal prey. Second, we predicted that activeC. v. viridis use prey chemicals, as well as other cues, to decide whether or not rodents are present in an area. Third, we hypothesized that vernally active males would be more responsive to rodent prey and their odors than females, given observed differences in behavior in the field. Fourth, we predicted that rattlesnakes captured in Colorado would be more sensitive to prey odors than those obtained in Wyoming, because of disparate community structure and, hence, small mammal spatial distributions. As expected, snakes exhibited reduced activity, as well as certain other dependent measures reflecting predatory investigation, in arena zones containing either live rodents or their chemicals. However, responses to the latter were reduced in Wyoming rattlesnakes tested with chemicals from deer mice (Peromyscus maniculatus), relative to Colorado animals tested with chemicals obtained from house mice (Mus musculus). In contrast to patterns observed in nature, males and females exhibited almost no differences in overall responsiveness. Results are discussed in the context of simulation modeling and ongoing studies of prairie rattlesnake behavior.

Evolution of nuclear gene families in primates. Copy-number variation in the argininosuccinate synthetase (ASS) pseudogene family and the anonymous DNA sequence, D1S1.

Changes in the copy number of nuclear genes provide the raw material for the creation of new gene functions. To better understand the mechanisms for such events, and their physiologic and evolutionary consequences, it is valuable to study a well characterized and closely related group of species such as primates. Fortuitously, most of the powerful molecular techniques and DNA probes developed for research in humans are equally applicable to non-human primates. We review what is known of copy number variation in primates and describe two informative DNA probes: pAS-1, a cDNA probe to the human urea cycle enzyme argininosuccinate synthetase (ASS), and an anonymous DNA probe, D1S1. In addition to the ASS structural locus on human chromosome 9, pAS-1 detects at least 14 dispersed, processed pseudogenes in humans. The number of pseudogene copies appears to be approximately the same in humans, chimpanzees, gorillas, orangutans and baboons; less in marmosets; and least in some rodents. Chimpanzees and gorillas appear to have all of the human pseudogenes though an Xp copy may be missing from gorillas. The Y pseudogene is apparently absent from orangutans and baboons, and, finally, a comparison of humans and chimpanzees revealed that the number of nucleotide substitutions in the Y chromosome pseudogenes is approximately 1 per 100. D1S1 maps to human chromosome 3 but also detects a high homology copy on chromosome 1. Chimpanzees, gorillas and orangutans all appear to have only the chromosome 3 homolog suggesting that this is the ancestral sequence and that the duplication occurred after separation of humans and the great apes. Both the ASS pseudogene family and the D1S1 system provide valuable information on the evolution of nuclear gene families in primates.

The anonymous polymorphic DNA clone D1S1, previously mapped to human chromosome 1p36 by in situ hybridization, is from chromosome 3 and is duplicated on chromosome 1.

D1S1, a human anonymous DNA clone originally called lambda Ch4A-H3 or lambda H3, was mapped by two other laboratories to human chromosome 1p36 by in situ hybridization but its localization was not confirmed using a different mapping method. We used a panel of human-hamster somatic cell hybrids to show that there are copies of D1S1 on both chromosomes 1 and 3. The D1S1 clone itself is from chromosome 3, and part of it is duplicated at least twice on chromosome 1. A high frequency HindIII polymorphism detected by D1S1, believed to be at chromosome 1p36 on the basis of the in situ hybridization data, maps instead to chromosome 3. This finding demonstrates the importance of using two mapping methods to verify the localization of a gene or DNA segment, particularly a polymorphic one which itself may be used in mapping studies. It also raises the question of why in situ hybridization detected a duplicated portion of a clone but not the chromosomal origin of the clone itself.

Laser microbeam studies of role of amphid receptors in chemosensory behavior of nematodeCaenorhabditis elegans.

Amphid sensilla, historically considered the primary chemosensory structures of nematodes, were found to be necessary for the detection of only one of the six chemical stimuli that were tested. Only the attraction to cAMP was eliminated by damaging the two lateral lips, which bear the amphid sensilla. The inner labial sensilla, one of which occurs on each of the six lips, are probably the primary receptor structures for the other chemical stimuli. Damaging all six lips, which should destroy all anterior chemosensory input, not only eliminted the attraction to sodium and chloride ions, but reversed the nematodes' response to them. Nematodes with all six lips destroyed showed reversal behavior when exposed to these attractants. Nematodes with damage to all six lips appeared to recover much of their normal chemosensory function within 24 hr after treatment.