Determining Pneumocystis jirovecii Colonisation from Infection Using PCR-Based Diagnostics in HIV-Negative Individuals.

BACKGROUND: Pneumocystis jirovecii pneumonia is increasingly diagnosed with highly sensitive PCR diagnostics in immunocompromised, HIV-negative individuals. We assessed the performance of our in-house quantitative PCR with the aim to optimise interpretation. METHODS: Retrospective audit of all positive P. jirovecii qPCRs on induced sputum or BAL fluid at a single centre from 2012 to 2023. Medical and laboratory records were analysed and people with HIV were excluded. Cases were categorised as colonisation, high-probability PCP or uncertain PCP infection against a clinical gold standard incorporating clinico-radiological data. Quantitative PCR assay targeting the 5s gene was utilised throughout the time period. RESULTS: Of the 82 positive qPCRs, 28 were categorised as high-probability PCP infection, 30 as uncertain PCP and 24 as colonisation. There was a significant difference in qPCR values stratified by clinical category but not respiratory sample type. Current assay performance with a cutoff of 2.5 × 105 copies/mL had a sensitivity of 50% (95% CI, 30.65-69.35%) and specificity of 83.33% (95% CI, 62.62-95.26%). Youden Index calculated at 6.5 × 104 copies/mL had a sensitivity of 75% (56.64-87.32%, 95% CI) and specificity of 66.67% (46.71-82.03%, 95% CI). High and low cutoffs were explored. Significant variables associated with infection were age > 70 years old, the presence of fever, hypoxia or ground glass changes. CONCLUSIONS: A single qPCR cutoff cannot reliably determine P. jirovecii infection from colonisation. Low and high cutoffs are useful, however, a large "possible infection" cohort will remain where interpretation of clinic-radiological factors remains essential. Standardisation of assays with prospective validation in specific immunocompromised groups will allow greater generalisability and allow large-scale prospective assay validation to be performed.

False-negative Chlamydia polymerase chain reaction result caused by a cryptic plasmid-deficient Chlamydia trachomatis strain in Australia.

Background The 7.5-kb chlamydial cryptic plasmid remains a widely used sequence target for Chlamydia trachomatis nucleic acid amplification tests, but sequence variation in this plasmid, particularly a previously reported 377-bp deletion, can cause false-negative results. Here we report the presence in Australia of a C. trachomatis strain lacking the cryptic plasmid. METHODS: A rectal swab from a male in his 50s provided a positive result for C. trachomatis using the Roche Cobas 4800 test, but a negative result in our confirmatory in-house polymerase chain reaction (PCR) method targeting the chlamydial cryptic plasmid. This result was unexpected given our in-house PCR assay targeted a region of sequence outside the recognised 377-bp deletion. To further investigate this discrepancy, the sample was retested using a second in-house PCR targeting a chromosomal (ompA) gene as well as six primer sets flanking various regions of the cryptic plasmid. RESULTS: The sample provided positive results in the second in-house method, confirming the presence of C. trachomatis DNA. All other primer sets targeting the cryptic plasmid failed to amplify, indicating a lack of the chlamydial cryptic plasmid in this sample. CONCLUSIONS: The recognition of a plasmid-deficient strain of C. trachomatis within Australia highlights further limitations of using the chlamydial cryptic plasmid for C. trachomatis diagnostics and re-emphasises the benefits of using multitarget assays to avoid false-negative results.

60 YEARS OF NEUROENDOCRINOLOGY: Glucocorticoid dynamics: insights from mathematical, experimental and clinical studies.

A pulsatile pattern of secretion is a characteristic of many hormonal systems, including the glucocorticoid-producing hypothalamic-pituitary-adrenal (HPA) axis. Despite recent evidence supporting its importance for behavioral, neuroendocrine and transcriptional effects of glucocorticoids, there has been a paucity of information regarding the origin of glucocorticoid pulsatility. In this review we discuss the mechanisms regulating pulsatile dynamics of the HPA axis, and how these dynamics become disrupted in disease. Our recent mathematical, experimental and clinical studies show that glucocorticoid pulsatility can be generated and maintained by dynamic processes at the level of the pituitary-adrenal axis, and that an intra-adrenal negative feedback may contribute to these dynamics.

Dairy milk fat augments paclitaxel therapy to suppress tumour metastasis in mice, and protects against the side-effects of chemotherapy.

Milk fat is a natural product containing essential nutrients as well as fatty acids and other food factors with reported anti-cancer potential. Here bovine milk fat was tested for its ability to inhibit the growth of breast and colon cancers and their metastasis to the lung and liver; either alone or in combination with the chemotherapeutic agent paclitaxel. A diet containing 5% typical anhydrous milk fat (representing ~70% of the total dietary fat component) fed to Balb/c mice delayed the appearance of subcutaneous 4T1 breast and CT26 colon cancer tumours and inhibited their metastasis to the lung and liver, when compared to the control diet containing soybean oil as the only fat component. It augmented the inhibitory effects of paclitaxel on tumour growth and metastasis, and reduced the microvessel density of tumours. It displayed no apparent organ toxicity, but instead was beneficial for well-being of tumour-bearing mice by maintaining gastrocnemius muscle and epididymal adipose tissue that were otherwise depleted by cachexia. The milk fat diet ameliorated gut damage caused by paclitaxel in non-tumour-bearing mice, as evidenced by retention of jejunal morphology, villi length and intestinal γ-glutamyl transpeptidase activity, and inhibition of crypt apoptosis. It prevented loss of red and white blood cells due to both cancer-mediated immunosuppression and the cytotoxic effects of chemotherapy. The present study warrants the use of milk fat as an adjuvant to inhibit tumour metastasis during cancer chemotherapy, and to spare patients from the debilitating side-effects of cytotoxic drugs.

A computational framework for gene regulatory network inference that combines multiple methods and datasets.

BACKGROUND: Reverse engineering in systems biology entails inference of gene regulatory networks from observational data. This data typically include gene expression measurements of wild type and mutant cells in response to a given stimulus. It has been shown that when more than one type of experiment is used in the network inference process the accuracy is higher. Therefore the development of generally applicable and effective methodologies that embed multiple sources of information in a single computational framework is a worthwhile objective. RESULTS: This paper presents a new method for network inference, which uses multi-objective optimisation (MOO) to integrate multiple inference methods and experiments. We illustrate the potential of the methodology by combining ODE and correlation-based network inference procedures as well as time course and gene inactivation experiments. Here we show that our methodology is effective for a wide spectrum of data sets and method integration strategies. CONCLUSIONS: The approach we present in this paper is flexible and can be used in any scenario that benefits from integration of multiple sources of information and modelling procedures in the inference process. Moreover, the application of this method to two case studies representative of bacteria and vertebrate systems has shown potential in identifying key regulators of important biological processes.

Adenosine amine congener mitigates noise-induced cochlear injury.

Hearing loss from noise exposure is a leading occupational disease, with up to 5% of the population at risk world-wide. Here, we present a novel purine-based pharmacological intervention that can ameliorate noise-induced cochlear injury. Wistar rats were exposed to narrow-band noise (8-12 kHz, 110 dB SPL, 2-24 h) to induce cochlear damage and permanent hearing loss. The selective adenosine A(1) receptor agonist, adenosine amine congener (ADAC), was administered intraperitoneally (100 microg/kg/day) at time intervals after noise exposure. Hearing thresholds were assessed using auditory brainstem responses and the hair cell loss was evaluated by quantitative histology. Free radical damage in the organ of Corti was assessed using nitrotyrosine immunohistochemistry. The treatment with ADAC after noise exposure led to a significantly greater recovery of hearing thresholds compared with controls. These results were upheld by increased survival of sensory hair cells and reduced nitrotyrosine immunoreactivity in ADAC-treated cochlea. We propose that ADAC could be a valuable treatment for noise-induced cochlear injury in instances of both acute and extended noise exposures.

Post exposure administration of A(1) adenosine receptor agonists attenuates noise-induced hearing loss.

Adenosine is a constitutive cell metabolite with a putative role in protection and regeneration in many tissues. This study was undertaken to determine if adenosine signalling pathways are involved in protection against noise injury. A(1) adenosine receptor expression levels were altered in the cochlea exposed to loud sound, suggesting their involvement in the development of noise injury. Adenosine and selective adenosine receptor agonists (CCPA, CGS-21680 and Cl-IB-MECA) were applied to the round window membrane of the cochlea 6h after noise exposure. Auditory brainstem responses measured 48h after drug administration demonstrated partial recovery of hearing thresholds (up to 20dB) in the cochleae treated with adenosine (non-selective adenosine receptor agonist) or CCPA (selective A(1) adenosine receptor agonist). In contrast, the selective A(2A) adenosine receptor agonist CGS-21680 and A(3) adenosine receptor agonist Cl-IB-MECA did not protect the cochlea from hearing loss. Sound-evoked cochlear potentials in control rats exposed to ambient noise were minimally altered by local administration of the adenosine receptor agonists used in the noise study. Free radical generation in the cochlea exposed to noise was reduced by administration of adenosine and CCPA. This study pinpoints A(1) adenosine receptors as attractive targets for pharmacological interventions to reduce noise-induced cochlear injury after exposure.

Increased osteoporosis screening rates associated with the provision of a preventive health examination.

OBJECTIVES: We determined the frequencies of (1) female patients aged 65 years and older having bone density measurement performed and (2) prescription therapy use among osteoporotic women. METHODS: We completed a retrospective chart audit to assess our adherence to Physician Quality Reporting Initiative guidelines. Women aged 65 to 75 with an office visit between June 1 to November 30, 2007, were divided into 3 subgroups: those who had a recent preventive general medical examination (GME), those who received one in the last 10 years, and those who had not. We determined osteoporosis screening rates for all 3 groups. The first group then underwent electronic medical record review to obtain patient demographics, determine bone mineral density results, and review if those with osteoporosis were receiving prescription treatment. RESULTS: Ninety-six percent of 305 female patients seen for a GME during the study period had completed bone mineral density testing. This was a screening rate significantly greater than that for patients with an earlier GME and those who never had one in our offices (70% and 50%, respectively). Seventy-seven percent of recent GME patients had abnormal T scores. Low weight and body mass index were significantly associated with osteoporotic T scores. Seventy-four percent of patients whose latest T scores were less than -2.5 were receiving prescription therapy. CONCLUSIONS: Female patients who completed a recent GME had extraordinarily high rates of screening for osteoporosis. We believe this demonstrates the importance of a dedicated preventive health examination as well as the increased significance that physicians and patients currently place on this behavior.

Preservation of cochlear function in Cd39 deficient mice.

Signalling actions of extracellular nucleotides via P2 receptors influence cellular function in most tissues. In the inner ear, P2 receptor signaling is involved in many processes including the regulation of hearing sensitivity and the cochlea's response to noise stress. CD39 (NTPDase1/ENTPD1) is an ectonucleotidase (ecto-nucleoside triphosphate diphosphohydrolase) that can hydrolyse purine and pyrimidine nucleoside tri- and di-phosphates to generate monophosphate nucleosides. Mice null for Cd39 exhibit major alterations in haemostasis and profound alterations in inflammatory and thrombotic reactions. Studies in the cochlea have suggested the involvement of purinergic-type signals that could be modulated by CD39 in regulation of cochlear blood flow and also auditory neurotransmission. This study aimed to determine the auditory phenotype of adult Cd39 null mice on the C57BL6 background. Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were unaffected in Cd39-deficient mice across the range of test frequencies, suggesting normal neural and outer hair cell function. Mutant mice also showed little difference to wild type mice in vulnerability to acoustic trauma. Gene expression analysis of other membrane-bound NTPDases with comparable hydrolytic activity demonstrated an up-regulation of Entpd2 and Entpd8 in the cochleae of Cd39 deficient mice. These findings suggest that Cd39 deletion alone does not adversely affect cochlear function, possibly as compensatory up-regulation of other surface located NTPDases may offset predicted alterations in cochlear homeostasis.

'Iron-saturated' lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy.

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.

The impact of poverty on the current and future health status of children.

Child poverty in Canada is a significant public health concern. Because child development during the early years lays the foundation for later health and development, children must be given the best possible start in life. Family income is a key determinant of healthy child development. Children in families with greater material resources enjoy more secure living conditions and greater access to a range of opportunities that are often unavailable to children from low-income families. On average, children living in low-income families or neighbourhoods have poorer health outcomes. Furthermore, poverty affects children's health not only when they are young, but also later in their lives as adults. The health sector should provide services to mitigate the health effects of poverty, and articulate the health-related significance of child poverty, in collaboration with other sectors to advance healthy public policy.

Effect of PKCbeta on retinal oxygenation response in experimental diabetes.

PURPOSE: To test the hypotheses that, in mice, breathing carbogen (95% O(2)-5% CO(2)) oxygenates the retina better than breathing 100% oxygen, the superior hemiretinal oxygenation response to carbogen inhalation is subnormal early in diabetes, and diabetes-induced elevation of retinal protein kinase C (PKC)-beta contributes to this pathophysiology. METHODS: Retinal oxygenation response (DeltaPO(2)) was measured using functional magnetic resonance imaging (MRI) and either carbogen or 100% oxygen inhalation challenge in C57BL/6J control (C) mice. Retinal DeltaPO(2) during carbogen breathing was also measured in PKCbeta knockout (C57BL6-Prkcb1; [KO]), 4 month C57BL/6J diabetic (D), and 4-month diabetic PKCbeta KO (D+KO) mice. Retinal PKCbeta protein expression was assessed by Western analysis. RESULTS: In C mice, retinal DeltaPO(2) during carbogen breathing was significantly greater (P < 0.05) than during oxygen breathing. In D mice, DeltaPO(2) during carbogen breathing was significantly lower than normal in the superior, but not the inferior, hemiretina and was normal (P > 0.0 5) in the KO group. In the D+KO mice DeltaPO(2) was normal (P > 0.05) only at distances less than 1.5 mm from the optic nerve head. PKCbeta expression was elevated in the retina in diabetes (P < 0.05), but was significantly decreased (P < 0.05) in D+KO mice. CONCLUSIONS: The present study confirms that, in the mouse, retinal DeltaPO(2) patterns during different inhalation challenges or in the presence of diabetes are similar to what has been reported in rats. Diabetes-induced elevation of PKC appears to contribute only focally to subnormal retinal DeltaPO(2). This raises the possibility that PKC inhibition therapy may be only regionally effective in treating retinal pathophysiology associated with diabetic retinopathy.

Regulation of the early subnormal retinal oxygenation response in experimental diabetes by inducible nitric oxide synthase.

We aimed to test the hypothesis that the inducible form of nitric oxide synthase (iNOS) contributes to the development of an early subnormal retinal oxygenation response in preclinical models of diabetic retinopathy. In urethane anesthetized Sprague Dawley rats or C57BL/6 mice, functional magnetic resonance imaging was used to noninvasively measure the change in retinal oxygen tension (Delta PO(2)) during a carbogen-inhalation challenge. In the rat experiments, the retinal Delta PO(2) of the following groups were compared: control rats (n = 9), 3-month diabetic rats (n = 5), and 3-month diabetic rats treated orally with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of an inhibitor of iNOS (n = 6). In addition, the retinal Delta PO(2) of the following mouse groups were compared: C57BL/6 mice (n = 20), C57BL/6-Nos2(tm1 Lau) mice (n = 10), 4-month diabetic mice (n = 13), and 4-month diabetic knockout mice (n = 6). Only the Delta PO(2) of the superior hemiretina of the diabetic rat and mice groups were significantly subnormal (P < 0.05). The superior Delta PO(2) of the diabetic rats treated with the prodrug was not significantly (P > 0.05) different from their respective normal controls. In the mice experiments, the superior retinal Delta PO(2) of the iNOS null mice was not statistically different (P > 0.05) from that of normal control mice. iNOS is required for the development of an early subnormal Delta PO(2) in experimental diabetic retinopathy.

Patient with bilateral pleural effusion: are the findings the same in each fluid?

STUDY OBJECTIVES: To determine whether, in patients with bilateral pleural effusions, the main cellular and biochemical features of the pleural fluid on the right side differ from or correlate with those on the left side. We examined lactate dehydrogenase (LDH), glucose, and total protein (TP) levels, RBC count, nucleated cell count (NCC), and differential cell count. PATIENTS AND METHODS: Twenty-seven patients with bilateral pleural effusions, including 13 patients with effusions after coronary artery bypass graft surgery, 12 patients with congestive heart failure, 1 patient with malignant pericarditis, and 1 patient with renal failure, were studied retrospectively. RESULTS: The right-sided and the left-sided pleural effusions did not differ in the mean TP (p = 0.38), glucose (p = 0.31), and LDH (p = 0.39) levels, RBC count (p = 0.31), NCC (p = 0.96), and the percentage of neutrophils (p = 0.22), lymphocytes (p = 0.73), mononuclear cells (MNCs) [p = 0.49], and eosinophils (p = 0.65). The bias +/- precision was 0.1 +/- 0.64 g/dL for TP, - 2.7 +/- 23 mg/dL for glucose, 41 +/- 362 IU/L for LDH, 6,100 +/- 62,900 cells/ micro L for RBC count, - 36 +/- 1,043 cells/ micro L for NCC, - 2.9 +/- 11.6% for the percentage of neutrophils, 1.15 +/- 17% for the percentage of lymphocytes, 2.3 +/- 17% for the percentage of the MNCs, and - 0.15 +/- 5.4% for the percentage of eosinophils. Moreover, there was a close correlation between the right-sided and the left-sided pleural effusions concerning TP level (r = 0.85, p < 0.001), glucose level (r = 0.78, p < 0.001), LDH level (r = 0.71, p < 0.001), RBC count (r = 0.66, p < 0.001), NCC (r = 0.60, p = 0.001), and the percentage of neutrophils (r = 0.77, p < 0.001), lymphocytes (r = 0.77, p < 0.001), MNCs (r = 0.74, p < 0.001), and eosinophils (r = 0.84, p < 0.001). CONCLUSION: Since the pleural fluid findings tend to be similar in both sides of patients with bilateral pleural effusion, we suggest that diagnostic thoracentesis may not need to be performed on both sides, unless there is a specific clinical indication.