An Evaluation of the OLM PneumID Real-Time Polymerase Chain Reaction to Aid in the Diagnosis of Pneumocystis Pneumonia.

BACKGROUND: The use of the PCR to aid in the diagnosis of Pneumocystis pneumonia (PcP) has demonstrated excellent clinical performance, as evidenced through various systematic reviews and meta-analyses, yet there are concerns over the interpretation of positive results due to the potential presence of Pneumocystis colonization of the airways. While this can be overcome by applying designated positivity thresholds to PCR testing, the shear number of assays described limits the development of a universal threshold. Commercial assays provide the opportunity to overcome this problem, provided satisfactory performance is determined through large-scale, multi-centre evaluations. METHODS: Retrospective case/control and consecutive cohort performance evaluations of the OLM PneumID real-time PCR assay were performed on DNA eluates from a range of samples sent from patients where "in-house" PCR had been performed as part of routine diagnostic testing. The clinical performance of the PneumID assay was determined before including it in a diagnostic algorithm to provide the probability of PcP (dependent on diagnostic evidence). RESULTS: After being used to test 317 patients (32 with PcP), the overall performance of the PneumID assay was found to be excellent (Sensitivity/Specificity: 96.9%/95.1%). False positivity could be removed by applying a threshold specific to sample type (<33.1 cycles for BAL fluid; <37.0 cycles for throat swabs), whereas considering any positive respiratory samples as significant generated 100% sensitivity, making absolute negativity sufficient to exclude PcP. Incorporating the PneumID assay into diagnostic algorithms alongside (1-3)-ß-D-Glucan testing provided high probabilities of PcP (up to 85.2%) when both were positive and very low probabilities (<1%) when both were negative. CONCLUSIONS: The OLM PneumID qPCR provides a commercial option for the accurate diagnosis of PcP, generating excellent sensitivity and specificity, particularly when testing respiratory specimens. The combination of PcP PCR with serum (1-3)-ß-D-Glucan provides excellent clinical utility for diagnosing PcP.

Pneumocystis jirovecii pneumonia in intensive care units: a multicenter study by ESGCIP and EFISG.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) is an opportunistic, life-threatening disease commonly affecting immunocompromised patients. The distribution of predisposing diseases or conditions in critically ill patients admitted to intensive care unit (ICU) and subjected to diagnostic work-up for PJP has seldom been explored. MATERIALS AND METHODS: The primary objective of the study was to describe the characteristics of ICU patients subjected to diagnostic workup for PJP. The secondary objectives were: (i) to assess demographic and clinical variables associated with PJP; (ii) to assess the performance of Pneumocystis PCR on respiratory specimens and serum BDG for the diagnosis of PJP; (iii) to describe 30-day and 90-day mortality in the study population. RESULTS: Overall, 600 patients were included in the study, of whom 115 had presumptive/proven PJP (19.2%). Only 8.8% of ICU patients subjected to diagnostic workup for PJP had HIV infection, whereas hematological malignancy, solid tumor, inflammatory diseases, and solid organ transplants were present in 23.2%, 16.2%, 15.5%, and 10.0% of tested patients, respectively. In multivariable analysis, AIDS (odds ratio [OR] 3.31; 95% confidence interval [CI] 1.13-9.64, p = 0.029), non-Hodgkin lymphoma (OR 3.71; 95% CI 1.23-11.18, p = 0.020), vasculitis (OR 5.95; 95% CI 1.07-33.22, p = 0.042), metastatic solid tumor (OR 4.31; 95% CI 1.76-10.53, p = 0.001), and bilateral ground glass on CT scan (OR 2.19; 95% CI 1.01-4.78, p = 0.048) were associated with PJP, whereas an inverse association was observed for increasing lymphocyte cell count (OR 0.64; 95% CI 0.42-1.00, p = 0.049). For the diagnosis of PJP, higher positive predictive value (PPV) was observed when both respiratory Pneumocystis PCR and serum BDG were positive compared to individual assay positivity (72% for the combination vs. 63% for PCR and 39% for BDG). Cumulative 30-day mortality and 90-day mortality in patients with presumptive/proven PJP were 52% and 67%, respectively. CONCLUSION: PJP in critically ill patients admitted to ICU is nowadays most encountered in non-HIV patients. Serum BDG when used in combination with respiratory Pneumocystis PCR could help improve the certainty of PJP diagnosis.

An Evaluation of the OLM CandID Real-Time PCR to Aid in the Diagnosis of Invasive Candidiasis When Testing Serum Samples.

Background: Treatment for invasive candidiasis (IC) is time-critical, and culture-based tests can limit clinical utility. Nonculture-based methods such as Candida PCR represent a promising approach to improving patient management but require further evaluation to understand their optimal role and incorporation into clinical algorithms. This study determined the performance of the commercially available OLM CandID real-time PCR when testing serum and developed a diagnostic algorithm for IC. Methods: The study comprised a retrospective performance evaluation of the CandID real-time PCR assay when testing surplus serum (n = 83 patients, 38 with IC), followed by a prospective consecutive cohort evaluation (n = 103 patients, 24 with IC) post incorporation into routine service. A combined diagnostic algorithm, also including (1-3)-ß-D-Glucan testing, was generated. Results: Prospective CandID testing generated a sensitivity/specificity of 88%/82%, respectively. Specificity was improved (>95%) when both PCR replicates were positive and/or the patient had multiple positive samples. When combining CandID with (1-3)-ß-D-Glucan testing, the probability of IC when both were positive or negative was >69% or <1%, respectively. Conclusions: The CandID provides excellent performance and a rapid time-to-result using methods widely available in generic molecular diagnostic laboratories. By combining nonculture diagnostics, it may be possible to accurately confirm or exclude IC.

A Composite System Based upon Hydroxypropyl Cyclodextrins and Soft Hydrogel Contact Lenses for the Delivery of Therapeutic Doses of Econazole to the Cornea, In Vitro.

Fungal keratitis, a disease in which the cornea becomes inflamed due to an invasive fungal infection, remains difficult to treat due in part to limited choices of available treatments. Topical eye drops are first-line treatment, but can be ineffective as low levels of drug reach the target site due to precorneal losses and the impenetrability of the cornea. The aim of this study was to determine the corneal delivery of econazole using a novel topical enhancement approach using a composite delivery system based upon cyclodextrins and soft hydrogel contact lenses. Excess econazole nitrate was added to hydroxypropyl-α-cyclodextrin (HP-α-CD) and hydroxypropyl-ß-cyclodextrin (HP-ß-CD) solutions, and the solubility determined using HPLC. Proprietary soft hydrogel contact lenses were then impregnated with saturated solutions and applied to freshly enucleated porcine eyeballs. Econazole nitrate 'eye drops' at the same concentrations served as the control. After 6 h, the corneas were excised and drug-extracted, prior to quantification using HPLC. Molecular dynamic simulations were performed to examine econazole−HP-ß-CD inclusion complexation and dissociation. The minimum inhibitory concentration (MIC) of econazole was determined against four fungal species associated with keratitis, and these data were then related to the amount of drug delivered to the cornea, using an average corneal volume of 0.19 mL. The solubility of econazole increased greatly in the presence of HP-ß-CD and more so with HP-α-CD (p < 0.001), with ratios >> 2. Hydrogel contact lenses delivered ×2.8 more drug across the corneas in comparison to eye drops alone, and ×5 more drug delivered to the cornea when cyclodextrin was present. Molecular graphics demonstrated dynamic econazole release, which would create transient enhanced drug concentration at the cornea surface. The solution-only drops achieved the least satisfactory result, producing sub-MIC levels with factors of ×0.81 for both Fusarium semitectum and Fusarium solani and ×0.40 for both Scolecobasidium tshawytschae and Bipolaris hawaiiensis. All other treatments delivered econazole at > MIC for all four fungal species. The efficacies of the delivery platforms evaluated were ranked: HP-α-CD contact lens > HP-ß-CD contact lens > contact lens = HP-α-CD drops > HP-ß-CD drops > solution-only drops. In summary, the results in this study have demonstrated that a composite drug delivery system based upon econazole−HP-ß-CD inclusion complexes loaded into contact lenses can achieve significantly greater corneal drug delivery with the potential for improved clinical responses.

In Vitro Topical Delivery of Chlorhexidine to the Cornea: Enhancement Using Drug-Loaded Contact Lenses and ß-Cyclodextrin Complexation, and the Importance of Simulating Tear Irrigation.

Microbial keratitis is a severe, sight-threatening condition caused by various pathogens. Eyedrops are the standard delivery modality for treating these disorders; however, blinking reflex, elevated tear production, and nasolacrimal drainage eliminate much of the instilled dose within a few seconds. Therefore, eyedrops must be applied repeatedly for prolonged periods. The present study aimed to probe more effective ocular delivery of chlorhexidine based upon drug-loaded hydrogel contact lenses and ß-cyclodextrin (ß-CD), while also determining the effect of constant irrigation with simulated tear fluid (STF) in in vitro experiments. Chlorhexidine digluconate (as 0.2 and 2% solutions, ß-CD inclusion complexes, and loaded hydrogel contact lenses) were applied to enucleated porcine eyes as single or multiple 10 µL doses, or as drug-loaded contact lenses, with and without ß-CD. The corneas were then excised and drug-extracted quantified by high-performance liquid chromatography (HPLC). The effect of constant irrigation by STF was evaluated to test the effect of increased tear production on corneal delivery. Potential antimicrobial activity of the delivered drug was also assessed. Results showed that drug-loaded contact lenses delivered the greatest amount of chlorhexidine into the cornea over a 24 h period, while the eyedrop solution comparator delivered the least. The ß-CD significantly enhanced chlorhexidine delivery to the cornea from eyedrop solution, although contact lenses loaded with chlorhexidine-ß-CD failed to enhance delivery. ß-CD within the hydrogel matrix impeded drug release. Constant irrigation with STF significantly reduced the amount of drug delivered to the cornea in all cases. Chlorhexidine retained antimicrobial activity in all delivery methods. Hydrogel contact lenses loaded with chlorhexidine delivered significantly higher levels to the cornea compared to eyedrops, either multiple hourly doses or a single dose. They also offer reduced application, in particular, to a nonulcerated corneal infection. Finally, the importance of fully accounting for tear production in in vitro ocular delivery experiments was highlighted.

Length of time domestic dogs (Canis familiaris) spend smelling urine of gonadectomised and intact conspecifics.

Domestic dogs (Canis familiaris) use urine to communicate among themselves, however, it is unknown whether the gonadectomy (neutering or spaying) of a dog affects this communication in anyway. Urine samples from 10 intact and 10 gonadectomised, unfamiliar dogs were presented to 12 tester dogs to sniff under controlled conditions in a pilot study. The amount of time the tester dogs spent sniffing each sample was recorded. Overall, tester dogs were recorded smelling the urine of gonadectomised individuals for a longer time. In addition to the type of urine sample, the result is likely to have been influenced by the sex and status (gonadectomised or intact) of the tester dogs. The observed increase in the length of time spent sniffing urine from gonadectomised individuals could be explained by the tester dogs experiencing more difficulty in gaining information from the urine or facing more confusion while analysing the urine compared to the intact urine they have evolved to smell.