Comparison of Three ß-Glucan Tests for the Diagnosis of Invasive Candidiasis in Intensive Care Units.

The (1→3)-ß-d-glucan (BDG) is a marker of invasive fungal infection that can be detected in serum by different commercial kits. In this study, we compared the performance of the Fungitell assay (FA), the Fungitell STAT assay (STAT), and the Wako ß-glucan test (WA) for the diagnosis of invasive candidiasis (IC) in the intensive care unit (ICU). Patients for whom at least one BDG testing was required for a clinical suspicion of IC were retrospectively enrolled. A total of 85 serum samples from 56 patients were tested by the three BDG tests. The rate of IC was 23% (13/56) with a predominance of noncandidemic (intra-abdominal) IC. STAT and WA results exhibited overall good correlation with those obtained by FA (Spearman's coefficient R = 0.90 and R = 0.89, respectively). For the recommended cutoffs of positivity, sensitivity and specificity for IC diagnosis were 77%/51% (FA, 80 pg/mL), 69%/53% (STAT, ratio 1.2), and 54%/65% (WA, 7 pg/mL), respectively. Optimal performance was obtained at 50 pg/mL (FA), ratio 1.3 (STAT), and 3.3 pg/mL (WA) with sensitivity/specificity of 85%/51%, 69%/57%, and 77%/58%, respectively. Overall, the three BDG tests showed comparable but limited performance in this setting with positive and negative predictive values for an estimated IC prevalence of 20% that were in the range of 30 to 35% and 85 to 95%, respectively.

Rational Use of Microbiological Tests in the Diagnosis of Central Nervous System Infections Using Restrictive Criteria: a Retrospective Study.

Central nervous infections, mostly represented by meningitis and encephalitis, remain a diagnostic challenge despite substantial advances in microbiological tools in recent years. Meanwhile, extensive microbiological workups, which often prove to be irrelevant retrospectively, continue to be processed on a large scale, therefore leading to unnecessary costs. The main goal of this study was to evaluate a systematic approach enabling more rational use of microbiological tools in the setting of community-acquired central nervous system infection diagnosis. In this single-center descriptive study, the modified Reller criteria were retrospectively extended to all neuropathogens tested in cerebrospinal fluid (CSF) samples with the FilmArray meningitis/encephalitis panel (BioFire Diagnostics, LLC) and bacterial culture. The inclusion period was 30 months. In total, 1,714 fluid (CSF) samples analyzed from 1,665 patients over 2 and a half years were reported. According to the retrospective application of the modified Reller criteria, microbiological testing was considered unnecessary in 544 CSF samples. Fifteen positive microbiological results were found among these samples, interpreted either as inherited chromosomally integrated human herpesvirus 6 (HHV-6), a false-positive result, or a true microbial detection without clinical relevance. No CNS infection case would have been missed if these analyses were not carried out, while about one-third of all meningitis/encephalitis multiplex PCR panels would have been saved. Our retrospective analysis suggests that the modified Reller criteria could be safely applied to all microbiological tests performed in CSF, thereby saving substantial costs. IMPORTANCE Microbiological testing in general and in the setting of central nervous system (CNS) infection in particular are often excessive, leading to superfluous laboratory work and costs. In this regard, restrictive criteria, named Reller criteria, have been developed to reduce unnecessary CSF herpes simplex virus 1 (HSV-1) PCR testing when suspecting encephalitis. These criteria were then adapted for increased safety to become the modified Reller criteria. This retrospective study aims at evaluating the safety of these criteria when applied to CSF microbiological testing in general, including multiplex PCR, direct examination, and bacterial culture. The postulate was that a CNS infection can be excluded if none of these criteria is present. According to our data set, no CNS infection would have been missed if the modified Reller criteria would have been applied to save microbiological tests. This study therefore proposes a simple way to reduce unnecessary microbiological testing in the context of CNS infection suspicion.

Causes of fever in Tanzanian adults attending outpatient clinics: a prospective cohort study.

OBJECTIVES: Exploring fever aetiologies improves patient management. Most febrile adults are outpatients, but all previous studies were conducted in inpatients. This study describes the spectrum of diseases in adults attending outpatient clinics in urban Tanzania. METHODS: We recruited consecutive adults with temperature ≥38°C in a prospective cohort study. We collected medical history and performed a clinical examination. We performed 27 364 microbiological diagnostic tests (rapid tests, serologies, cultures and molecular analyses) for a large range of pathogens on blood and nasopharyngeal samples. We based our diagnosis on predefined clinical and microbiological criteria. RESULTS: Of 519 individuals, 469 (89%) had a clinically or microbiologically documented infection and 128 (25%) were human immunodeficiency virus (HIV) -infected. We identified 643 diagnoses: 264 (41%) acute respiratory infections (36 (5.6%) pneumonia, 39 (6.1%) tuberculosis), 71 (11%) infections with another focus (31 (4.8%) gastrointestinal, 26 (4.0%) urogenital, 8 (1.2%) central nervous system) and 252 (39%) infections without focus (134 (21%) dengue, 30 (4.7%) malaria, 28 (4.4%) typhoid). Of the 519 individuals, 318 (61%), 179 (34%), 30 (6%) and 15 (3%), respectively, had a viral, bacterial, parasitic and fungal acute infection. HIV-infected individuals had more bacterial infections than HIV-negative (80/122 (66%) versus 100/391 (26%); p < 0.001). Patients with advanced HIV disease had a higher proportion of bacterial infections (55/76 (72%) if CD4 ≤200 cells/mm3 and 25/52 (48%) if CD4 >200 cells/mm3, p 0.02). CONCLUSIONS: Viral diseases caused most febrile episodes in adults attending outpatient clinics except in HIV-infected patients. HIV status and a low CD4 level strongly determined the need for antibiotics. Systematic HIV screening is essential to appropriately manage febrile patients.

Accuracy of comprehensive PCR analysis of nasopharyngeal and oropharyngeal swabs for CT-scan-confirmed pneumonia in elderly patients: a prospective cohort study.

OBJECTIVES: We aimed to assess the accuracy of PCR detection of viruses and bacteria on nasopharyngeal and oropharyngeal swabs (NPS) for the diagnosis of pneumonia in elderly individuals. METHODS: We included consecutive hospitalized elderly individuals suspected of having pneumonia. At inclusion, NPS were collected from all participants and tested by PCR for the presence of viral and bacterial respiratory pathogens (index test, defined as comprehensive molecular testing). Routine diagnostic tests (blood and sputum culture, urine antigen detection) were also performed. The reference standard was the presence of pneumonia on a low-dose CT scan as assessed by two independent expert radiologists. RESULTS: The diagnosis of pneumonia was confirmed in 127 of 199 (64%) included patients (mean age 83 years, community-acquired pneumonia in 105 (83%)). A pathogen was identified by comprehensive molecular testing in 114 patients (57%) and by routine methods in 22 (11%). Comprehensive molecular testing was positive for viruses in 62 patients (31%) and for bacteria in 73 (37%). The sensitivity and specificity were 61% (95% CI 53%-69%) and 50% (95% CI 39%-61%) for comprehensive molecular testing, and 14% (95% CI 82%-21%) and 94% (95% CI 86%-98%) for routine testing, respectively. Positive likelihood ratio was 2.55 for routine methods and 1.23 for comprehensive molecular testing. CONCLUSION: Comprehensive molecular testing of NPS increases the number of pathogens detected compared with routine methods, but results are poorly predictive of the presence of pneumonia. Hence, comprehensive molecular testing is unlikely to impact clinical decision-making (NCT02467192). CLINICAL TRIALS REGISTRATION: NCT02467192.

Low-conductance high selective inositol (1,4,5)-trisphosphate activated Ca2+ channels in plasma membrane of A431 carcinoma cells.

In many cells, activation of receptors coupled to PIP2 turnover results in Ca2+ release from the intracellular stores accompanied by Ca2+ influx across the PM. It is not well established yet whether Ca2+ influx is activated by IP3 or by an unknown signal generated upon Ca2+ store depletion. We report here a single-channel study of low-conductance IP3-activated channels of very high selectivity for Ca2+ in the PM of A431 carcinoma cells. The channels are strongly potential dependent and sensitive to [Ca2+]i within the physiological range. The data obtained argues for IP3 acting directly on plasma membrane Ca2+ channels.

[Activation of mechanosensitive ion channels in plasma membrane of K562 cells]. / Aktivatsiia mekhanochuvstvitel'nykh ionnykh kanalov v plazmaticheskoi membrane kletok K562.

Patch clamp method in cell-attached configuration was used to search for mechanogated ion channels in plasma membrane of human myeloid leukemia K562 cells. A reversible activation of transmembrane currents in response to negative pressure applied to membrane patch was observed. Four types of mechanosensitive channels were identified in K562 cells: two main types were characterized with conductance values of 16 and 25 pS; while two others, showing higher conductance values (about 35 and 50 pS), were rarely met. In terms of gating, all channels described here could be assigned to the stretch-activated type. No inactivation of mechanosensitive channels at the sustained stimulation was observed. The activation of mechanosensitive channels in K562 cells was not dependent upon the presence of bivalent cations in the extracellular solution.

[A new type of IP3-sensitive highly selective calcium channels of low conductance in the plasma membrane of carcinoma A 431 cells]. / Novyi tip IP3-chuvstvitel'nykh vysokoselektivnykh kal'tsievykh kanalov nizkoi provodimosti v plazmaticheskoi membrane kletok kartsinomy A 431.

Application of 0.2-15.0 mM inositol 1,4,5-trisphosphate (IP3) to excised plasma membrane patches from A 431 cells induced activity of low conductance channels that were highly selective for Ca2+ and Ba2+. With 100 mM Ca2+ or 105 mM Ba2+, the channel conductance was 1.1 pS for the minor conductance substrate. The dose response curve gave an apparent binding constant of 0.18 microM IP3. The channel open probability displayed a strong potential dependence: it decreased markedly upon depolarization, and the half-maximum value was achieved at 73 mV. The dependence of channel activity on the intracellular free Ca2+ concentration was bell shaped, but markedly different from that reported for endoplasmic IP3 receptor. The same channels were detected in intact cells upon application of calcium mobilizing reagents. The activity induced in cell attached mode disappeared after patch excision but could be resumed by application of IP3. The data obtained argue for IP3 acting directly on plasma membrane Ca2+ channels.

Epidemiology of viral respiratory infections in a tertiary care centre in the era of molecular diagnosis, Geneva, Switzerland, 2011-2012.

Few studies have examined the epidemiology of respiratory viral infections in large tertiary centres over more than one season in the era of molecular diagnosis. Respiratory clinical specimens received between 1 January 2011 and 31 December 2012 were analysed. Respiratory virus testing was performed using a large panel of real-time PCR or RT-PCR. Results were analysed according to sample type (upper versus lower respiratory tract) and age group. In all, 2996 (2469 (82.4%) upper; 527 (17.6%) lower) specimens were analysed. Overall positivity rate was 47.4% and 23.7% for upper and lower respiratory samples, respectively. The highest positivity rate was observed in patients under 18 years old (p <0.001); picornaviruses were the most frequent viruses detected over the year. Influenza virus, respiratory syncytial virus, human metapneumovirus and coronaviruses showed a seasonal peak during the winter season, while picornaviruses and adenoviruses were less frequently detected in these periods. Multiple viral infections were identified in 12% of positive cases and were significantly more frequent in children (p <0.001). In conclusion, we observed significant differences in viral infection rates and virus types among age groups, clinical sample types and seasons. Follow-up of viral detection over several seasons allows a better understanding of respiratory viral epidemiology.

Long-term hepatitis E viral load kinetics in an immunocompromised patient treated with ribavirin.

Hepatitis E virus (HEV) can cause chronic infections in immunocompromised hosts. Viral kinetics in plasma and stools are poorly understood, particularly during antiviral treatment. Prolonged faecal shedding may be a concern for transmission. We describe HEV kinetics in an immunocompromised patient with prolonged faecal shedding despite undetectable viraemia on ribavirin treatment.

Detection of insulin mRNA in the peripheral blood after human islet transplantion predicts deterioration of metabolic control.

Recent updates of the Edmonton trial have shown that insulin independence is progressively lost in approximately 90% of islet transplant recipients over the first 5 years. Early prediction of islet graft injury could prompt the implementation of strategies attempting to salvage the transplanted islets. We hypothesize that islet damage is associated with the release and detection of insulin mRNA in the circulating blood. Whole blood samples were prospectively taken from 19 patients with type 1 diabetes receiving 31 islet transplants, immediately prior to transplantation and at regular time-points thereafter. After RNA extraction, levels of insulin mRNA were determined by quantitative reverse tran-scriptase-polymerase chain reaction. All patients exhibited a primary peak of insulin mRNA immediately after transplantation, without correlation of duration and amplitude with graft size or outcome. Twenty-five subsequent peaks were observed during the follow-up of 17 transplantations. Fourteen secondary peaks (56%) were closely followed by events related to islet graft function. Duration and amplitude of peaks were higher when they heralded occurrence of an adverse event. Peaks of insulin mRNA can be detected and are often associated with alterations of islet graft function. These data suggest that insulin mRNA detection in the peripheral blood is a promising method for the prediction of islet graft damage.

Effect of intracellular calcium on ATP-activated, GTP-dependent calcium channels in rat macrophages.

1. In order to study the effect of intracellular free Ca2+ concentration ([Ca2+]i) on the activity of ATP-activated, GTP-dependent Ca2+ channels in rat macrophages, experiments were performed using the inside-out configuration of the patch-clamp technique. 2. Channel activity was observed in the cell-attached mode when 100 microM ATP was added to the pipette solution containing 105 mM Ba2+, but it disappeared rapidly after patch excision. The activity could be restored by the application of 100 microM GTP or GTP gamma S onto the internal surface of the plasma membrane. 3. The properties of the GTP gamma S-evoked channels are identical to those of channels activated by extracellular application of ATP. The channels exhibited four current sublevels with conductances of about 3.5, 7, 10 and 15 pS when 105 mM Ba2+ was the only permeant cation. The extrapolated reversal potentials were similar for all the sublevels and averaged about +40 mV. 4. Elevation of [Ca2+]i within the range 0.01-1 microM resulted in a decrease in mean inward current. The half-maximal value of the mean current was about 0.08 microM. 5. This decreases in mean current resulted from a redistribution of sublevel occupancies: the 1st sublevel tended to be come more abundant with elevation of [Ca2+]i, while the relative weights of the high-conductance 3rd and 4th sublevels decreased. 6. The open-channel current fell with an increase in [Ca2+]i as quickly as the mean current did, indicating that the sublevel redistribution alone is sufficient to produce the revealed decrease in net inward current. 7. It is concluded that [Ca2+]i elevation does not fix the channel in a closed state but rather decreases the ability of the channel to operate in high-conductance states.

Nonselective cation channels in endothelial cells derived from human umbilical vein.

(i) We have used a combined patch-clamp and fura-2 fluorescence technique to characterize a nonselective cation channel (NSC) in Ea. hy926 (EA) cells, an endothelial cell line derived from human umbilical vein. (ii) Stimulation with ATP, histamine and bradykinin activated slowly and with a long delay after application of the agonist, a nonselective cation current (INSC) which is time- and voltage-independent. The permeability sequence for cations was PNa > PCs >> PNMDG, PCa. In the absence of external Ca2+ and at rather high concentrations, La3+ and Gd3+ blocked INSC. (iii) Single channel analysis revealed that ATP activates in the cell-attached configuration a nonselective cation channel with a conductance of approximately 24 pS and a permeation sequence identical to that of the macroscopic current. The channel activity disappeared after membrane excision. (iv) Activation of NSC required physiological intracellular Ca2+ levels (100 nm or higher). All agonists failed to activate NSC if cytosolic Ca2+ ([Ca2+]i) was lowered by 10 mm BAPTA. Clamping internal Ca2+ at 1 microm sometimes (8 out of 17 cells) spontaneously activated INSC in the absence of any additional stimulus. (v) Application of 2,5-di-tert-butylhydroquinone and internal perfusion of inositol 1,4,5-trisphosphate also activated INSC. The phospholipase C inhibitor, U-73122 inhibited INSC and the sustained Ca2+ plateau during agonist stimulation whereas the inactive analogue, U-73343 had no effect. (vi) These results indicate NSC may act as a Ca2+ entry pathway in endothelium. [Ca2+]i and inositol 1,4,5-trisphosphate play a role in the activation cascade of NSC, and possibly also store depletion.

The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6.

AIMS/HYPOTHESIS: The paired-homeobox genes pax-4 and pax-6 are crucial for islet development; whereas the null mutation of pax-6 results in the nearly absence of glucagon-producing alpha cells, pax-4 homozygous mutant mice lack insulin and somatostatin-producing beta and delta cells but contain an increased number of alpha cells suggesting that alpha cells could develop by a default mechanism. METHODS: To investigate whether beta-cell specific factors act negatively on glucagon gene transcription, we ectopically expressed pax-4 in glucagon producing InR1G9 cells; Pax-4 inhibited basal transcription of the glucagon gene promoter by 60%. To assess the mechanism of this inhibition, we cotransfected the non-islet cell line BHK-21 with Pax-4 and various transcription factors present in alpha cells. RESULTS: In addition to a general repressor activity on basal glucagon gene promoter activity of 30-50%, a specific 90% inhibition of Pax-6 mediated transactivation was observed. In contrast, Pax-4 had no effect on Cdx-2/3 or HNF3alpha mediated transcriptional activation. Pax-4 showed similar affinity to the Pax-6 binding sites on the glucagon gene promoter compared to Pax-6, but varying with KCl concentrations. CONCLUSION/INTERPRETATION: Pax-4 impairs glucagon gene transcription specifically through inhibition of Pax-6 mediated transactivation. Transcriptional inhibition seems to be mediated by direct DNA binding competition with Pax-6 and potentially additional mechanisms such as protein-protein interactions and a general repressor activity of Pax-4. Glucagon gene expression in alpha cells could thus result from both the presence of islet cell specific transcription factors and the absence of Pax-4.

Miniature Ca2+ channels in excised plasma-membrane patches: activation by IP3.

In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5'o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels (Icrac). These conductive properties include extremely low conductance (approximately 1 pS), very high selectivity for Ca2+ over K+ (PCa/PK>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains Icrac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane.

ATP-activated inward current and calcium-permeable channels in rat macrophage plasma membranes.

1. To study mechanisms of receptor-operated Ca2+ influx in non-excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole-cell cell-attached and outside-out configurations of the patch clamp technique. Under whole-cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP-induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4-. The nucleotide potency order was ATP > ATP gamma S > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type. 3. The macrophage plasma membrane was impermeable to Tris+ during the ATP-induced current at ATP4- concentrations varying from 0.07 to 500 microM. At higher concentrations, ATP produced a large inward steady-state current, which could be attributed to membrane permeabilization. 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell-attached and outside-out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mM Ba2+ as the permeant cation, the conductance sublevels were 3.5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole-cell current noise analysis (3.5-4.5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current-voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mM Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: PCa:PBa:PK = 71.:29:1. Thus, ATP-activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca2+.

ATP-operated calcium-permeable channels activated via a guanine nucleotide-dependent mechanism in rat macrophages.

1. To elucidate the possible involvement of a G protein in ATP-evoked Ca(2+)-permeable channel activity, membrane currents of rat peritoneal macrophages were recorded using inside-out and cell-attached configurations of the patch clamp technique. 2. In inside-out experiments with a pipette solution containing 105 mM Ba2+, application of 100 microM GTP or GTP gamma S to the internal surface of the membrane elicited a rise in channel activity. This effect was observed in 49% of the patches investigated (n = 69). The mean value of NPo (N, number of open channels; Po, channel open probability) was equal to 0.49 +/- 0.27 (mean +/- S.E.M.; n = 16). The delay in the activity development was 21 +/- 8 s (n = 18) with 200 microM ATP added to the pipette solution and about 4 min (n = 5) without agonist in the pipette. Similar results were obtained with 10 mM Ca2+ as the only permeant cation. 3. Properties of GTP gamma S-evoked channels were identical to those of channels activated by extracellular application of ATP. The channels exhibited at least four conductance sublevels, the 4th one being the least frequent. With 105 mM Ba2+ as a permeant cation, sublevel conductances were 3.5, 7, 10 and 15 pS. Corresponding values for 10 mM Ca2+ were about 4, 9, 13 and 17 pS. Extrapolated reversal potential (Er) values were about +40 and +25 mV for Ba2+ and Ca2+, respectively. 4. The activity of channels with similar characteristics could be induced by the extracellular application of fluoride in cell-attached experiments without any agonist in the pipette solution.(ABSTRACT TRUNCATED AT 250 WORDS)