Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens

The present report describes an alternative method for in vitro detection of HIV-1 -specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analized. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection.

Withholding antimalarials in febrile children who have a negative result for a rapid diagnostic test.

BACKGROUND: The availability of a rapid diagnostic test for malaria (RDTm) allows accurate diagnosis at all levels of health facilities. The objective of the present study was to evaluate the safety of withholding antimalarials in febrile children who have a negative test result. METHODS: We conducted a prospective 2-arm longitudinal study in areas of Tanzania that are moderately and highly endemic for malaria. Children with a history of fever were managed routinely by resident clinicians of 2 health facilities, except that no antimalarials were prescribed if the RDTm result was negative. Children were followed up at home on day 7. The main outcome was the occurrence of complications in children with negative RDTm results; children with positive RDTm results were followed up for the same outcomes for indirect comparison. RESULTS: One thousand children (median age, 24 months) were recruited. Six hundred three children (60%) had a negative RDTm result. Five hundred seventy-three (97%) of these children were cured on day 7. Forty-nine (8%) of the children with negative RDTm results spontaneously visited the dispensary before day 7, compared with 10 (3%) of the children with positive RDTm results. All children who had negative initial results had negative results again when they were tested either at spontaneous attendance or on day 7 because they were not cured clinically, except for 3 who gave positive results on days 2, 4, and 7 respectively but who did not experience any complication. Four children who had negative initial results were admitted to the hospital subsequently, all with negative results for malaria tests upon admission. Two of them died, of causes other than malaria. CONCLUSIONS: Not giving antimalarial drugs in febrile children who had a negative RDTm result was safe, even in an area highly endemic for malaria. Our study provides evidence for treatment recommendations based on parasitological diagnosis in children <5 years old.

Genotype 3 is associated with rapid histological progression in chronic HCV infection

While several risk factors for the histological progression of chronic hepatitis C have been identified, the contribution of HCV genotypes to liver fibrosis evolution remains controversial. The aim of the present study was to assess independent predictors for fibrosis progression. Methods: We identified 1540 patients from the Swiss Hepatitis C Cohort database with at least one liver biopsy prior to antiviral treatment. Factors associated with fibrosis stage, steatosis and histological activity were assessed in univariate and multivariate regression models. Fibrosis progression rate per year was calculated in a subgroup of 1263 patients, in whom risk factors were assessed by cumulative incidence curves, logistic and linear regression models. Results: Independent risk factors for rapid fibrosis progression included male sex (OR = 1.66, 95% CI 1.25-2.21, P <0.001), age at infection (OR = 1.08, 95% CI 1.06-1.10, P <0.001), histological activity (OR = 2.14, 95% CI 1.61-2.85, P <0.001) and genotype 3 (OR = 1.97, 95% CI 1.43-2.72, P <0.001). Genotype 2 was associated with slow progression (OR = 0.51, 95% CI 0.30-0.89, P = 0.02), but this observation may be due to the decreased prevalence of genotype 2 over the last decades, leading to an overrepresentation of subjects with genotype 2 with a slow progression rate. Conclusion: This study shows a significant association of genotype 3 with accelerated fibrosis. While assessing risk factors for fibrosis progression, the changing epidemiology of HCV genotypes over time needs to be taken into account.

Rapid diagnostic tests for non-malarial febrile illness in the tropics.

The recent roll-out of rapid diagnostic tests (RDTs) for malaria has highlighted the decreasing proportion of malaria-attributable illness in endemic areas. Unfortunately, once malaria is excluded, there are few accessible diagnostic tools to guide the management of severe febrile illnesses in low resource settings. This review summarizes the current state of RDT development for several key infections, including dengue fever, enteric fever, leptospirosis, brucellosis, visceral leishmaniasis and human African trypanosomiasis, and highlights many remaining gaps. Most RDTs for non-malarial tropical infections currently rely on the detection of host antibodies against a single infectious agent. The sensitivity and specificity of host-antibody detection tests are both inherently limited. Moreover, prolonged antibody responses to many infections preclude the use of most serological RDTs for monitoring response to treatment and/or for diagnosing relapse. Considering these limitations, there is a pressing need for sensitive pathogen-detection-based RDTs, as have been successfully developed for malaria and dengue. Ultimately, integration of RDTs into a validated syndromic approach to tropical fevers is urgently needed. Related research priorities are to define the evolving epidemiology of fever in the tropics, and to determine how combinations of RDTs could be best used to improve the management of severe and treatable infections requiring specific therapy.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethiedine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrone, ethidium. Following uptake of the dye, erythrocytes contamine viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.

Rapid bacterial genome sequencing: methods and applications in clinical microbiology.

The recent advances in sequencing technologies have given all microbiology laboratories access to whole genome sequencing. Providing that tools for the automated analysis of sequence data and databases for associated meta-data are developed, whole genome sequencing will become a routine tool for large clinical microbiology laboratories. Indeed, the continuing reduction in sequencing costs and the shortening of the 'time to result' makes it an attractive strategy in both research and diagnostics. Here, we review how high-throughput sequencing is revolutionizing clinical microbiology and the promise that it still holds. We discuss major applications, which include: (i) identification of target DNA sequences and antigens to rapidly develop diagnostic tools; (ii) precise strain identification for epidemiological typing and pathogen monitoring during outbreaks; and (iii) investigation of strain properties, such as the presence of antibiotic resistance or virulence factors. In addition, recent developments in comparative metagenomics and single-cell sequencing offer the prospect of a better understanding of complex microbial communities at the global and individual levels, providing a new perspective for understanding host-pathogen interactions. Being a high-resolution tool, high-throughput sequencing will increasingly influence diagnostics, epidemiology, risk management, and patient care.

Rapid detection of bacterial resistance to antibiotics using AFM cantilevers as nanomechanical sensors.

The widespread misuse of drugs has increased the number of multiresistant bacteria, and this means that tools that can rapidly detect and characterize bacterial response to antibiotics are much needed in the management of infections. Various techniques, such as the resazurin-reduction assays, the mycobacterial growth indicator tube or polymerase chain reaction-based methods, have been used to investigate bacterial metabolism and its response to drugs. However, many are relatively expensive or unable to distinguish between living and dead bacteria. Here we show that the fluctuations of highly sensitive atomic force microscope cantilevers can be used to detect low concentrations of bacteria, characterize their metabolism and quantitatively screen (within minutes) their response to antibiotics. We applied this methodology to Escherichia coli and Staphylococcus aureus, showing that live bacteria produced larger cantilever fluctuations than bacteria exposed to antibiotics. Our preliminary experiments suggest that the fluctuation is associated with bacterial metabolism.

Exercise induces rapid interstitial lung water accumulation in patients with chronic mountain sickness.

BACKGROUND: Chronic mountain sickness (CMS) is a major public health problem in mountainous regions of the world. In its more advanced stages, exercise intolerance is often found, but the underlying mechanism is not known. Recent evidence indicates that exercise-induced pulmonary hypertension is markedly exaggerated in CMS. We speculated that this problem may cause pulmonary fluid accumulation and aggravate hypoxemia during exercise. METHODS: We assessed extravascular lung water (chest ultrasonography), pulmonary artery pressure, and left ventricular function in 15 patients with CMS and 20 control subjects at rest and during exercise at 3,600 m. RESULTS: Exercise at high altitude rapidly induced pulmonary interstitial fluid accumulation in all patients but one (14 of 15) with CMS and further aggravated the preexisting hypoxemia. In contrast, in healthy high-altitude dwellers exercise did not induce fluid accumulation in the majority of subjects (16 of 20) (P = .002 vs CMS) and did not alter arterial oxygenation. Exercise-induced pulmonary interstitial fluid accumulation and hypoxemia in patients with CMS was accompanied by a more than two times larger increase of pulmonary artery pressure than in control subjects (P < .001), but no evidence of left ventricular dysfunction. Oxygen inhalation markedly attenuated the exercise-induced pulmonary hypertension (P < .01) and interstitial fluid accumulation (P < .05) in patients with CMS but had no detectable effects in control subjects. CONCLUSIONS: To our knowledge, these findings provide the first direct evidence that exercise induces rapid interstitial lung fluid accumulation and hypoxemia in patients with CMS that appear to be related to exaggerated pulmonary hypertension. We suggest that this problem contributes to exercise intolerance in patients with CMS. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01182792; URL: www.clinicaltrials.gov.

The rapid urinary drug toxicology screen in the emergency room: a useful tool ?

Intoxications are a frequent problem in the ER. In the vast majorityof cases, supportive treatment is sufficient. Severe intoxications withunknown agents are considered an indication for a urinary drug screen,and are recommended by several toxicology centers. However, theirusefulness for patient management remains uncertain.Study objectives: Evaluation of the impact of a urinary drug screen(Biosite Triage TOX Drug Screen) testing 11 substances(acetaminophen, amphetamines, methamphetamines, barbiturates,benzodiazepines, cocaïne, methadone, opioids, phencyclidine,cannabis, tricyclic antidepressants) on initial adult patient managementin the emergency department of a university hospital with ~35.000annual admissions.Methods: Observational retrospective analysis of all tests performedbetween 09/2009 and 09/2010. A test utility was defined as useful if itresulted in the administration of a specific antidote (Flumazenil/Naloxone), the use of a quantitative confirmatory toxicologic test, or achange in patient's disposition.Results: 57 tests were performed. Patient age was 32 ± 11 (SD) years;58% were men; 30% were also intoxicated with alcohol. Two patientsdied (3.5%): the first one of a diphenhydramin overdose, the other of ahypertensive intracerebral hemorrhage believed to be caused cocaineabuse but a negative urine test. Test indications were: 54% firstpsychotic episode; 25% acute respiratory failure; 18% coma; 12%seizure; 11% opioids toxidrome; 7% sympathicomimetic toxidrome; 5%hypotension; 4% ventricular arrhythmia (VT, VF, torsades de pointes)or long QT. 75% of tests were positives for >=1 substance (mean 1.7 ±0.9). 47% of results were unexpected by history. 18% of resultsinfluenced patient management: 7% had a negative test that confirmedthe diagnosis of endogenous psychosis in a first psychotic episode, andallowed transfer to psychiatry; 5% received flumazenil/naloxone;2% had an acetaminophen blood level after a positive screen; finally,4% had an unexpected methadone abuse that required prolongationof hospital stay.Conclusions: A rapid urinary toxicologic screen was seldom used inour emergency department, and its impact on patient managementwas marginal: only one in 6 tests influenced treatment decisions.

Naltrexone-assisted rapid methadone discontinuation : a pilot study

Rapport de Synthèse : Un sevrage lent comme méthode élective pour l'interruption de la méthadone est coûteux en termes de temps, le plus souvent associé à un taux élevé d'abandon. Bien que les méthodes ultrarapides de désintoxication des opiacés aient gagné en popularité récemment, elles sont chères et posent les problèmes spécifiques liés aux patients traités par la méthadone. Méthodologie: ont été inclus dans l'étude dix patients en traitement de substitution avec de la méthadone. La dernière dose de méthadone a été administrée le matin même du jour de l'admission, en préalable à l'hospitalisation. Les médicaments suivants ont été administrés le jour suivant l'admission: ondansetron 36mg, ranitidine 40mg, loperamide 8m., clonazepam 4m., promazine 1OOmg, metoclopramide 70mg, naltrexone 5Omg. L'échelle objective de sevrage des opiacés (Objective Opiate Withdrawal Scale) a été appliquée au deuxième, troisième et quatrième jour d'hospitalisation, deux fois par jour, à 8h00 et 18h00. Un suivi a été réalisé sous la forme d'entretiens téléphoniques pendant une semaine, respectivement six mois après la date de sortie de l'hôpital, faisant suite à la désintoxication. Un autre entretient téléphonique a été réalisé dans les six mois suivant le "post-sevrage", avec pour objectif d'investiguer la continuité du traitément, une éventuelle rechute dans l'abus de drogues et une possible réintroduction de la méthadone. Résultats: nous avons pu déterminer quatre groupes de symptômes, sur la base d'une observation de trois jours d'évolution: 1) Les signes typiques du syndrome de sevrage de retrait des opiacés, symptôme de froid et chaud, pilo-érection, anxiété caractérisée par une intensité initiale élevée et une disparition relativement continue. 2) Hyperactivité neurovégétative caractérisée par une intensité initiale élevée et une rapide disparition. 3) Phénomènes neurovégétatifs dont l'intensité s'est maintenue durant toute la période d'observation. 4) Contractions musculaires, insomnies et anorexie, manque d'appétit, réapparaissant chez certains patients au 2ème et au début du 3ème jour. Conclusions: une procédure courte de désintoxication utilisant une dose unique de naltrexone s'avère être une méthode alternative valable pour un sevrage de la méthadone. Cette méthode semble accélérer et écourter la symptomatologie associée au sevrage. Le cours des symptômes peut être interprété comme biphasique. Une première phase de retrait est éminemment caractérisée par tous les symptômes typiques eux-mêmes et probablement induits par la naltrexone. La seconde phase, pour un plus petit nombre de patients, peut être interprétée comme en corrélation avec une concentration de méthadone en diminution significative ultérieurement.

Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.

Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non-toxic expression. We demonstrate the power of this multicolor 'Wave' marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immunoelectron microscopy. Among other findings, our systematic co-localization analysis revealed that a class of plant Rab1-homologs has a much more extended localization than was previously assumed, and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.