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.

The correlation of maxillomandibular advancement and airway volume change in obstructive sleep apnea using cone beam computed tomography.

The purpose of this retrospective study was to evaluate the correlation of maxillomandibular advancement (MMA) and airway volume changes in patients with obstructive sleep apnea (OSA), and to determine the surgical skeletal movements necessary to achieve an increase in total airway volume (TAV) of ≥70%. Thirty patients with OSA treated by MMA were evaluated. Pre- and postoperative cone beam computed tomography images were used to determine the horizontal distance and angular changes in surgical parameters and linear, area, and volumetric airway parameters. Postoperatively, the horizontal distance of surgical parameters (A-point, UI, B-point, pogonion, and menton) and craniofacial angulation (SNA and SNB) increased significantly, similar to total surface area, TAV, and minimum cross-sectional area of the airway (p<0.0001). The total airway length decreased significantly (p<0.0001). The mean increase in TAV was 67.2%. There were positive correlations between linear surgical changes and the percentage change in TAV. All surgical parameters were predictive of a change in TAV ≥70%. The optimal surgical change was 6mm for A-point, 7.9mm for UI, 7.6mm for B-point, 11.2mm for pogonion, and 10mm for menton. In conclusion, maxillary advancement of less than 10mm was adequate in this study to obtain an increase in the TAV of at least 70%.

Diagnostic challenges of central nervous system infection: extensive multiplex panels versus stepwise guided approach.

BACKGROUND: Cerebrospinal fluid (CSF) testing is a key component for the diagnosis of central nervous system (CNS) infections. Current meningitis and encephalitis management guidelines agree on the need for CSF molecular testing in combination with other direct and indirect biological testing, both in CSF and blood. Multiplex molecular tests have been developed to reduce turnaround times and facilitate the diagnostic approach. OBJECTIVES: We aim to discuss the role of multiplex molecular panels in the management of CNS infections. SOURCES: The MEDLINE database and the grey literature have been searched for relevant articles. CONTENT: New molecular multiplex panels are being developed to simultaneously detect a large array of neuropathogens in CSF. Although one of these assays has been US Food and Drug Administration-approved, extensive analytical and clinical validation is still missing, and suboptimal performance related issues have been raised. Its use has been associated with decreased costs, reduced length of hospital stay and reduced antiviral therapy administration in retrospective, industry-sponsored studies. The pros and cons of this multiplex syndromic approach are discussed in this narrative review. IMPLICATIONS: Molecular multiplex CNS infection diagnosis panels have been developed and present several attractive features, including ease of use and low turnaround time. However, suboptimal analytical performances render these tests difficult to use without additional confirmatory tests. Such panels are not comprehensive nor adapted to all situations, depending on the epidemiological or clinical context. Overall, available data in the literature currently do not support the use of a multiplex PCR panel in clinical routine as a 'stand-alone' molecular assay. Except in restricted laboratory capacity settings where such easy-to-use multiplex panels offer the diagnostic means that would otherwise not be available, the stepwise testing approach remains a more rational option. Serological testing both in blood and CSF should not be neglected, but it represents essential complementary tools regarding some neuropathogens.

A molecular based diagnosis of positive blood culture in the context of viral haemorrhagic fever: proof of concept.

OBJECTIVES: The aim of this study was to evaluate the possibility of using a PCR-based panel to identify bacterial and fungal bloodstream infections in the setting of suspected or confirmed viral haemorrhagic fever. METHODS: The accuracy of the FilmArray® Blood Culture Identification Panel (BCID) assay was assessed to identify the common bacterial and fungal pathogens associated with bloodstream infections after positive blood culture inactivation using a guanidinium thiocyanate containing buffer lysis that is commonly used for viral haemorrhagic fever molecular diagnostics. RESULTS: The FilmArray® BCID panel assay detected 95% (19/20) of the pathogens analysed in this study by using both protocols with and without inactivation. Absolute consistency (100%) was observed in all isolates with phenotypes compatible with the presence of the antibiotic resistance genes mecA, vanA, vanB and blaKPC. CONCLUSIONS: The FilmArray® BCID panel assay coupled to inactivation using a guanidinium thiocyanate containing buffer lysis represents a convenient, sensitive and specific diagnostic tool to detect some of the most pathogens associated with bloodstream infections in the context of a suspected or confirmed viral haemorrhagic fever.

Diagnostic approach to encephalitis and meningoencephalitis in adult returning travellers.

BACKGROUND: Encephalitis and meningoencephalitis are severe, sometime life-threatening infections of the central nervous system. Travellers may be exposed to a variety of neurotropic pathogens. AIMS: We propose to review known infectious causes of encephalitis in adults acquired outside Europe, and how to identify them. SOURCES: We used Pubmed and Embase, to search the most relevant publications over the last years. CONTENT: Microbiologic tests and radiological tools to best identify the causative pathogen in travellers presenting with encephalitis and ME are presented in this narrative review, as well as a diagnostic approach tailored to the visited area and types of exposures. IMPLICATIONS: This review highlights the diagnostic difficulties inherent to exotic causes of central nervous system infections, and attempts to guide clinicians with respect to which microbiological tests to consider, in addition to brain MRI, when approaching a returning traveller presenting with encephalitis.

Diagnostic tools to tackle infectious causes of encephalitis and meningoencephalitis in immunocompetent adults in Europe.

BACKGROUND: Encephalitis and meningoencephalitis imply inflammation of the brain parenchyma, and comprise many diagnostic entities, such as various infections and causes of dysimmunity. The cause remains unknown in around 50% of cases. OBJECTIVES: To summarize the main infectious causes of encephalitis and meningoencephalitis acquired in Europe, and the diagnostic means to identify them. SOURCES: PubMed, ECDC and WHO websites, personal experience. CONTENT: The principal infectious causes of encephalitis and meningoencephalitis acquired in Europe in adults are discussed in this review, with special emphasis on the microbiological and imaging diagnostic approaches. The role of electroencephalography in diagnosing encephalitis is also mentioned. Among infections, viruses are more frequent than other pathogen types, and their epidemiology varies according to geographic area. A few bacteria, such as Listeria monocytogenes and Mycobacterium tuberculosis, are also to be considered. In contrast, parasites and fungi are rare encephalitis causes in Europe. IMPLICATIONS: Identifying the causative pathogen of infectious encephalitis and meningoencephalitis is complex because of the variety of pathogens, the epidemiology of which is determined by geography and environmental factors. Furthermore, despite extensive microbiological testing, many cases of encephalitis remain of unknown origin. Brain magnetic resonance imaging and electroencephalography are useful complementary diagnostic tools, and newer unbiased sequencing technologies might help to fill in the diagnostic gap.

Comparative analysis of viral shedding in pediatric and adult subjects with central nervous system-associated enterovirus infections from 2013 to 2015 in Switzerland.

BACKGROUND: Several enterovirus (EV) genotypes can result in aseptic meningitis, but their routes of access to the central nervous system remain to be elucidated and may differ between the pediatric and adult populations. OBJECTIVE: To assess the pattern of viral shedding in pediatric and adult subjects with acute EV meningitis and to generate EV surveillance data for Switzerland. STUDY DESIGN: All pediatric and adult subjects admitted to the University Hospitals of Geneva with a diagnosis of EV meningitis between 2013 and 2015 were enrolled. A quantitative EV real-time reverse transcriptase (rRT)-PCR was performed on the cerebrospinal fluid (CSF), blood, stool, urine and respiratory specimens to assess viral shedding and provide a comparative analysis of pediatric and adult populations. EV genotyping was systematically performed. RESULTS: EV positivity rates differed significantly between pediatric and adult subjects; 62.5% of pediatric cases (no adult case) were EV-positive in stool and blood for subjects for whom these samples were all collected. Similarly, the EV viral load in blood was significantly higher in pediatric subjects. Blood C-reactive protein levels were lower and the number of leucocytes/mm3 in the CSF were higher in non-viremic than in viremic pediatric subjects, respectively. A greater diversity of EV genotypes was observed in pediatric cases, with a predominance of echovirus 30 in children ≥3 years old and adults. CONCLUSION: In contrast to adults, EV-disseminated infections are predominant in pediatric subjects and show different patterns of EV viral shedding. This observation may be useful for clinicians and contribute to modify current practices of patient care.

[Ebola: the unexpected emerging disease]. / Ebola: le point au crépuscule d'une épidémie inattendue.

In one year, Ebola virus disease has already been responsible of around 10000 deaths. 24 patients have been medically evacuated in different University Hospitals in Europe or in the United States. One medical doctor, infected during a humanitarian mission in the field has been treated in Geneva at the end of 2014. This review aims to summarize the epidemiology of the current outbreak, to describe the main virological and clinical characteristics of Ebola virus disease, and to address the most important experimental treatments available. Although the number of cases has fallen the last two months, the outbreak is not over. A safe and proctective vaccine is still needed in the race to fight this emerging viral disease.

[New viruses: myth, fantasy or reality?]. / Nouveaux virus: mythe, fantasme ou réalité?

Emerging viruses previously unknown or partially known that infect repeatedly the human population are more than ever in the medias actuality headlines. Multiple factors may explain this dynamic. The most important is certainly the rapid evolution and the adaptation capacity of these viruses. Note that the increase in travel and international trade or climate change also play an important role. On the other hand, laboratory tests and current surveillance systems are more efficient. Thus, transmission of virus from an animal reservoir to human are more easily detected, accentuating the feeling of increasing phenomenon. Virological predictions have very low reliability in epidemiology. It is a reality that we have to accept.

[Intracerebral HIV replication despite an efficient antiretroviral treatment: clinical impact and management]. / Réplication cérébrale du VIH malgré une thérapie antirétrovirale efficace: impact clinique et prise en charge.

Various neurological and neuropsychological manifestations are still relatively frequently reported in HIV infected patients in the highly active antiretroviral therapy era. A fraction of them could be related to HIV replication in the central nervous system (CNS) despite adequate peripheral viral suppression. This hypothesis is supported by numerous reports of detectable HIV RNA in the cerebrospinal fluid in the context of a low or undetectable viremia in association with neurological or neuropsychological complaints. In addition, some antiviral molecules may not achieve adequate levels in the CNS, thus potentially favoring intracerebral HIV replication and even antiretroviral resistance. Neurologic manifestations in the presence of CNS HIV replication often decrease after antiretroviral treatment CNS penetration optimization.

[Corticosteroids in the treatment of infectious diseases]. / Quelle place pour les stéroïdes dans le traitement des maladies infectieuses?

The addition of a corticosteroid has become a common practice for the treatment of some infectious diseases, such as meningitis, septic shock, moderate to severe Pneumocystis jirovecii pneumonia. The belief that steroids may have a beneficial effect in the early stage of pro-inflammatory infections explains the renewed interest for these treatments. This review of recent literature helps determine the use of steroids in the treatment of infectious diseases as formal guidance, questionable or rather contraindicated. When there is a clear scientific indication for the use of corticosteroids regardless of the current infection, the latter is never a formal contraindication.

[Where are we in the treatment of chronic osteomyelitis?]. / Où en est-on dans le traitement médical des ostéomyélites chroniques?

Chronic osteomyelitis is a multifaceted bacterial infection with common features, which requires surgery for remission. The duration and modality of concomitant administration of antibiotic agents for adult patients is still based on expert opinions. The traditional recommendation of 6 to 12 weeks of antibiotic therapy with intravenous administration for at least the first 2 weeks is more and more challenged in favor of an oral antibiotic treatment with selected agents from the start. There is no evidence that the total duration of antibiotic therapy for more than 6-12 weeks improves outcome, when compared with shorter regimens. External advice from an expert team with combined surgeons and infectious disease physicians may help to reduce antibiotic consumption in a cost-effective way.

[Clinical and genetic diversity of rhinoviruses]. / Rhinovirus : diversité clinique et génomique.

Human rhinoviruses are the most common cause of viral respiratory infections across all age groups, from the neonate to the elderly patient. The benign nature of most of these infections as well as the difficulty to isolate the causative agent limits our perception of its real clinical impact. Molecular diagnostic tools have allowed to better characterize the variety of clinical presentations which are not limited to the common cold alone. It is now clearly established that rhinoviruses infect both the upper and lower tracheobronchial tree which may also be the site of viral replication. Moreover, the virus is the cause of significant complications in patients at risk such as those with asthma or highly immunocompromised hosts. The use of molecular screening techniques shows the very high diversity of circulating strains which are not only limited to known serotypes and has allowed the identification of new subgroups previously unknown. Detailed analysis of the genomic organization shows a common phylogeny between certain subgroups of rhinoviruses and enteroviruses and sheds light on the constraints modelling the evolution of human Picornaviridae. Furthermore, detailed analysis of the CRE structures shows that this structure is not only conserved for each species, but is also located on a specific region for each of these species.

Immunolocalization of electrogenic sodium-bicarbonate cotransporters pNBC1 and kNBC1 in the rat eye.

The human NBC1 gene encodes two electrogenic sodium-bicarbonate cotransport proteins, pNBC1 and kNBC1, which are candidate proteins for mediating electrogenic sodium-bicarbonate cotransport in ocular cells. Mutations in the coding region of the human NBC1 gene in exons common to both pNBC1 and kNBC1 result in a syndrome with a severe ocular and renal phenotype (blindness, band keratopathy, glaucoma, cataracts, and proximal renal tubular acidosis). In the present study, we determined the pattern of electrogenic sodium-bicarbonate cotransporter protein expression in rat eye. For this purpose, pNBC1- and kNBC1-specific antibodies were generated and used to detect these NBC1 protein variants by immunoblotting and immunocytochemistry. pNBC1 is expressed in cornea, conjunctiva, lens, ciliary body, and retina, whereas the expression of kNBC1 is restricted to the conjunctiva. These results provide the first evidence for extrarenal kNBC1 protein expression. The data in this study will serve as a basis for understanding the molecular mechanisms responsible for abnormalities in ocular electrogenic sodium-bicarbonate cotransport in patients with mutations in the NBC1 gene.

Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes.

A multidisciplinary approach was taken to investigate the intracellular locations of the 26-kDa integral membrane protein encoded by the bcl-2 gene. Subcellular fractionation analysis of a t(14;18)-containing lymphoma cell line revealed the presence of Bcl-2 protein in nuclear, heavy-membrane, and light-membrane fractions but not in cytosol. Sedimentation of heavy-membrane fractions in Nycodenz and Percoll continuous gradients demonstrated comigration of p26-Bcl-2 with mitochondrial but not other organelle-associated proteins. Fractionation of light-membrane fractions using discontinuous sucrose-gradients revealed association of Bcl-2 protein primarily with lighter-density microsomes (smooth endoplasmic reticulum) as opposed to heavy-density microsomes (rough endoplasmic reticulum). Immune microscopy studies using laser-scanning microscopy, pre- and postembedding electron microscopic methods, and six different anti-Bcl-2 antibodies demonstrated Bcl-2 immunoreactivity in the nuclear envelope and outer mitochondrial membrane in a patchy distribution. Furthermore, anti-Bcl-2 antibody immunoreactivity generally appeared to directly overlie the nuclear envelope in high magnification electron microscopic studies, reminiscent of nuclear pore complexes. Addition of in vitro translated p26-Bcl-2 to isolated translocation-competent mitochondria revealed transmembrane domain-dependent association of Bcl-2 protein with mitochondria but provided no evidence for import into a protease-resistant compartment, consistent with immunomicroscopic localization to the outer mitochondrial membrane. Taken together, the findings demonstrate that p26-Bcl-2 resides primarily in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membrane in a nonuniform distribution suggestive of participation in protein complexes perhaps involved in some aspect of transport.

The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.

Elimination of permeability mutants from selections for drug resistance in mammalian cells.

Chinese hamster ovary (CHO) cells exhibit increased sensitivity to a wide variety of microtubule inhibitory drugs when verapamil is present in the growth medium. The extent of this increased sensitivity is drug specific: some drugs such as taxol and vinblastine respond greatly to the presence of verapamil, whereas other drugs such as griseofulvin respond very poorly. For the majority of drugs examined, however, a 2- to 10-fold increase in drug sensitivity is observed in the presence of verapamil at 5 micrograms/ml. The effects of verapamil are even more dramatic when drug-resistant mutant cells with a presumed alteration in membrane permeability are examined. In the presence of appropriate levels of verapamil, these mutants demonstrate a level of drug sensitivity comparable to that of the wild-type parental cells. Drug-resistant cells from similar selections but with well-defined alterations in alpha- or beta-tubulin and no evidence of alterations in membrane permeability, however, continue to exhibit increased resistance to the selecting drug even in the presence of verapamil. These studies support the conclusion that verapamil affects the membrane permeability to or transport of a wide variety of hydrophobic drugs. In addition, we have used this information to devise selections that virtually eliminate the isolation of drug-resistant permeability mutants. This methodology should be generally applicable to genetic studies of drug action that are complicated by the isolation of large numbers of mutants with permeability alterations.

Purification and characterization of a basal body-associated Ca2+-binding protein.

Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.