Ilka Engelmann

Virus and cystic fibrosis: Rhinoviruses are associated with exacerbations in adult patients

Abstract Background Few studies have suggested the potential role of respiratory viruses in cystic fibrosis (CF) exacerbation, but their real impact is probably underestimated. Method Sixty-four sputum samples collected from 46 adult patients were included in the study: 33 samples were collected during exacerbation of CF, and 31 during the stable phase. After extraction, nucleic acids were tested for the presence of respiratory viruses. When rhinovirus (HRV) was detected, the 5′UTR and VP4/2 regions were sequenced, and phylogenetically analyzed. The characteristics of patients in exacerbation and stable phase were compared. Results Viruses were found in 25% of samples. The HRV viruses were the most frequently detected followed by coronaviruses. Only the HRV detection was significantly associated with the occurrence of CF pulmonary exacerbation (p <0.027). Characterization of 5′UTR and VP4/2 regions of the HRV genome specified that HRV-A, -B, -C were detected. All HRV-C were recombinant HRV-Ca. Conclusions HRV were the most frequently detected viruses; their detection was significantly associated with the occurrence of an exacerbation. The reality of viral recombination between HRV was demonstrated in CF patients for the first time, raising the role of viruses in lung microbiota. Further studies are now warranted to decipher virus impact in CF.

Diagnosis of Viral Infections Using Myxovirus Resistance Protein A (MxA)

BACKGROUND: Myxoma resistance protein 1 (MxA) is induced during viral infections. MxA testing could be helpful to differentiate between viral and bacterial infections. METHODS: A prospective multicenter cohort study was performed in pediatric emergency departments. MxA blood values were measured in children with confirmed viral or bacterial infections, uninfected controls, and infections of unknown origin. First patients were used to determine MxA threshold for viral infection. The diagnostic performance of MxA was determined by using receiver operating characteristic (ROC) analysis. Sensitivities (Se), specificities (Sp), and positive and negative likelihood ratios (LR+, LR–) were calculated. RESULTS: The study included 553 children; 44 uninfected controls and 77 confirmed viral infections (mainly respiratory syncytial virus and rotavirus) were used to determine an MxA threshold at 200 ng/mL. In the 193 other patients with confirmed infections and uninfected controls (validation group), MxA was significantly higher in patients with viral than in those with bacterial infections and uninfected controls (P < .0001). The area under the ROC curve (AUC) were 0.98, with 96.4% Se and 85.4% Sp, for differentiating uninfected from virus-infected patients and 0.89, with 96.4% Se and 66.7% Sp, for differentiating bacterial and viral infections. MxA levels were significantly higher in patients with clinically diagnosed viral versus clinically diagnosed bacterial infections (P < .001). Some patients with Streptococcus pneumonia infections had high MxA levels. Additional studies are required to elucidate whether this was due to undiagnosed viral coinfections. CONCLUSIONS: MxA is viral infection marker in children, at least with RSV and rotavirus. MxA could improve the management of children with signs of infection.

Serum-Dependent Enhancement of Coxsackievirus B4-Induced Production of IFNα, IL-6 and TNFα by Peripheral Blood Mononuclear Cells

Only a few reports have been published on the interactions between Coxsackievirus B4 (CVB4) and human peripheral blood mononuclear cells (PBMC) but have not been extensively documented. Human serum containing non-neutralizing anti-CVB4 antibodies increased CVB4-induced synthesis of IFNα by PBMC. In this study, we determined if CVB4 and human serum have the ability to activate inflammatory cytokines in addition to IFNα in PBMC cultures. PBMC from healthy donors were inoculated with infectious, inactivated CVB4 or with CVB4 incubated with dilutions of human serum or polyvalent IgG with anti-CVB4 activity. Levels of IFNα, TNFα, IL-6, IL-12, IFNγ and IL-10 in the cell-free supernatants of PBMC cultures were measured using ELISA. Infection was assessed by real-time PCR. PBMC inoculated with CVB4 produced inflammatory cytokines but not IFNα. When CVB4 was incubated with serum or IgG, IFNα was detected in the culture supernatants, and high concentrations of TNFα and IL-6 were measured. The concentrations of TNFα and IL-6 were not reduced in cultures inoculated with inactivated CVB4, whereas the IgG-dependent enhancement of IFNα, IL-6 and TNFα production with inactivated virus was suppressed. The potentiation of IFNα production was associated with a high intracellular viral load. Infectious and non-infectious CVB4 can induce the production of inflammatory cytokines but not IFNα by PBMC. High levels of IFNα, in addition to TNFα and IL-6, in culture supernatants were obtained when infectious CVB4 was combined with immune serum or IgG, and they were associated with high amounts of intracellular viral RNA.

Rhinovirus and Asthma: Reinfection, Not Persistence

and therefore not different from the sensitivity reported usingEBUSTBNA in our article. They state that all non–small cell lung cancer– not otherwise specified (NSCLC-NOS) samples in their institution have immunocytochemistry (ICC) performed. However, in their illustrative retrospective cohort, only 19 of 24 NSCLC-NOS cases underwent ICC, implying that 21% of cases of NSCLC-NOS were inadequate for further testing despite ROSE. In our multicenter study of 774 patients, 101 had a diagnosis of NSCLC-NOS and in only 6 patients was immunohistochemistry not possible. Dr. Craig and colleagues unfortunately also do not refer to the randomized controlled trials of ROSE that also do not support its routine use. Even in patients undergoing conventional TBNA, the presence of ROSE does not appear to improve yield (2). In a randomized controlled trial of patients undergoing EBUS-TBNA with and without ROSE, the sensitivity was not significantly different between the arms (3). Dr. Craig and colleagues also suggest that the presence of malignant cells in one lymph node station as assessed by ROSE can preclude further investigation of other diagnoses such as tuberculosis in other lymph node stations. This is certainly not our practice in our high-tuberculosis-prevalence region (4). Dr. Craig and colleagues suggest some potential advantages of ROSE. They omit that it may improve the negative predictive value of a negative EBUS sample; however, they also do not include any potential disadvantages that must also be considered. For example, the process of ROSE will use up vital tissue that could be better used for further tumor characterization in the laboratory. The number of passes submitted to the pathologist in the room before a lymph node is labeled as benign is unknown, and the risk of false-positive diagnoses in the bronchoscopy suite must also be clarified. The cost-effectiveness of ROSE is also unknown; however, the number of cases reported by a pathologist in an EBUS session is less than if the equivalent time was spent in the cytology laboratory. Routine use of ROSEwill have enormous impact on pathology service provision and costs to the United Kingdom National Health Service. Dr. Craig and colleagues’ analysis of our data (225 patients with “lymphoid cells only” on EBUS-TBNA) also highlights a key point surrounding terminology of EBUS-TBNA samples. Dr. Craig and colleagues refer to this group of patients with lymphoid cells only or an inadequate sample as the “nondiagnostic” group. We believe this is an unhelpful and inaccurate term and should be restricted to the group of patients where EBUS-TBNA has provided an insufficient sample only. Lymphoid cells only may be entirely diagnostic of a reactive lymph node, which was the final diagnosis in 71% in this group. We currently classify these results as negative, appreciating that this negative result may be diagnostic (true negative) or a false negative if malignancy is subsequently confirmed. We agree with the authors that when a phenotypic diagnosis is not clear on initial assessment, ICC plays an important role in subtyping, and it is also our routine practice that this is performed as well as epidermal growth factor receptor mutation testing where appropriate. In addition to the tests routinely used in Dr. Craig and colleagues’ institution, we routinely analyze adenocarcinomas for the EML4-ALK gene rearrangement and are about to embark on multiplex testing of lung cancers, with a view to aiding recruitment to early-phase clinical trials. This emphasizes the pressure that specimens from EBUS-TBNA are likely to be subjected to in the future and highlights that using specimen at the bedside for ROSE may not be the best use of lung cancer tissue. The evidence is not currently available to suggest that ROSE is effective or cost-effective, and therefore, we do not agree with Dr. Craig and colleagues’ conclusion that availability of ROSE should be default. Although we agree that ROSE is helpful in the learning phase of the procedure (5), a recommendation for routine use of ROSE will add to the already high capital costs of embarking on EBUS-TBNA and may deter further centers from being commissioned. Finally, we agree that all EBUS-TBNA services should be subject to audit with a review of service specifications if standards are not being met.

Clinically severe Epstein-Barr virus encephalitis with mild cerebrospinal fluid abnormalities in an immunocompetent adolescent: a case report

A 15-year-old boy developed Epstein-Barr virus (EBV) encephalitis, a rare complication of infectious mononucleosis. The severe clinical picture and the marked neuroimaging changes were in contrast with mild cerebrospinal fluid abnormalities: leukocyte count was normal and protein level was only slightly elevated. EBV DNA was detected in cerebrospinal fluid by polymerase chain reaction.

Enterovirus D68 detection in respiratory specimens: Association with severe disease

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)‐D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV‐D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT‐PCR. This multiplex RT‐PCR does not detect EV‐D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV‐specific‐RT‐PCR. EV‐positive specimens were tested with an EV‐D68‐specific‐RT‐PCR and genotyped. Eleven specimens of 2014 tested positive in the EV‐specific‐RT‐PCR and of these seven were positive in the EV‐D68‐specific‐RT‐PCR. Typing confirmed these as EV‐D68. Median age of EV‐D68‐positive patients was 3 years (1 month‐91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV‐D68‐positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV‐specific‐RT‐PCR but negative in the EV‐D68‐specific‐RT‐PCR. In conclusion, use of an EV‐specific‐RT‐PCR allowed us to detect EV‐D68 circulation in autumn 2014 that was not detected by the multiplex RT‐PCR and was associated with severe disease.

Necessity to critically review the automatic results of the Xpert Flu assay

Abstract While using the Xpert Flu assay we became aware of false-negative results. The study aimed to analyze the causes of these false-negative results. One hundred fifty-nine respiratory specimens were tested in the Xpert Flu assay and in multiplex reverse transcription–polymerase chain reactions (RT-PCRs) for respiratory viruses. Discordant specimens were tested in the Influenza A/B r-gene assay. One hundred fifty-two (96%) and 151 (95%) specimens yielded concordant results for influenza A and B, respectively. Fifteen specimens tested negative in the Xpert Flu assay and positive in a multiplex RT-PCR. Positive results were confirmed for 12 of these specimens in the Influenza A/B r-gene assay. Xpert Flu assay amplification curves and endpoints suggested that the false-negative results were mainly due to erroneous automatic result interpretation. We report false-negative results of the Xpert Flu assay due to erroneous automatic result interpretation. Careful analysis of amplification curves and endpoints is needed to avoid reporting of false-negative results.

Persistent viral DNA detection in blood after primary herpes simplex 1 infection revealed by hepatitis with hemophagocytic syndrome.

We here report the case of a 52-year old man who presented hepatitis with hemophagocytic syndrome triggered by herpes simplex 1 (HSV-1) primary infection. A high-level viremia was observed, and HSV-1 DNA remained detectable in blood for a long time after patients recovery.

Comparison of two commercial quantitative PCR assays and correlation with the first WHO International Standard for human CMV

Comparability between CMV assays could be facilitated by the first WHO International Standard for human CMV (standard). Standard dilutions were submitted to nucleic acid extraction with Versant kPCR Molecular systems SP or MagNA Pure LC System followed by the kPCR PLX™ CMV DNA (kPCR) or the CMV R-gene™ assay (R-gene), respectively; 139 clinical specimens were tested. Both assays correlated well with the standard (R2 > 0.96) and a matrix effect was observed. Quantitative results correlated reasonably between both assays for whole blood (R2 = 0.79) and well for other specimen types (R2 = 0.93). Quantification differences were within one log10 of the averaged log10 results for 25/27 blood specimens and for 32/33 other specimens. Calibration to the standard did not increase this percentage. In conclusion, results of both assays showed reasonable correlation with each other and good correlation with the standard. Calibration to the standard did not improve comparability of quantitative results.

Relapsing Pityriasis Rosea With HHV-7 Reactivation in an 11-Year-Old Girl

We describe an 11-year-old girl with recurrent episodes of PR over several years that was associated with HHV-7 DNA detection. Pityriasis rosea (PR) usually presents as acute exanthema with oval erythematous-squamous lesions localized on the trunk, arms, and legs with spontaneous remission. We present an unusual case of PR with frequent relapses during a period of 7 years. An 11-year-old white female patient presented with many pruritic erythematous oval lesions on her trunk. A second episode followed 2 years later with several pruritic erythematous lesions on her lower limbs. During the following 5 years, the patient had several relapses per year, with 1 to 3 lesions on changing localizations. PR was diagnosed on the basis of the clinical presentation and detection of human herpesvirus 7 DNA. Spontaneous remission occurred without treatment in each episode. Relapsing PR is a rare form of PR characterized by a lower number of lesions and smaller sized lesions compared with the classic form of PR. Pediatricians should consider the diagnosis of relapsing PR even if only a single or few erythematous lesions are present.