Theresia Popow-Kraupp

Effectiveness of Reverse Transcription-PCR, Virus Isolation, and Enzyme-Linked Immunosorbent Assay for Diagnosis of Influenza A Virus Infection in Different Age Groups

ABSTRACT The degrees of effectiveness of reverse transcription (RT)-PCR, virus isolation, and antigen enzyme-linked immunosorbent assay (ELISA) for the detection of influenza A virus were evaluated with nasopharyngeal swabs from 150 patients (1 week to 86 years old) with influenza A virus infection. RT-PCR had a sensitivity for influenza A virus in stock virus preparations 103 times higher than virus isolation and 106 to 107 times higher than ELISA. The detection rate achieved by RT-PCR in clinical samples was clearly higher (93%) than that by virus isolation (80%) and ELISA (62%). Despite low overall detection rates achieved by antigen ELISA, samples from patients younger than 5 years old yielded higher-than-average rates in this rapid assay (88%). The likelihood of negative results in the ELISA increased significantly with increasing age of the patient (P < 0.01). The degrees of effectiveness of RT-PCR and virus isolation were not influenced by the age of the patient. Neither influenza immunizations nor the interval between onset of symptoms and laboratory investigation (mean, 4.7 days; standard deviation, 3.3 days) affected results obtained by the three test systems. Our results demonstrate that the ELISA is reliable for rapid laboratory diagnosis of influenza in infants and young children, but for older patients application of RT-PCR or virus isolation is necessary to avoid false negative results.

Acute Encephalopathy Associated with Influenza A Virus Infection

Abstract Twenty-one patients aged 4–78 years with influenza A virus–associated acute encephalopathy were studied. Influenza A virus could be detected only in a cerebrospinal fluid (CSF) specimen obtained from 1 of 18 patients, despite the use of a highly sensitive polymerase chain reaction assay. Six patients experienced influenzal encephalopathy during the course of respiratory illness. Five of these patients had hypoprothrombinemia and 4 had increased serum creatinine levels, indicating hepatic and/or renal dysfunction. Fourteen patients experienced postinfluenzal encephalopathy ⩽3 weeks after resolution of acute respiratory symptoms. In 6 patients, focal areas of high signal intensity were visible on T2-weighted magnetic resonance images of the brain. Adenovirus DNA was detected in CSF specimens obtained from 4 (36%) of 11 patients with postinfluenzal encephalopathy. Thus, influenzal encephalopathy is frequently associated with metabolic disorders, whereas postinfluenzal encephalopathy appears to have different possible etiologies.

A Combination Vaccine for Allergy and Rhinovirus Infections Based on Rhinovirus-Derived Surface Protein VP1 and a Nonallergenic Peptide of the Major Timothy Grass Pollen Allergen Phl p 1

Allergens and rhinovirus infections are among the most common elicitors of respiratory diseases. We report the construction of a recombinant combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived VP1, the surface protein which is critically involved in infection of respiratory cells, and a nonallergenic peptide of the major grass pollen allergen Phl p 1. Recombinant hybrid molecules consisting of VP1 and a Phl p 1-derived peptide of 31 aa were expressed in Escherichia coli. The hybrid molecules did not react with IgE Abs from grass pollen allergic patients and lacked allergenic activity when exposed to basophils from allergic patients. Upon immunization of mice and rabbits, the hybrids did not sensitize against Phl p 1 but induced protective IgG Abs that cross-reacted with group 1 allergens from different grass species and blocked allergic patients’ IgE reactivity to Phl p 1 as well as Phl p 1-induced basophil degranulation. Moreover, hybrid-induced IgG Abs inhibited rhinovirus infection of cultured human epithelial cells. The principle of fusing nonallergenic allergen-derived peptides onto viral carrier proteins may be used for the engineering of safe allergy vaccines which also protect against viral infections.

Early detection of acute rhinovirus infections by a rapid reverse transcription-PCR assay.

ABSTRACT The development of a rhinovirus (RV)-RNA-specific reverse transcription (RT)-PCR assay is complicated by the close homology between the RV and enterovirus (EV) genomes in the highly conserved 5′-noncoding region, which is chosen for primer design in most RT-PCR assays. We have developed a sensitive, rapid, and RV-specific nested RT-PCR assay and have used it to test nasopharyngeal aspirates from 556 patients presenting with acute respiratory tract infections. RV RNA was detected by nested RT-PCR not only in all of 52 samples that were RV positive by virus isolation methods but also in 124 of 367 samples that were negative by virus isolation methods and enzyme-linked immunosorbent assay (ELISA). In addition, in 23 of 137 samples that were positive for a different respiratory virus by virus isolation and/or ELISA, RV RNA was detected by RT-PCR. EVs, adenoviruses, respiratory syncytial viruses, coronaviruses, and influenza and parainfluenza viruses, including clinical isolates as well as stock viruses, were not amplified in our RV-specific RT-PCR assay, indicating that this assay was highly specific. The processing time was less than 2 days for the RT-PCR, as opposed to up to 2 weeks for virus isolation. These results indicate that nested RT-PCR is more sensitive than conventional methods for the detection of RV in patients experiencing acute respiratory tract infections and represents the only reliable tool for the early laboratory diagnosis of RV infections. This is especially important in light of new opportunities for therapy currently being developed.

Antibody Maturation and Viremia after Primary Cytomegalovirus Infection, in Immunocompetent Patients and Kidney-Transplant Patients

To investigate antibody maturation and serum levels of cytomegalovirus (CMV) DNA after primary CMV infection, we studied 51 immunocompetent and 27 kidney-transplant patients. Compared with the immunocompetent patients, the transplant patients had significantly more-prolonged and -variable antibody maturation, clearly longer durations of viremia, and higher levels of CMV DNA; however, antibody maturation continued for >1 year even in immunocompetent patients. Long-term ganciclovir prophylaxis in the transplant patients was associated with either delayed immunoglobulin-G seroconversion, inhibition of antibody maturation (n=2), or immunoglobulin-class switching (n=1). In conclusion, antibody maturation continues in immunocompetent patients for a period longer than previously had been thought and is significantly delayed or even inhibited in kidney-transplant patients.

Presence of Cytomegalovirus in Cerebrospinal Fluid of Patients with Guillain-Barré Syndrome

BACKGROUND Because poliomyelitis has been almost completely eradicated, Guillain-Barre syndrome (GBS) now accounts for most cases of acute flaccid paralysis. Understanding of the role of cytomegalovirus (CMV) in the pathogenesis of GBS is still very limited. METHODS We identified 42 CMV-seropositive patients with GBS between 1998 and 2001. Cerebrospinal fluid (CSF) and serum samples obtained from these patients were tested by CMV-specific polymerase chain reaction, and the glycoprotein B (gB) segment of the detected CMV genome was analyzed. Virological findings were compared with clinical characteristics and CSF laboratory values. RESULTS CMV DNA was detected in 13 (31%) of 42 CSF samples from patients with GBS but was not detected in 42 CSF samples from age-matched control subjects with acute encephalopathy. CSF samples obtained early after the onset of GBS were significantly more likely to be positive for CMV DNA (P=.048). gB1 was the most prevalent genotype detected in patients with GBS (88%), followed by gB3 (8%) and gB2 (4%). CONCLUSIONS CMV DNA was detected frequently in CSF samples from CMV-seropositive patients with GBS, especially early during the course of the disease. The clinical significance of this finding has yet to be elucidated, but early administration of antiviral therapy might prove to be beneficial for selected patients with GBS.

Near-patient assays for diagnosis of influenza virus infection in adult patients

Abstract Rapid and reliable diagnosis of influenza is essential for identification of contagious patients and effective patient management. Near-patient assays allow establishment of the diagnosis within minutes in young children, and this study aimed to evaluate near-patient assays in relation to the patient’s age. A total of 194 patients with laboratory-confirmed influenza A/H3N2 virus infection, diagnosed within a prospective cohort study, were included. Cryopreserved nasopharyngeal swabs collected from these patients were tested by four near-patient assays (Binax Now Influenza A&B, Quick S-Influ A/B, Influ-A&B Respi-Strip, and Actim Influenza A&B). The main outcome measure was sensitivity of the near-patient assays in relation to the age of patients. The Binax Now, Quick S-Influ, Influ-A&B Respi-Strip and Actim assays had overall sensitivities of 19%, 18%, 26%, and 40%, respectively. The estimated sensitivity for influenza A/H3N2 virus detection in nasopharyngeal swabs was 17–56% in children 1 year of age and decreased to 8–22% in patients 80 years of age (logistic regression). The sensitivity of the Influ-A&B Respi-Strip and Actim assays decreased significantly with increasing age (p 0.014 and p 0.033, respectively (logistic regression)), a trend for decrease was observed for the Binax Now assay (p 0.074 (logistic regression)), and the low sensitivity of the Quick S-Influ assay was similar in children and adults. Less than one-fourth of diagnosed influenza A/H3N2 virus infections can be identified in elderly patients using a near-patient assay. Consequently, near-patient assays are of limited value for confirming the diagnosis when influenza is clinically suspected in adults. Antiviral therapy and additional diagnostic procedures cannot be withheld on the basis of a negative near-patient assay result, particularly in adult patients.

Decreased interferon-gamma response in respiratory syncytial virus compared to other respiratory viral infections in infants.

An inappropriate interferon‐gamma response has been implicated in the pathogenesis of severe respiratory syncytial virus (RSV) lower respiratory tract illness (LRTI). To assess whether this is unique for RSV primary LRTI compared to a first non‐RSV LRTI, intracellular interferon‐gamma was determined by flow cytometry in peripheral blood mononuclear cells from 32 infants with a primary RSV infection, 28 with a first non‐RSV LRTI due to adenoviral, parainfluenzaviral and rhinoviral infection and 13 healthy infants. Interferon‐γ responses were increased significantly during adenoviral, parainfluenzaviral and the majority of the rhinoviral infections, but remained low during RSV and severe rhinoviral infection. Low interferon‐γ responses were associated with a more severe clinical course of LRTI. This indicates that depending on the nature of the viral pathogen, respiratory virus infections in infants differ significantly with regard to the quantity of the interferon‐γ production and that this may contribute to the clinical course of the disease.

Influenza A and B Viruses but Not MERS-CoV in Hajj Pilgrims, Austria, 2014

To the Editor: The World Health Organization recommends that persons who return from pilgrimages to the Middle East with acute severe respiratory infections be tested to determine the cause of infection; the aim is to identify infections with the Middle East respiratory syndrome coronavirus (MERS-CoV), which have been occurring in Saudi Arabia since 2012. Each year, >2.5 million persons from >180 countries, including 240,000 pilgrims from Europe, participate in Hajj, the Muslim pilgrimage to Mecca, Saudi Arabia. The gathering of mass numbers of persons during the Hajj increases the risk for the spread of respiratory infections among participants, and this risk has raised global concern that travelers returning from this pilgrimage could contribute to the international spread of MERS-CoV. During the 2012 and 2013 Hajj and Umrah (a minor pilgrimage) pilgrimages, no MERS cases in pilgrims were reported (1,2). However, in 2014, cases of MERS-CoV infection were confirmed in 2 returning pilgrims in the Netherlands (3). The International Health Regulations Emergency Committee advised all countries to improve awareness about MERS-CoV among pilgrims and to conduct surveillance for MERS-CoV among pilgrims during and after Hajj (4). According to data from the International Air Transport Association, Austria received an estimated 68,000 air travelers from Saudi Arabia, Jordan, Qatar, and the United Arab Emirates during June–November 2012 (a period encompassing 1 month before Ramadan and 1 month after the Hajj) (5); of these travelers, 1,000 were pilgrims performing the Hajj. Relatively constant travel volumes to Austria on commercial flights out of these countries during 2010–July 2014 have been confirmed by an analysis of air traffic statistics for Austria (A. Herndler, M. Rudolf, pers. comm.). We report on the investigation of illness among Austrian residents just after their return home from the 2014 Hajj pilgrimage, which ended in early October. As of October 27, 2014, a total of 7 Hajj pilgrims from Austria had sought medical care in different Austrian hospitals/medical centers just after returning from Saudi Arabia. The patients had fever and/or respiratory symptoms. A summary of the patients’ characteristics is presented in the Table. Of the 7 patients, 4 had cough, 1 had dyspnea, and 4 had fever. Patients 1, 2, and 7 had an acute febrile illness and clinical and/or radiologic evidence of pulmonary parenchymal disease. Patient 1 had sought medical care in Saudi Arabia, and patient 2 had been hospitalized for 10 days in Saudi Arabia. Table Characteristics of pilgrims who returned from the Hajj with acute respiratory illness and detectable virus in respiratory specimens, Austria, 2014* For the diagnosis of viral infection, a serum sample and a sputum, throat swab, or bronchoalveolar lavage sample were collected and sent to the Department of Virology, Medical University of Vienna, Austria, for analysis. All samples were tested for MERS-CoV by using reverse transcription PCR targeting regions upstream of the envelope gene (6). Respiratory and serum samples from all 7 patients were negative for MERS-CoV. The respiratory samples were also tested for influenza A and B viruses and for rhinoviruses, as previously described (7–9). Of the 7 patients, 3 were positive for influenza B virus, 2 for influenza A(H3N2) virus, and 2 for rhinoviruses (Table). Subsequent phylogenetic analysis showed that the influenza A(H3N2) strains belonged to the A/Hong Kong/146/2013-like viruses and the influenza B strains belonged to the B/Phuket/3073/2013-like viruses of the Yamagata lineage, both of which are subtype H3N2 and B strains included in the 2014–15 seasonal influenza vaccine for the Northern Hemisphere. Our results showed that MERS-CoV was not detected in any of these patients, and our findings support those from reports investigating illness among 2013 Hajj pilgrims (2). Our data indicate that all patients had a respiratory virus infection, and 5 of the 7 patients were infected with influenza virus. Six of the patients had not received seasonal influenza vaccine before traveling; for the seventh patient, influenza vaccine uptake data were not available. In 2014 in Austria, the incidence of influenza-like illnesses was below the epidemic threshold during weeks 40–43: no cases of influenza were reported by the National Influenza Surveillance network, Austria (http://www.influenza.at). Therefore, it is likely that these 7 patients became infected while at the Hajj, as reported for patients during previous Hajj seasons (10), or while traveling through international airports. Our preliminary data indicate that influenza virus infection was the cause of severe respiratory illness in 5 of 7 Austrian pilgrims returning from the 2014 Hajj. Because influenza is a serious and potentially life-threatening illness, seasonal influenza vaccination is recommended for Hajj pilgrims. Accurate and rapid testing for MERS-CoV and other respiratory viruses, including influenza, is essential for identifying persons who may be contagious and for timely and effective antiviral drug treatment and, thus, should be considered for pilgrims with severe respiratory illness.

Cytomegalovirus DNA Load Patterns Developing After Lung Transplantation Are Significantly Correlated With Long-Term Patient Survival

Background. Cytomegalovirus (CMV) causes complications after lung transplantation (LuTX). We have investigated whether patient survival is associated with CMV DNA load patterns developing in EDTA-plasma and bronchoalveolar lavage (BAL) during the first year after LuTX and whether patients’ predispositions influence this development. Materials and Methods. CMV DNA loads in plasma and BAL were investigated serially in 84 LuTX recipients. Various patient characteristics and variants in mannose-binding lectin and toll-like receptor 2 genes were analyzed. Patient survival beyond 1 year posttransplantation and influence of patient-specific factors on viral load development were assessed by Kaplan-Meier survival and Cox regression methods. Results. Four distinct group patterns were observed: (a) less than 1000 copies CMV DNA/mL in plasma and BAL (42%); (b) more than 1000 copies/mL in BAL only (21%); (c) one limited episode (25%), or (d) more than one episode of more than 1000 copies/mL plasma (12%). The risk of death after the first year was elevated 14-fold in group D and sixfold in group B. CMV DNA load patterns were not associated with patient-specific factors except CMV-D+/R− status. Initial and peak plasma CMV DNA levels were significantly increased in group D. Conclusions. Active CMV replication that is not limited after one episode of CMV DNAemia leads to significantly decreased long-term survival after LuTX.