Lars Öhrmalm

Clinical Utility of PCR for Common Viruses in Acute Respiratory Illness

BACKGROUND: Acute respiratory illness (ARI) accounts for a large proportion of all visits to pediatric health facilities. Quantitative real-time polymerase chain reaction (qPCR) analyses allow sensitive detection of viral nucleic acids, but it is not clear to what extent specific viruses contribute to disease because many viruses have been detected in asymptomatic children. Better understanding of how to interpret viral findings is important to reduce unnecessary use of antibiotics. OBJECTIVE: To compare viral qPCR findings from children with ARI versus asymptomatic control subjects. METHODS: Nasopharyngeal aspirates were collected from children aged ≤5 years with ARI and from individually matched, asymptomatic, population-based control subjects during a noninfluenza season. Samples were analyzed by using qPCR for 16 viruses. RESULTS: Respiratory viruses were detected in 72.3% of the case patients (n = 151) and 35.4% of the control subjects (n = 74) (P = .001). Rhinovirus was the most common finding in both case patients and control subjects (47.9% and 21.5%, respectively), with a population-attributable proportion of 0.39 (95% confidence interval: 0.01 to 0.62). Metapneumovirus, parainfluenza viruses, and respiratory syncytial virus were highly overrepresented in case patients. Bocavirus was associated with ARI even after adjustment for coinfections with other viruses and was associated with severe disease. Enterovirus and coronavirus were equally common in case patients and control subjects. CONCLUSIONS: qPCR detection of respiratory syncytial virus, metapneumovirus, or parainfluenza viruses in children with ARI is likely to be causative of disease; detection of several other respiratory viruses must be interpreted with caution due to high detection rates in asymptomatic children.

Respiratory viruses associated with community-acquired pneumonia in children: matched case–control study

Background Community-acquired pneumonia (CAP) is the leading cause of death in children worldwide and a substantial proportion of childhood CAP is caused by viruses. A better understanding of the role of virus infections in this condition is needed to improve clinical management and preventive measures. The aim of the study was therefore to assess the association between specific respiratory viruses and childhood CAP. Methods A case–control study was conducted during 3 years in Stockholm, Sweden. Cases were children aged ≤5 years with radiological CAP. Healthy controls were consecutively enrolled at child health units during routine visits and matched to cases on age and calendar time. Nasopharyngeal aspirates were obtained and analysed by real-time PCR for 15 viruses. Multivariate conditional logistic regression was used to account for coinfections with other viruses and baseline characteristics. Results A total of 121 cases, of which 93 cases met the WHO criteria for radiological pneumonia, and 240 controls were included in the study. Viruses were detected in 81% of the cases (n=98) and 56% of the controls (n=134). Influenza virus, metapneumovirus and respiratory syncytial virus were detected in 60% of cases and were significantly associated with CAP with ORs >10. There was no association with parainfluenza virus, human enterovirus or rhinovirus and coronavirus and bocavirus were negatively associated with CAP. Conclusions Our study indicates viral CAP is an underestimated disease and points out hMPV as a new important target for the prevention of childhood CAP.

Respiratory viruses, a common microbiological finding in neutropenic children with fever.

Abstract Background Febrile neutropenia is a common complication in children undergoing chemotherapy for malignancies. A microbial agent is only identified in 15–30% of the fever episodes and corresponds mostly to bacterial findings. Objective To investigate viral infections as possible etiologic agents in episodes of febrile neutropenia. Study design Nasopharyngeal aspirates (NPAs) from patients presenting with neutropenic fever at two pediatric oncology wards in Sweden and Australia were analyzed with a conventional virus-diagnostic approach and RT-PCR. Coupled blood samples were analyzed for the detection of CMV, EBV, adenovirus and erythrovirus. Bacterial blood culture was performed routinely. Results Conventional virus-diagnostic approach coupled to routinely performed bacterial analyzes revealed an infectious agent in 29% compared to 60% when using PCR. By adding PCR, a viral pathogen was detected in 46% of the NPAs and in 4% of the blood samples collected. In half of the patients with bacteremia, respiratory tract viruses were co-detected. Conclusion Respiratory viruses were frequently detected in NPAs suggesting a significant role of viral infections in children presenting with neutropenic fever. The meaning of these findings needs to be further evaluated but has the potential to individualize infection treatment and to reduce the extensive use of antibiotics in immunocompromised children with neutropenia.

Flocked nasal swab versus nasopharyngeal aspirate for detection of respiratory tract viruses in immunocompromised adults: a matched comparative study

BackgroundSeveral studies have compared nasal swabs to the more invasive nasopharyngeal aspirate (NPA) for detection of respiratory viruses. Mostly, the comparisons have been performed on immunocompetent children with upper respiratory tract symptoms. The results range from a relatively poor sensitivity for the swabs to an even higher sensitivity than for the NPA. We aimed to investigate the sensitivity of a flocked nasal swab (fNS) on immunocompromised adults with febrile neutropenia.MethodsDuring 16 months, adults with a hematological disorder presenting with febrile neutropenia were enrolled in the study. Paired samples of the fNS and NPA were collected in the outer part of the nasal cavity and the nasopharynx, respectively. The samples were analyzed regarding a panel of 15 respiratory viruses by means of quantitative polymerase chain reaction. Furthermore, as an indirect measure of cell yield by either method, the copy number of the human beta actin gene was also determined. Cohens kappa was calculated as a measure of agreement of the results obtained from either method. Wilcoxon signed-rank test was used for comparison of cell yield.ResultsA total of 98 paired samples from a total of 89 patients were collected. Twenty of the pairs had virus detected in at least one of the specimens; 11 in both, 7 in NPA only, and 2 in fNS only. For the fNS, the overall sensitivity for any virus and for rhinovirus only was 65% and 78%, respectively. NPA was significantly superior to the fNS in collecting epithelial cells.ConclusionWe found the overall sensitivity of 65% to be too low to replace NPA with this sampling technique in this patient category.

Metagenomic analysis of bloodstream infections in patients with acute leukemia and therapy-induced neutropenia

Leukemic patients are often immunocompromised due to underlying conditions, comorbidities and the effects of chemotherapy, and thus at risk for developing systemic infections. Bloodstream infection (BSI) is a severe complication in neutropenic patients, and is associated with increased mortality. BSI is routinely diagnosed with blood culture, which only detects culturable pathogens. We analyzed 27 blood samples from 9 patients with acute leukemia and suspected BSI at different time points of their antimicrobial treatment using shotgun metagenomics sequencing in order to detect unculturable and non-bacterial pathogens. Our findings confirm the presence of bacterial, fungal and viral pathogens alongside antimicrobial resistance genes. Decreased white blood cell (WBC) counts were associated with the presence of microbial DNA, and was inversely proportional to the number of sequencing reads. This study could indicate the use of high-throughput sequencing for personalized antimicrobial treatments in BSIs.

Bacterial Landscape of Bloodstream Infections in Neutropenic Patients via High Throughput Sequencing

Background Bloodstream infection (BSI) is a common and potentially life-threatening complication in patients with hematological malignancies and therapy-induced neutropenia. Administration of broad spectrum antibiotics has substantially decreased the mortality rate in febrile neutropenia, but bacterial infection is documented in only one-third or fewer of the cases. BSI is typically diagnosed by blood culture; however, this method can detect only culturable pathogens. Methods In the present study, a total of 130 blood samples from hematological patients receiving dose-intensive antitumoural treatment were subjected to 16S rRNA PCR and 62 of them were cultured. PCR positive samples were processed to high throughput sequencing by amplifying the V1-V3 regions of the 16S rRNA gene to obtain a full spectrum of bacteria present in BSI. Results Five phyla and 30 genera were identified with sequencing compared to 2 phyla and 4 genera with culture. The largest proportion of bacteria detected by sequencing belonged to Proteobacteria (55.2%), Firmicutes (33.4%) and Actinobacteria (8.6%), while Fusobacteria (0.4%) and Bacteroidetes (0.1%) were also detected. Ninety-eight percent of the bacteria identified by sequencing were opportunistic human pathogens and 65% belonged to the normal human microbiota. Conclusions The present study indicates that BSIs in neutropenic hosts contain a much broader diversity of bacteria, likely with host origin, than previously realized. The elevated ratio of Proteobacteria in BSI corroborates the results found in other systemic inflammatory diseases, such as inflammatory bowel disease or mucosal infections. This knowledge may become of value for tailoring antimicrobial drug administration.

Mannose-binding lectin 2 polymorphisms do not influence frequency or type of infection in adults with chemotherapy induced neutropaenia.

Background Mannose-binding Lectin protein (MBL) has been suggested to be relevant in the defence against infections in immunosuppressed individuals. In a Swedish adult cohort immunosuppressed from both the underlying disease and from iatrogenic treatments for their underlying disease we investigated the role of MBL in susceptibility to infection. Methods In this cross sectional, prospective study, blood samples obtained from 96 neutropaenic febrile episodes, representing 82 individuals were analysed for single nucleotide polymorphism (SNP) in the MBL2 gene. Concurrent measurement of plasma MBL protein concentrations was also performed for observation of acute response during febrile episodes. Findings No association was observed between MBL2 genotype or plasma MBL concentrations, and the type or frequency of infection. Adding to the literature, we found no evidence that viral infections or co-infections with virus and bacteria would be predisposed by MBL deficiency. We further saw no correlation between MBL2 genotype and the risk of fever. However, fever duration in febrile neutropaenic episodes was negatively associated with MBL2 SNP mutations (p<0.05). Patients with MBL2 SNP mutations presented a median febrile duration of 1.8 days compared with 3 days amongst patients with wildtype MBL2 genotype. Interpretation We found no clear association between infection, or infection type to MBL2 genotypes or plasma MBL concentration, and add to the reports casting doubts on the benefit of recombinant MBL replacement therapy use during iatrogenic neutropaenia.

Bacteremia in Swedish hematological patients with febrile neutropenia: Bacterial spectrum and antimicrobial resistance patterns

Abstract Background: The etiology of bacteremia in hematological patients with febrile neutropenia differs geographically and changes over time. Since efficient empirical antibiotic treatment depends on relevant knowledge of the bacterial panorama, the aim of this study was to describe the prevalence of bacteremia, the bacterial spectrum, and the resistance patterns of the isolates in this group today. Methods: In a cross-sectional study, routine blood cultures from febrile episodes occurring in adult patients with hematological disorders and neutropenia presenting to Karolinska University Hospital, Stockholm, Sweden during a 24-month period, were analyzed. Results: A total of 142 febrile neutropenic episodes occurring in 124 hematological patients were included in the study. Bacteremia was documented in 27% of the episodes, and of these, 58% were due to Gram-positive pathogens. The most common isolates were viridans streptococci, coagulase-negative staphylococci, and Escherichia coli. Low levels of antibiotic resistance were detected. The underlying diagnosis of non-Hodgkins lymphoma (NHL) was independently negatively associated with documented bacteremia (p < 0.01). Conclusions: The prevalence of bacteremia and the bacterial spectrum were consistent with recent Scandinavian reports. Substantially lower levels of antimicrobial resistance were registered compared to those found in other European centers. Patients with NHL were less likely to have documented bacteremia in this study.

Microbial Translocation Contribute to Febrile Episodes in Adults with Chemotherapy-Induced Neutropenia

In this study we sought to determine the contribution of microbial translocation to febrile episodes with no attributable microbiological cause (Fever of Unknown Origin, FUO) in an adult febrile neutropaenic cohort. Endotoxin concentrations were measured with the chromogenic Limulus Amoebocyte Assay and used as a direct measure of bacterial products whilst soluble CD14 (sCD14), measured with ELISA was selected as an indicator of the early host response to endotoxins. Endotoxin concentrations in this cohort were generally elevated but did not differ with the presentation of fever. Further stratification of the febrile episodes based on the microbiological findings revealed significantly (p = 0.0077) elevated endotoxin concentrations in FUO episodes compared with episodes with documented bacterial and viral findings. sCD14 concentrations were however, elevated in febrile episodes (p = 0.0066) and no association was observed between sCD14 concentration and microbiological findings. However, FUO episodes and episodes with Gram-negative bacteraemia were associated with higher median sCD14 concentrations than episodes with Gram-positive bacteraemia (p = 0.030). In conclusion, our findings suggest that in the absence of microbiological findings, microbial translocation could contribute to febrile episodes in an adult neutropaenic cohort. We further observed an association between prophylactic antibiotic use and increased plasma endotoxin concentrations (p = 0.0212).

Endotoxemia as a Diagnostic Tool for Patients with Suspected Bacteremia Caused by Gram-Negative Organisms: a Meta-Analysis of 4 Decades of Studies

ABSTRACT The clinical significance of endotoxin detection in blood has been evaluated for a broad range of patient groups in over 40 studies published over 4 decades. The influences of Gram-negative (GN) bacteremia species type and patient inclusion criteria on endotoxemia detection rates in published studies remain unclear. Studies were identified after a literature search and manual reviews of article bibliographies, together with a direct approach to authors of potentially eligible studies for data clarifications. The concordance between GN bacteremia and endotoxemia expressed as the summary diagnostic odds ratios (DORs) was derived for three GN bacteremia categories across eligible studies by using a hierarchical summary receiver operating characteristic (HSROC) method. Forty-two studies met broad inclusion criteria, with between 2 and 173 GN bacteremias in each study. Among all 42 studies, the DORs (95% confidence interval) were 3.2 (1.7 to 6.0) and 5.8 (2.4 to 13.7) in association with GN bacteremias with Escherichia coli and those with Pseudomonas aeruginosa, respectively. Among 12 studies of patients with sepsis, the proportion of endotoxemia positivity (95% confidence interval) among patients with P. aeruginosa bacteremia (69% [57 to 79%]; P = 0.004) or with Proteus bacteremia (76% [51 to 91%]; P = 0.04) was significantly higher than that among patients without GN bacteremia (49% [33 to 64%]), but this was not so for patients bacteremic with E. coli (57% [40 to 73%]; P = 0.55). Among studies of the sepsis patient group, the concordance of endotoxemia with GN bacteremia was surprisingly weak, especially for E. coli GN bacteremia.