Burden of cytomegalovirus DNAemia among pediatric renal transplant patients on antiviral prophylaxis: A hospital-based analysis.

INTRODUCTION: We examined the burden of CMV DNAemia and time to such events among renal transplant patients receiving CMV prophylaxis. We targeted the first year after transplantation, with the primary focus being on the first 3 months. METHODS: We conducted a retrospective review of renal transplant patients (<18 years) who were transplanted and followed at our center between January 2007, and December 2017. Clinical and laboratory data were obtained from the medical records and laboratory databases. RESULTS: Among 141 patients, the median age at transplant was 12.7 years (range 0.87-17.83 years). CMV DNAemia was detected in 33 of 77 patients eligible for prophylaxis (42.9%; 95% CI 31.6-54.6) during the first post-transplant year. Proportionately more D+R- patients were present among patients with DNAemia compared with those without DNAemia (15/38, 39.5% vs 16/103, 15.5%, P = .005). Median time to first positivity was 134 days (range 0-304 days). Eight patients had a positive PCR during the first 3 months (5.7% of all patients). Among those eligible for prophylaxis, 6.5% had DNAemia during the first 3 months while on prophylaxis. Among patients whose first positive PCR was after 3 months post-transplant, the median time to positivity was 52 days (range 13-214 days) after the end of prophylaxis. CONCLUSIONS: Breakthrough CMV DNAemia was documented among children receiving antiviral prophylaxis. While routine monitoring while on prophylaxis might not be warranted for the majority of patients, studies are needed to determine the optimal indications for CMV PCR testing while on prophylaxis.

Diagnostic Interpretation Guidance for Pediatric Enteric Pathogens: A Modified Delphi Consensus Process.

BACKGROUND: We sought to develop diagnostic test guidance definitions for pediatric enteric infections to facilitate the interpretation of positive test results in the era of multianalyte molecular diagnostic test platforms. METHODS: We employed a systematic, two-phase, modified Delphi consensus process consisting of three web-based surveys and an expert panel face-to-face meeting. In phase 1, we surveyed an advisory panel of North American experts to select pathogens requiring diagnostic test guidance definition development. In phase 2, we convened a 14-member expert panel to develop, refine, and select the final definitions through two web-based questionnaires interspersed with a face-to-face meeting. Both questionnaires asked panelists to rate the degree to which they agreed that if the definition is met the pathogen is likely to be causative of clinical illness. RESULTS: The advisory panel survey identified 19 pathogens requiring definitions. In the expert panel premeeting survey, 13 of the 19 definitions evaluated were rated as being highly likely ("agree" or "strongly agree") to be responsible for acute gastroenteritis symptoms by ≥67% of respondent panel members. The definitions for the remaining six pathogens (Aeromonas, Clostridium difficile, Edwardsiella, nonenteric adenovirus, astrovirus, and Entamoeba histolytica) were indeterminate. After the expert panel meeting, only two of the modified definitions, C. difficile and E. histolytica/dispar, failed to achieve the a priori specified threshold of ≥67% agreement. CONCLUSIONS: We developed diagnostic test guidance definitions to assist healthcare providers for 17 enteric pathogens. We identified two pathogens that require further research and definition development.

Measuring influenza RNA quantity after prolonged storage or multiple freeze/thaw cycles.

In this study, we aim to determine what effects prolonged storage and repeated freeze/thaw cycles have on the stability of influenza A(H1N1)pdm09 (influenza A/H1N1)RNA. Cloned influenza A/H1N1 RNA transcripts were serially diluted from 8.0-1.0 log10 copies/µl. RT-qPCR was used to measure RNA loss in transcripts stored at -80°C, -20°C, 4°C and 25°C for up to 84days or transcripts undergoing a total of 10 freeze/thaw cycles. Viral load was measured in clinical specimens stored at-80°C for three years (n=89 influenza A RNA extracts; n=35 primary specimens) and in 10 clinical specimens from the 2015/2016 influenza season that underwent 7 freeze/thaw cycles. RNA stored at -80°C, -20°C, 4°C and 25°C is stable for up to 56, 56, 21, and 7days respectively or up to 9 freeze/thaw cycles when stored at -80°C. There is no difference in viral load in clinical specimens that have been stored for up to three years at -80°C if they are re-extracted. Similarly, clinical specimens undergoing up to 7 freeze/thaw cycles are stable if they are re-extracted between cycles. Influenza specimens can be stored for up to three years at -80°C or undergo up to 7 freeze/thaw cycles without loss of RNA quantity if re-extracted.

Mycobacterium Fortuitum Bloodstream Infection in a Very Low Birth Weight Preterm Neonate.

Mycobacterium fortuitum is a rapidly growing Mycobacterium species that is a rare cause of disease, primarily in immunocompromised patients. We present a very low birth weight preterm neonate who developed M. fortuitum bloodstream infection, where 16S rDNA sequencing allowed accurate identification. Cure was achieved by line removal and adjuvant combination treatment with amikacin, ciprofloxacin and clarithromycin.

Childhood exposures to discarded needles and other objects potentially contaminated with blood-borne pathogens in Toronto, Canada.

BACKGROUND: Exposure to discarded needles or other objects put children at risk for infection with blood-borne pathogens (BBP), including human immunodeficiency virus (HIV), hepatitis B (HBV) and hepatitis C. OBJECTIVE: The purpose of this study was to retrospectively analyze the epidemiology, management and outcome of children following such exposures in the greater Toronto community setting. METHODS: A retrospective study of children <19 years of age who had community-based exposure to objects that could contain BBP between January 2001 and December 2014. Sexual and hospital inpatient exposures were excluded. Patients were identified by medical record review of all children who had HIV testing performed. RESULTS: Sixty-six community-based exposures to objects potentially contaminated with BBP were identified (71.2% needlesticks). The median age was 6.3 years (interquartile range 3.8, 7.8). Exposures occurred outdoors in the community (45.5%), in schools (30.3%), homes (15.2%) and community/outpatient clinics (9.0%). Of 11 (16.7%) identified source subjects, 7 were known to be HIV infected. HIV post-exposure prophylaxis was prescribed to 22 (33.3%) children; 15 (71.4%) completed the course. Only 41.2% of previously unvaccinated children were documented to have completed a full HBV vaccine series post-exposure. No blood-borne infections were documented, but only 60.6% had documentation of adequate follow-up testing. CONCLUSIONS: Enhanced public health interventions in schools and other community settings are needed to reduce childhood risk of exposure to needlesticks or other objects potentially contaminated with BBP.

Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples.

BACKGROUND: Interassay harmonization of cytomegalovirus (CMV) DNA measurement is important for infection management. Uncertainty exists regarding the result harmonization achievable in patient plasma samples using quantitative polymerase chain reaction (qPCR) assays with calibrators now traceable to the First World Health Organization International Standard (IS) for CMV DNA. METHOD: Serial dilutions of the IS and a blinded panel of 40 genotyped CMV DNA-positive pooled plasma samples and 10 negative plasma samples were tested by 6 laboratories using 10 qPCR assays calibrated to the IS. Each clinical sample was constructed using plasma from a single unique transplant recipient. RESULTS: The variance for individual CMV DNA-positive samples was greater for clinical samples (median, 1.50 [range, 1.22-2.82] log10 IU/mL) than for IS dilutions (median, 0.94 [range, 0.69-1.35] log10 IU/mL) (P < .001); 58.9% of all clinical sample results and 93.6% of IS dilution results fell within ±0.5 log10 IU/mL of the mean viral load of each sample. Result variability was not impacted by either genotype or quantitative levels of CMV DNA. Testing procedure differences can significantly influence results, even when analyte-specific reagents are identical. For clinical samples, all assays demonstrated result bias (P < .008). Assays with amplicon sizes ≤86 bp had significantly higher results compared to assays with larger amplicon sizes (≥105 bp) (P < .001). CONCLUSIONS: The variability in CMV DNA results reported on individual samples has been reduced by the IS, but ongoing clinically relevant variability persists, preventing meaningful interassay result comparison.

Crib Mattress Investigation: A quality improvement study to assess mattress cover permeability and bacterial growth in crib mattresses.

PURPOSE: Any opening in a medical bed mattress cover may allow bodily fluids to enter the mattress, leading to contamination and potential nosocomial infection. This study's purpose was to assess permeability of crib mattress covers and measure bacterial growth within and on crib mattress surfaces. METHOD: Mattresses were selected randomly from hospital inventory. Bonney's blue dye was applied over mattress covers to assess permeability. Mattress cover surface swabs were acquired from standardized locations. Samples of mattress foam were acquired under sterile conditions. All samples were collected with the Eswab and eMRSA systems (Copan Diagnostics Inc, Brescia, Italy). Total aerobic bacteria count and colony types were assessed. Results are presented as mean ± standard error of the mean, independent t tests and analysis of variance were used to analyze data, and significance was achieved with P < .05. RESULTS: All mattresses (n = 7) had Bonney's blue dye visible on underlying mattress foam. There were 77 samples and 44 had bacterial growth. Total bacterial count ranged from 0.2-11.6 CFU/cm(2) with mean of 1.7 ± 0.38 CFU/cm(2). There was no relative differences between mattress sample location and colony type. All samples were negative for Staphylococcus aureus, including methicillin-resistant S aureus. CONCLUSIONS: Any crack in a mattress cover renders it permeable to fluid entering the mattress. Bacterial growth was present on mattress covers and within mattress foam. Mattresses support microbial viability from which nosocomial infection may occur.

Detection of enterovirus D68 in Canadian laboratories.

The recent emergence of a severe respiratory disease caused by enterovirus D68 prompted investigation into whether Canadian hospital and provincial laboratories can detect this virus using commercial and laboratory-developed assays. This study demonstrated analytical sensitivity differences between commercial and laboratory-developed assays for the detection of enterovirus D68.

Clinical severity of rhinovirus/enterovirus compared to other respiratory viruses in children.

BACKGROUND: Human rhinovirus/enterovirus (HRV/ENT) infections are commonly identified in children with acute respiratory infections (ARIs), but data on their clinical severity remain limited. OBJECTIVES: We compared the clinical severity of HRV/ENT to respiratory syncytial virus (RSV), influenza A/B (FLU), and other common respiratory viruses in children. PATIENTS/METHODS: Retrospective study of children with ARIs and confirmed single positive viral infections on mid-turbinate swabs by molecular assays. Outcome measures included hospital admission and, for inpatients, a composite endpoint consisting of intensive care admission, hospitalization >5 days, oxygen requirements or death. RESULTS: A total of 116 HRV/ENT, 102 RSV, 99 FLU, and 64 other common respiratory viruses were identified. Children with single HRV/ENT infections presented with significantly higher rates of underlying immunosuppressive conditions compared to those with RSV (37.9% versus 13.6%; P < 0.001), FLU (37.9% versus 22%; P = 0.018) or any other single viral infection (37.9% versus 22.5%; P = 0.024). In multivariable analysis adjusted for underlying conditions and age, children with HRV/ENT infections had increased odds of hospitalization compared to children with RSV infections (OR 2.6; 95% CI 1.4, 4.8; P < 0.003) or FLU infections (OR 3.0; 95% CI 1.6, 5.8; <0.001) and increased odds of severe clinical disease among inpatients (OR 3.0; 95% CI 1.6,5.6; P = 0.001) when compared to those with FLU infections. CONCLUSIONS: Children with HRV/ENT had a more severe clinical course than those with RSV and FLUA/B infections and often had significant comorbidities. These findings emphasize the importance of considering HRV/ENT infection in children presenting with severe acute respiratory tract infections.

Co-circulation of multiple genotypes of human rhinovirus during a large outbreak of respiratory illness in a veterans' long-term care home.

BACKGROUND: Human rhinoviruses (HRVs) are a well-recognized cause of long-term care home (LTCH) outbreaks of respiratory illness. However, there are limited data on the molecular epidemiology of the HRV types involved. OBJECTIVES: To determine whether a large respiratory outbreak in a LTCH was caused by a single type of HRV, and to describe the clinical impact of the outbreak. STUDY DESIGN: Nasopharyngeal swabs were collected from residents with one or more of the following: fever, cough, rhinitis, or congestion. Specimens were interrogated by multiplex PCR using the ResPlex II assay. Samples positive for HRV were then submitted for genotyping by partial sequence analysis of the 5' untranslated (UTR) and viral protein (VP) 1 capsid regions. RESULTS: Of 71 screened, 56 residents were positive for a HRV during an outbreak that lasted 5.5 weeks; 27 healthcare workers also had respiratory symptoms. Three residents were transferred to hospital and 2 died. Seven units in two wings of the LTCH were affected, resulting in 3152.5 resident unit closure days. Three different HRV genotypes were identified, although HRV-A1 dominated. CONCLUSIONS: This large outbreak of HRVs among residents and healthcare workers in a LTCH was associated with substantial resident and staff morbidity as well as significant unit closures. Multiple types of HRV were implicated but an HRV-A1 type dominated, warranting further investigation into viral determinants for virulence and transmission.

Evaluation of MALDI-TOF mass spectrometry and Sepsityper Kit™ for the direct identification of organisms from sterile body fluids in a Canadian pediatric hospital.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit - the Sepsityper Kit (Bruker Daltonik, Germany) - and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8%) and 39 of 50 (78%) blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%. MALDI-TOF MS is a promising tool for the direct identification of organisms cultured from sterile sites.

La spectrométrie de masse à temps de vol par désorption-ionisation par impact laser assisté par matrice (MALDI-TOF MS) peut être utilisée pour identifier les bactéries directement dans le sang et les cultures positives de liquide stérile. Les auteurs ont évalué une trousse commerciale, la Sepsityper Kit (Bruker Daltonik, Allemagne), et la MALDI-TOF MS pour identifier rapidement des organismes dans 80 bouillons d'hémoculture signalés comme positifs, dont 73 (91,2 %) étaient des échantillons d'hémoculture et sept (8,7 %), des échantillons de liquide céphalorachidien, par rapport aux méthodes d'identification classiques. Les chercheurs ont obtenu la bonne identification du genre et de l'espèce dans 75 des 80 (93,8 %) et 39 des 50 (78 %) bouillons d'hémoculture, respectivement. La mise en application du module d'analyse d'hémoculture, un nouvel outil informatique, a fait passer l'identification des espèces d'organismes Gram négatif de 94,7 % à 100 % et des organismes Gram positif de 66,7 % à 70 %. La MALDI-TOF MS est un outil prometteur pour l'identification directe d'organismes cultivés à partir de foyers stériles.

Molecular diagnosis of respiratory virus infections.

The appearance of eight new respiratory viruses, including the SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009, in the human population in the past nine years has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid based amplification tests (NATs) for respiratory viruses were first introduced two decades ago and today are utilized for the detection of both conventional and emerging viruses. These tests are more sensitive than other diagnostic approaches, including virus isolation in cell culture, shell vial culture (SVC), antigen detection by direct fluorescent antibody (DFA) staining, and rapid enzyme immunoassay (EIA), and now form the backbone of clinical virology laboratory testing around the world. NATs not only provide fast, accurate and sensitive detection of respiratory viruses in clinical specimens but also have increased our understanding of the epidemiology of both new emerging viruses such as the pandemic H1N1 influenza virus of 2009, and conventional viruses such as the common cold viruses, including rhinovirus and coronavirus. Multiplex polymerase chain reaction (PCR) assays introduced in the last five years detect up to 19 different viruses in a single test. Several multiplex PCR tests are now commercially available and tests are working their way into clinical laboratories. The final chapter in the evolution of respiratory virus diagnostics has been the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance. These assays are now being multiplexed with primary detection and subtyping assays, especially in the case of influenza virus. These resistance assays, together with viral load assays, will enable clinical laboratories to provide physicians with new and important information for optimal treatment of respiratory virus infections.

Evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses.

The clinical and public health importance of influenza and other respiratory viruses has accelerated the development of highly sensitive molecular diagnostics, but data are limited regarding preanalytical stages of diagnostic testing. We evaluated CyMol, an alcohol-based transport medium, for its ability to maintain specimen integrity for up to 21 days of storage at various temperatures; for its ability to inactivate virus; and for its compatibility with antigen- or nucleic acid-based diagnostics for respiratory viruses in clinical samples. In mock-infected samples, both universal transport medium (UTM-RT) and CyMol maintained equivalent viral quantities for at least 14 days at room temperature or colder, whereas a dry swab collection maintained viral quantities only if refrigerated or frozen. CyMol inactivated influenza virus within 5 min of sample immersion. UTM-RT- and CyMol-collected nasal swab specimens from 73 symptomatic students attending a campus health clinic were positive for a respiratory virus in 56.2% of subjects by multiplex PCR testing, including influenza A and B viruses, rhinovirus/enteroviruses, coronaviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and adenovirus. Detection by PCR was equivalent in UTM-RT- and CyMol-collected specimens and in self- and staff-collected swabs. Direct fluorescent antibody (DFA) testing was substantially less sensitive (23.3%) than multiplex PCR, and DFA testing from UTM-RT-collected swabs was more sensitive than that from CyMol-collected swabs. These data indicate that an alcohol-based transport medium such as CyMol preserves respiratory virus integrity, rapidly inactivates viruses, and is compatible with PCR-based respiratory diagnostics.

Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories.

BACKGROUND: Noroviruses (NoVs) are the leading cause of infectious gastroenteritis worldwide. Real-time reverse transcription PCR (real-time RT-PCR) is the preferred method of NoV detection for the majority of testing laboratories. Although the accepted target region for molecular detection assays is the conserved ORF1/ORF2 junction, multiple variations have been published with differences in primers, probes, reagents, multiplexing, etc. OBJECTIVES: We assessed the detection limit for GII.4 NoV real-time RT-PCR assays as well as the ability to detect the non-GII.4 NoV genotypes in each participating laboratory. STUDY DESIGN: A panel of 25 RNA samples was circulated to 18 testing laboratories for comparison of their real-time RT-PCR procedures for NoV detection. RESULTS: Multiple protocols with slight differences in reagents or conditions successfully detected 10 genome equivalents or fewer of NoV per reaction. Multiplex procedures were significantly associated (p=0.04) with false negative results, particularly for a GI.2 strain. Sensitive detection was associated with false positive results (p=0.03). CONCLUSIONS: Overall, the data indicate that comparable results are produced under slightly different assay conditions.

Development of a novel bead-based multiplex PCR assay for combined subtyping and oseltamivir resistance genotyping (H275Y) of seasonal and pandemic H1N1 influenza A viruses.

BACKGROUND: The identification of influenza A virus subtypes in clinical specimens is becoming increasingly important for clinical laboratories since seasonal H1N1, H3N2 and pandemic H1N1 influenza A viruses can have defined antiviral resistance patterns and subtyping can be used as a surrogate for antiviral resistance testing. OBJECTIVES: To develop a novel multiplex PCR (M-PCR) assay for the combined identification of influenza A subtype and oseltamivir resistance (H275Y) genotype in a combined assay format using Luminex xMAP™ technology. STUDY DESIGN: The M-PCR assay employed five degenerate primers to amplify the hemagglutinin (HA) and neuraminidase (NA) genes and eight tagged primers in a target specific primer extension reaction (TSPE). Products were analysed using xTAG™ beads containing specific anti-tag oligonucleotides. RESULTS: M-PCR correctly identified the subtype for 54/54 specimens that were influenza A positive, including 13/13 seasonal H3N2, 17/17 seasonal H1N1 and 24/24 pandemic H1N1 for both HA and NA genes. For oseltamivir resistance the M-PCR assay correctly identified 41/41 H1N1 viruses as oseltamivir sensitive (H275) or resistant (H275Y). Analysis of sequential specimens from two immunocompromised patients revealed the appearance of the H275Y allele at earlier time points after infection compared with Sanger sequencing. CONCLUSIONS: The combined M-PCR assay correctly subtyped seasonal and pandemic influenza A viruses and accurately detected the H275Y oseltamivir resistance allele. This assay should provide useful information to clinicians for appropriate patient management.

Development and evaluation of a flocked nasal midturbinate swab for self-collection in respiratory virus infection diagnostic testing.

We developed and evaluated flocked nasal midturbinate swabs obtained from 55 asymptomatic and 108 symptomatic volunteers. Self-collected swabs obtained from asymptomatic volunteers yielded numbers of respiratory epithelial cells comparable to those of staff-collected nasal (n = 55) or nasopharyngeal (n = 20) swabs. Specific viruses were detected in swabs self-collected by 42/108 (38.9%) symptomatic volunteers by multiplex PCR.

Multiplex PCR tests sentinel the appearance of pandemic influenza viruses including H1N1 swine influenza.

BACKGROUND: Since the turn of the century seven new respiratory viruses have infected man and two of these have resulted in worldwide epidemics. Both SARS Coronavirus which quickly spread to 29 countries in February 2003 and H1N1 swine influenza that recently spread from Mexico to 30 countries in three weeks represent major pandemic threats for mankind. Diagnostic assays are required to detect novel influenza strains with pandemic potential. OBJECTIVE: In this report we evaluate the ability of a multiplex PCR test (xTAG RVP) to detect new, "non-seasonal" influenza viruses including the H1N1 swine influenza A/swine/California/04/2009. STUDY DESIGN: Laboratory based study using retrospective and prospective specimens. RESULTS: This multiplex PCR test detected the present of non-seasonal (non-H1, non-H3) influenza in 20 of 20 patients infected with H1N1 swine flu virus. In addition to detecting the current swine flu the xTAG RVP test detected the H5N1 A/Vietnam/1203/2004 high pathogenicity avian influenza virus that circulated in South East Asia in 2003 as well as 17 out of 17 influenza A viruses representing 11 HA subtypes isolated from birds, swine and horses not yet seen in the human population. CONCLUSION: Based on these results we believe that this molecular test can perform an important role as a sentinel test to detect novel non-seasonal influenza A viruses in patients presenting with influenza-like illness (ILI) and therefore act as an early warning system for the detection of future pandemic influenza threats.

Comparison of in-house and commercial 16S rRNA sequencing with high-performance liquid chromatography and genotype AS and CM for identification of nontuberculous mycobacteria.

Sequencing of the 16S gene or other targets and line probe assay are in wide use for the identification of nontuberculous mycobacteria. We compared in-house and commercial sequencing with 3 sequence databases against high-performance liquid chromatography (HPLC) and line probe assay (HAIN Genotype AS and CM) for the identification of 84 reference, clinical, and unique strains representing 41 species. Consensus of methods was used as reference standard. Sequencing identification was more specific and flexible than HPLC, but it was limited by database content and quality as well as fragment length. No one database satisfied all requirements. In-house sequencing was lower in cost than commercial sequencing or line probe assay.

Comparison of the RIDASCREEN norovirus enzyme immunoassay to IDEIA NLV GI/GII by testing stools also assayed by RT-PCR and electron microscopy.

RIDASCREEN norovirus enzyme immunoassay (EIA) detected 80.3% of norovirus-infected feces samples compared to 60.6% by IDEIA NLV GI/GII from 228 patients with no false positives by either assay. RT-PCR and electron microscopy percent sensitivity and specificity were 98.5, 100 and 36.4 and 96.9, respectively.