Astrid Petrich

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.

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.

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.

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.

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.

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.

Aches and Pains with a Shocking Rash

CASE PRESENTATION A 10-year-old girl presented to the emergency department with a three-day history of fever and generalized rash. Eleven days earlier, she had been camping in Ontario. Activities while camping included sleeping in a tent, hiking in the forest and swimming in a lake. She sustained multiple mosquito bites that were pruritic and subsequently formed scabs. Ten days before presentation, she developed left hip and thigh pain, which progressed in severity over the next three days. She also developed severe generalized myalgia. Three days before presentation she developed a fever and an erythematous, nonpruritic rash. The rash was first noticed on the thigh but soon involved the upper limbs, palms and soles. There was no sore throat or contact with others with fever or rash. Her general practitioner suspected scarlet fever and she was started on amoxicillin. After no improvement over the following day, she presented to the emergency room. On examination in the emergency department, the patient was febrile and her blood pressure was 100/50 mmHg. She had a generalized erythematous macular rash, bilateral injected conjunctiva, jaundice, peeling of the skin on the soles of her feet and multiple hyperpigmented crusted lesions on her legs. She had generalized myalgia with tenderness in the left hip and thigh region. Over the subsequent 6 h to 12 h, her blood pressure dropped to 85/37 mmHg and she had a peak temperature of 39.2°C. She was treated with fluid resuscitation and antimicrobials. Her white blood cell count was 40.4×109/L with a left shift and her platelet count was 72×109/L. She was in renal failure (serum creatinine level 400 μmol/L) and had conjugated hyperbilirubinemia (serum conjugated bilirubin level 138 μmol/L) and mild transaminitis (alanine aminotransferase level 157 U/L, aspartate aminotransferase level 264 U/L). Ultrasound of the left hip showed no joint effusion. X-rays of the femur and lumbosacral spine were unremarkable. What is the diagnosis?