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Blood viral load in the diagnostic workup of congenital cytomegalovirus infection
Journal Pre-proof Blood Viral Load in the Diagnostic Workup of Congenital Cytomegalovirus Infection Mina Smiljkovic (Conceptualization) (Investigation) (Writing original draft), Jean-Baptiste Le Meur (Methodology)Analysis), Brigitte Malette (Resources) (Investigation), Isabelle Boucoiran (Data curation), ´ erique ´ ´ Anne-Fred Minsart (Investigation), Valerie Lamarre (Data curation), Bruce Tapiero (Methodology), Christian Renaud (Resources) (Investigation), Fatima Kakkar (Conceptualization) (Supervision) (Writing - review and editing)
PII:
S1386-6532(19)30261-6
DOI:
https://doi.org/10.1016/j.jcv.2019.104231
Reference:
JCV 104231
To appear in:
Journal of Clinical Virology
Received Date:
3 September 2019
Revised Date:
22 November 2019
Accepted Date:
28 November 2019
Please cite this article as: Smiljkovic M, Le Meur J-Baptiste, Malette B, Boucoiran I, Minsart ´ erique, ´ A-Fred Lamarre V, Tapiero B, Renaud C, Kakkar F, Blood Viral Load in the Diagnostic Workup of Congenital Cytomegalovirus Infection, Journal of Clinical Virology (2019), doi: https://doi.org/10.1016/j.jcv.2019.104231
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
1 Blood Viral Load in the Diagnostic Workup of Congenital Cytomegalovirus Infection Mina Smiljkovic, MD1, Jean-Baptiste Le Meur MSc3, Brigitte Malette, PhD4, Isabelle Boucoiran MS, MSc5Anne-Frédérique Minsart MD, Msc5 , Valérie Lamarre MD 6, Bruce Tapiero, MD6, Christian Renaud MD, MSc4, Fatima Kakkar, MD, MPH6
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Division of Infectious Diseases, Department of Pediatrics, The Hospital for Sick Children,
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University of Toronto, Toronto, Canada Division of Infectious Diseases, Department of Pediatrics, Children’s Hospital of Eastern
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Ontario, University of Ottawa, Ottawa, Canada
Department of Social and Preventive Medicine, Université Laval, Québec, Canada
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Department of Microbiology, CHU Sainte-Justine, University of Montreal, Montreal, Canada
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Department of Obstetrics and Gynaecology, CHU Sainte-Justine, University of Montreal,
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Montreal, Canada
Division of Infectious Diseases, Department of Pediatrics, CHU Sainte-Justine, University of
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Montreal, Montreal, Canada
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Word count: 2500 words
Corresponding author Dr Fatima Kakkar
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2 Department of Pediatrics, Division of Infectious Diseases CHU Sainte-Justine, 3175 Côte Sainte-Catherine, Montreal, Quebec, Canada Email: [email protected] Tel: 514-345-4931 ext: 6885
Highlights Level of CMV viremia is associated with symptom severity in congenital CMV infection
Moderate to severely symptomatic newborns have a higher baseline viral load
Newborns with neurological involvement have higher viral load at time of diagnosis
No single viral load cutoff distinguishes asymptomatic from symptomatic infants.
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Abstract: Background: There is limited data on the role of cytomegalovirus (CMV) blood quantitative polymerase chain reaction (qPCR) in the diagnostic workup of congenital CMV (cCMV) infection. Objectives: The objective of this study was to determine if CMV blood qPCR at the time diagnosis could differentiate between symptomatic and asymptomatic infants according to the recent consensus classification. Study Design: Retrospective study of children diagnosed with cCMV infection at CHU SainteJustine, Montreal, Canada, between 2008 and 2016. Cases for whom qPCR was done at baseline (<4 weeks of age) alongside a complete diagnostic workup were included. The association between CMV blood viral load (VL) and clinical severity group was determined. The probability of having moderate to severe symptoms was assessed using univariate logistic regression analysis. Results: Forty-seven patients were included in the analysis. Median VL was significantly higher among infants with moderate to severely symptomatic disease vs. those asymptomatic or asymptomatic with isolated sensorineural hearing loss (SNHL) (13 736 vs. 1876 copies/ml, p=0.004), infants with moderate to severe disease or asymptomatic with isolated SNHL vs. asymptomatic (17 736 vs. 1496 copies/ml, p<0.001), and in infants with baseline neurological involvement vs. those without (17 317 vs. 2641 copies/ml, p=0.03). Using logistic regression, an infant would have a > 75% probability of being moderate to severely symptomatic above 18 770 copies/ml, with a threshold of 100 000 copies/ml approaching a 100% probability. Conclusions: Our baseline assessment of CMV blood VL suggests that that the level of CMV viremia correlates with symptom severity.
Keywords: cytomegalovirus; congenital; viral load; blood PCR; diagnosis
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BACKGROUND Congenital cytomegalovirus (cCMV) infection is the leading cause of congenital infection, and a major cause of sensorineural hearing loss (SNHL) and neurodevelopmental delay among children worldwide [1, 2]. In recent years, there have been a number of developments in the diagnosis and treatment of cCMV infection, with increased capacity to diagnose the disease
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using molecular based polymerase chain reaction (PCR) technique [3-5]. However, there are
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diagnostic workup of infants with cCMV infection [6].
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limited data on the role of cytomegalovirus (CMV) blood quantitative PCR (qPCR) in the
The spectrum of presentation of cCMV varies widely, from asymptomatic infection to very
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symptomatic at birth, with important clinical sequelae for both groups[6]. While only 10-15% of infants with cCMV infection have clinical symptoms at birth, symptomatic newborns may
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develop permanent SNHL, vision loss, cerebral palsy, seizures, cognitive and/or motor impairment as well as developmental delay [7, 8]. At the same time, up to 15% of asymptomatic
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newborns develop SNHL or other permanent neurologic sequelae [9]. Treatment with oral valganciclovir for 6 months has been shown to preserve or improve hearing in symptomatic newborns, and may improve neurodevelopmental outcomes [10].
Recent expert consensus
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guidelines have classified cCMV as moderate to severely symptomatic, asymptomatic and asymptomatic with isolated SNHL, for purposes of establishing treatment criteria [11]. Treatment is only currently recommended in moderate-severe cases. However, there is no consensus on the recommended investigations in cases of cCMV infection to determine level of symptomatology in an individual patient, and practice can vary by center and treating physician.
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4 There are currently no recommendations for the use baseline CMV qPCR in the initial assessment of infant with cCMV, with limited and conflicting data on whether CMV viral load (VL) reflects disease severity among symptomatic and asymptomatic infants[4, 9, 12-15]. While treatment initiation is recommended early (within the first month of life) [10] for moderate to severely symptomatic infants, the cascade of care from time of diagnosis to completion of all investigations necessary to determine level of symptoms can be long, resulting in delays in
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treatment initiation, or unnecessary treatment in some cases. Hearing loss detected at birth may take weeks to confirm, with some failed newborn hearing assessments being subsequently
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normal after weeks of follow-up [16]. The time to complete imaging studies, including head
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ultrasound (HUS), Computerized Tomography Scan (CT) or Magnetic Resonance Imaging
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(MRI) may be considerable, with discordant results between modalities [17, 18].
OBJECTIVES:
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Given the challenges in correctly identifying level of symptoms within the first month of life among newborns with cCMV, the objective of this study was to assess whether the level of CMV
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viremia at time of diagnosis could identify moderate to severely symptomatic newborns, according to criteria defined by recent consensus guidelines [11].
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STUDY DESIGN Study Population
This was a retrospective study of children diagnosed with cCMV infection at CHU SainteJustine between 2008 and 2016.
Cases of cCMV infection were identified through the
microbiology laboratory clinical database, based on positive testing by culture or shell vial assay on urine or saliva, or qPCR in blood, urine, saliva, within the first 21 days of life [19]. If the
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5 initial test was qPCR in saliva, confirmatory urine PCR was done to confirm diagnosis. In 2007, blood qPCR for CMV was introduced at the CHU Sainte-Justine laboratory, and its use in the management of cCMV infection was at individual physician discretion. For purposes of this study, patients diagnosed with cCMV infection were included if 1) CMV PCR was done in the blood at baseline (<28 days of life and prior to initiating treatment) and 2) complete baseline information was available on all clinical findings, laboratory results, specialized investigations
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and at least one neuroimaging modality (HUS, CT or MRI) (see standard testing below).
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Approval for the study was obtained from the CHU Sainte-Justine Research Ethics Board.
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Quantitative CMV PCR
Primers and TaqMan® MGB Hydrolysis probes were designed for the UL83 gene of CMV
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AD169 strain with internal control, the Arabidopsis thaliana CSG4 gene, using Primer express 2.0. The analytical limit of detection of this method is 200 copies per ml. (See supplementary
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material for full details on the extraction methods, primers and probes).
Audiology
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Standard testing for cases of cCMV infection
As part of the Quebec universal newborn screening program (Programme québecois de
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dépistage de la surdité chez les nouveau-nés), all infants were tested using a combined protocol of automated distorsion product otoacoustic emissions (DPOAE-A) and automated auditory brainstem response (A-ABR), followed by brainstem auditory evoked potentials by three weeks of age. Although definitions of SNHL vary in the literature from 20 to 40 decibels (dB) unilateral or bilateral threshold of hearing loss [1], at our center SNHL was defined as a unilateral or bilateral hearing threshold of >40 dB for at least 2 of the frequencies tested.
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Ophthalmology At the time of diagnosis, all infants underwent formal ophthalmological examination by a pediatric ophthalmologist. In the presence of any abnormal or uncertain findings, follow-up evaluations were conducted as clinically indicated.
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Neurological and Neuroimaging Evaluation Baseline neurological evaluation included physical examination by paediatrician or neurologist.
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The presence of microcephaly, seizures or hypotonia were listed. The findings of any
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neuroimaging (Head US, MRI or CT) were recorded for all infants. Any of the following was considered abnormal neuroimaging: intracranial calcifications, lenticulostriate vasculopathy,
Laboratory Testing
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and abnormal hyperintensity signals.
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white matter abnormalities, ventriculomegaly, defects of migration, periventricular leukomalacia,
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Baseline blood tests for all infants included complete blood count (CBC) and differential, alanine aminotransferase (ALT), bilirubin (total and direct). Lumbar puncture and cerebrospinal fluid (CSF) analyses, including CMV qPCR on CSF, were performed at individual physician
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discretion, and not routinely done in the diagnostic work-up for cCMV.
Clinical Definitions Cases were classified as “moderately to severely symptomatic” according to consensus guidelines[11] if they had multiple manifestations attributable to cCMV infection from among: thrombocytopenia, petechiae, hepatomegaly, splenomegaly, raised ALT or bilirubin, systemic
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7 signs such as intrauterine growth restriction (IUGR), or central nervous system involvement such as microcephaly, radiographic abnormalities consistent with CMV central nervous system disease as described above, chorioretinitis, SNHL, abnormal CSF indices for age in the absence of another cause, or the detection of CMV DNA in CSF. Patients were classified as “mildly symptomatic” if they had up to 2 isolated manifestations of cCMV infection that were mild and transient, such as mild hepatomegaly, a single measurement of low platelet count, or raised
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levels of alanine aminotransferase. Patients were categorized as “asymptomatic cCMV infection with isolated SNHL” if they had confirmed SNHL without other clinical or laboratory or
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imaging manifestation of cCMV infection. Children were considered to have “asymptomatic
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cCMV infection” if there were no manifestations of cCMV disease including normal hearing. Finally, patients were categorized as having “neurological involvement” at baseline if they had
Statistical Analysis
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retinal scarring or optic atrophy).
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any abnormal findings on neuroimaging, neurological exam, or eye involvement (chorioretinitis,
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Infants who underwent all of the above investigations were categorized into each of the 4 groups according to criteria described above (moderate to severely symptomatic, mildly symptomatic, asymptomatic with isolated SNHL hearing loss, and asymptomatic) and median viral load (VL)
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was compared between the groups. Given that equipoise exists regarding the need to treat asymptomatic patients with isolated SNHL[11, 20], we conducted separate analyses according to 1) “Consensus criteria” (treatment for moderate to severely symptomatic vs. no treatment for mildly symptomatic, asymptomatic, or asymptomatic with isolated SNHL) 2) “Expanded criteria” that included SNHL as a manifestation of moderate to severe symptoms for treatment purposes, and 3) Comparing infants with and without neurological involvement. Baseline VL
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8 was compared between infants according to each of these criteria using Wilcoxon rank-sum tests, and results plotted. The probability of having moderate to severe symptoms according to each criteria was determined using univariate logistic regression, with baseline VL as predictor.
RESULTS Demographic and Baseline Characteristics (Table 1)
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Among 57 cases of cCMV diagnosed during the study period, 47 were included in the analysis. Ten patients were excluded because baseline VL was not available within the first 28 days of
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life. Baseline VL was done at a median of 6 days (IQR 3-12 days). Reasons for testing for
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CMV in the newborns included symptoms suggestive of CMV infection (24 cases, 51%), suspected primary infection during pregnancy in 16 cases (34%), failed newborn hearing
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screening test in 5 cases (10.6%), and targeted screening of infants of human immunodeficiency
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virus (HIV) infected mothers in 2 cases (4.3%).
All patients completed standard testing as described above. Seventy-four percent of cases had at
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least one symptom suggestive of cCMV infection (n=35). The most common clinical findings were evidence of CNS involvement (microcephaly, seizures and brain abnormalities on neuroimaging) (n=24, 52.1%), followed by IUGR (n=18, 38.3%) and SNHL (n=18, 38.3%).
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Other clinical findings included thrombocytopenia (n=17, 36.3%), neonatal jaundice (n=10, 21.3%), petechiae or purpura (n=10, 21.3%), and hepatitis (n=9, 19.1%), hepatomegaly or splenomegaly (n=6, 12.8%), and lethargy, hypotonia or poor feeding (n=5, 10.6%). Pneumonitis and eye abnormalities were less frequent affecting only 2 (4.3%) and 1 (2.1%) patient respectively.
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9 Using consensus classification criteria, 31 (66%) of newborns were categorized as moderateseverely symptomatic, none as mildly symptomatic, 4 (8.5%) asymptomatic with isolated hearing loss, and 12 (26%) completely asymptomatic. Asymptomatic infants were identified due to targeted screening of HIV exposed infants (n=2), or due to proven or suspected primary infection during pregnancy (n=10). All 47 patients had baseline VL above the level of detection (>200 copies/ml). Median baseline VL in the moderate to severely symptomatic group was
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13736 copies/ml (IQR 9917- 187999) vs. 1876 copies/ml (IQR 835-17713) in the asymptomatic or asymptomatic with isolated SNHL group, p<0.004) (Figure 1a). When infants with isolated
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SNHL were included within the expanded definition of moderate to severe symptoms, median
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VL was again significantly higher among those with moderate-severe symptoms (VL 19360, IQR 3890-83951 copies/ml) vs. those asymptomatic (1496 copies/ml, IQR 835-2812 copies/ml)
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p<0.001 (Figure 1b). Among newborns with any abnormal neurological findings, median viral load was again significantly higher than those without (17 317, IQR 3467-80372 copies/ml vs.
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2461, IQR 1148-15796 copies/m, p=0.03) (Figure 1c).
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The likelihood of having moderate-to severe symptoms according to VL are presented in Figures 2a (for consensus criteria), 2b (expanded criteria), and 2c (neurological involvement). In all 3 scenarios, baseline VL in copies/ml was a predictor of symptom severity (p<0.02 for consensus
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criteria, p<0.006 for expanded treatment criteria and p=0.005 for neurological involvement). According to consensus guidelines, a patient at our center would have a > 75% probability of presenting moderate to severe symptoms above 18 770 copies/ml according to consensus criteria, 5 378 copies/ml according to the expanded criteria, and 43 815 copies/ml when considering neurological symptoms. In all three scenarios, a threshold of 100 000 copies/ml approached a near 100% probability of meeting symptomatic criteria.
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DISCUSSION Despite the significant morbidity and public health burden associated with cCMV disease, there are limited data on the role of blood qPCR in the diagnostic workup of cCMV infection, and mixed data on whether baseline VL can predict disease severity and long-term outcomes. While we have not yet assessed long-term outcomes among these patients, our baseline assessment of
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CMV blood viral load suggests that a quantitative measure of viremia is associated with symptom severity at birth. Our results demonstrate differences in baseline qPCR level among
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those with moderate to severe symptoms (whether including those with SNHL or not), and
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among those with evidence of neurological involvement. This is consistent with a previous study which found an association between baseline viral load and neurological involvement [13],
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though differs from another study which did not find an association between viral load and the presence of hearing loss at birth [15]. We suspect that these differences are due to different
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outcome measures. Where previous studies have assessed the association between a single symptom and viral load, these have not reached statistical significance [14, 15], however, where
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studies have considered the totality of symptoms, differences in viral load have been demonstrated [9,12,13].
While it is not possible to establish a clear viral load cut-off between
symptomatic and asymptomatic infants, as this will vary depending on the CMV assay used and
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the investigations conducted to determine level of symptoms, in our series, the probability of being moderate to severely symptomatic approached 100% among those with a baseline level above 100 000 copies/ml.
Between 1000 and 10 000 copies/ml, the probability of being
moderate to severely symptomatic increased in a linear fashion such that there is no clear cut-off.
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11 These results suggest that baseline qPCR could contribute to the initial evaluation of infants with congenital CMV infection, pending results of the complete diagnostic workup.
The rapid
turnaround time for qPCR results could allow for effective triage and assessment of patients prior to the availability of the final results on full diagnostic evaluation (confirmatory hearing or neuroimaging). Initial non-specific findings on HUS such as lenticulostriate vasculopathy and cystic lesions may subsequently be normal on MRI [18], while failed newborn hearing screens
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may be normal on confirmatory testing, which may take weeks to perform. In these instances, a low level CMV PCR in the blood may help avoid unnecessary treatment of infants based on This could also have
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preliminary findings on neuroimaging and hearing assessments.
While the
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implications for universal screening programs using dried blood spots (DBS).
sensitivity and specificity of the DBS for cCMV infection is not clear (ranging from 28-100%)
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[21-26], the sensitivity increases with increasing blood viral load [27, 28]. Depending on the sensitivity of the DBS assay used in a screening program, it could potential identify only the
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most symptomatic cases of cCMV infection with highest blood viral loads.
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Our study has a number of limitations, including its retrospective design, and the lack of a standardized assay for qPCR across laboratories. The identified thresholds of 10 and 100 000 copies/ml may not be applicable across laboratories and centers [29]. However, as is the case for
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treatment criteria for immunocompromised patients with CMV disease, it is possible that each center establishes its local thresholds based upon its qPCR technology and applies this to cCMV infection [30]. Furthermore, in the absence of a systematic screening program, our patient population comprises mainly symptomatic infants and children at risk of infection and is likely not representative of all cCMV cases. None of the infants in our study were classified as mildly symptomatic, while 26% were asymptomatic. Finally, while our definition of baseline was a
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12 positive CMV PCR measured in blood within the first 28 days of life, it is unclear if time elapsed between in utero infection and blood qPCR testing affects the VL at diagnosis, or how viral load evolves over the first weeks of life in the absence of antiviral treatment.
In summary, these results demonstrate a higher baseline VL within the first month of life was present among moderate to severely symptomatic infants with cCMV infection, as compared to
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those asymptomatic. The role of viral burden in cCMV-infected infants should be further investigated in prospective studies to better understand viral pathogenicity, predictors of long-
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term outcome, and response to therapy.
CREDIT Authorship Statement
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Mina Smiljkovic: Conceptualization, Investigation, Writing-original draft; Jean- Baptiste Le Meur: Methodology, Analysis; Brigitte Malette: Resources, investigation; Isabelle Boucoiran:
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Data curation; Anne Frederique Minsart: Investigation; Valerie Lamarre: Data curation; B.T: Methodology; Christian Renaud: Resources, investigation; Fatima Kakkar:
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Conceptualization, Supervision, Writing – review and editing. All authors contributed to interpreting the data, critically revising the manuscript, and approved the final version.
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Funding: This work was supported by a clinician investigator and infrastructure grant from the Fonds de Recherche Santé Québec (FRQS) to FK and IB.
ACKNOWLEDGEMENTS/ CONFLICTS OF INTEREST We thank Dorothée Leduc, Doris G Ransy, Hugo Soudeyns, Suzanne Taillefer, Silvie Valois and Nathalie Pichette for their work with the Centre d’Infectiologie Mère-Enfant (CIME) cohort, and
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13 all members of the CIME clinical and research teams.
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All authors declare no conflicts of interest.
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22 Table 1. Characteristics of Children Diagnosed with cCMV Infection (n = 47) n (%) 38 (80.9) 7 (14.9) 2 (4.3)
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16 (34) 5 (10.6) 2 (4.3) 24 (51.1) 25 (53.2) 14 (29.8) 8 (17.0)
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Baseline characteristics Gestational age (weeks) >=37 32-37 <32 Screening indication Maternal seroconversion Failed hearing test HIV infected mother Symptomatic infant Baseline blood CMV qPCR (copies/mL) < 10 000 10 000 - 100 000 > 100 000 Degree of symptoms Moderate-severe Mild Asymptomatic with isolated hearing loss Asymptomatic Identified clinical features Intrauterine growth retardation Microcephaly, seizures, and brain abnormalities on neuroimaging Lethargy, hypotonia or poor feeding Thrombocytopenia (platelet count <100 000/mm3) Petechiae or purpura Neonatal jaundice (direct bilirubin >50 μmol/L) Hepatitis (alanine aminotranferase >80 U/L) Hepatomegaly or splenomegaly Pneumonitis Chorioretinitis, retinal scarring or optic atrophy Sensorineural hearing loss
18 (38.3) 24 (52.1) 5 (10.6) 17 (36.3) 10 (21.3) 10 (21.3) 9 (19.1) 6 (12.8) 2 (4.3) 1(2.1) 18 (38.3)
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31 (66) 0 (0) 4 (8.5) 12 (25.8)
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PII:
S1386-6532(19)30261-6
DOI:
https://doi.org/10.1016/j.jcv.2019.104231
Reference:
JCV 104231
To appear in:
Journal of Clinical Virology
Received Date:
3 September 2019
Revised Date:
22 November 2019
Accepted Date:
28 November 2019
Please cite this article as: Smiljkovic M, Le Meur J-Baptiste, Malette B, Boucoiran I, Minsart ´ erique, ´ A-Fred Lamarre V, Tapiero B, Renaud C, Kakkar F, Blood Viral Load in the Diagnostic Workup of Congenital Cytomegalovirus Infection, Journal of Clinical Virology (2019), doi: https://doi.org/10.1016/j.jcv.2019.104231
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
1 Blood Viral Load in the Diagnostic Workup of Congenital Cytomegalovirus Infection Mina Smiljkovic, MD1, Jean-Baptiste Le Meur MSc3, Brigitte Malette, PhD4, Isabelle Boucoiran MS, MSc5Anne-Frédérique Minsart MD, Msc5 , Valérie Lamarre MD 6, Bruce Tapiero, MD6, Christian Renaud MD, MSc4, Fatima Kakkar, MD, MPH6
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Division of Infectious Diseases, Department of Pediatrics, The Hospital for Sick Children,
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University of Toronto, Toronto, Canada Division of Infectious Diseases, Department of Pediatrics, Children’s Hospital of Eastern
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Ontario, University of Ottawa, Ottawa, Canada
Department of Social and Preventive Medicine, Université Laval, Québec, Canada
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Department of Microbiology, CHU Sainte-Justine, University of Montreal, Montreal, Canada
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Department of Obstetrics and Gynaecology, CHU Sainte-Justine, University of Montreal,
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Montreal, Canada
Division of Infectious Diseases, Department of Pediatrics, CHU Sainte-Justine, University of
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Montreal, Montreal, Canada
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Word count: 2500 words
Corresponding author Dr Fatima Kakkar
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2 Department of Pediatrics, Division of Infectious Diseases CHU Sainte-Justine, 3175 Côte Sainte-Catherine, Montreal, Quebec, Canada Email: [email protected] Tel: 514-345-4931 ext: 6885
Highlights Level of CMV viremia is associated with symptom severity in congenital CMV infection
Moderate to severely symptomatic newborns have a higher baseline viral load
Newborns with neurological involvement have higher viral load at time of diagnosis
No single viral load cutoff distinguishes asymptomatic from symptomatic infants.
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Abstract: Background: There is limited data on the role of cytomegalovirus (CMV) blood quantitative polymerase chain reaction (qPCR) in the diagnostic workup of congenital CMV (cCMV) infection. Objectives: The objective of this study was to determine if CMV blood qPCR at the time diagnosis could differentiate between symptomatic and asymptomatic infants according to the recent consensus classification. Study Design: Retrospective study of children diagnosed with cCMV infection at CHU SainteJustine, Montreal, Canada, between 2008 and 2016. Cases for whom qPCR was done at baseline (<4 weeks of age) alongside a complete diagnostic workup were included. The association between CMV blood viral load (VL) and clinical severity group was determined. The probability of having moderate to severe symptoms was assessed using univariate logistic regression analysis. Results: Forty-seven patients were included in the analysis. Median VL was significantly higher among infants with moderate to severely symptomatic disease vs. those asymptomatic or asymptomatic with isolated sensorineural hearing loss (SNHL) (13 736 vs. 1876 copies/ml, p=0.004), infants with moderate to severe disease or asymptomatic with isolated SNHL vs. asymptomatic (17 736 vs. 1496 copies/ml, p<0.001), and in infants with baseline neurological involvement vs. those without (17 317 vs. 2641 copies/ml, p=0.03). Using logistic regression, an infant would have a > 75% probability of being moderate to severely symptomatic above 18 770 copies/ml, with a threshold of 100 000 copies/ml approaching a 100% probability. Conclusions: Our baseline assessment of CMV blood VL suggests that that the level of CMV viremia correlates with symptom severity.
Keywords: cytomegalovirus; congenital; viral load; blood PCR; diagnosis
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BACKGROUND Congenital cytomegalovirus (cCMV) infection is the leading cause of congenital infection, and a major cause of sensorineural hearing loss (SNHL) and neurodevelopmental delay among children worldwide [1, 2]. In recent years, there have been a number of developments in the diagnosis and treatment of cCMV infection, with increased capacity to diagnose the disease
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using molecular based polymerase chain reaction (PCR) technique [3-5]. However, there are
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diagnostic workup of infants with cCMV infection [6].
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limited data on the role of cytomegalovirus (CMV) blood quantitative PCR (qPCR) in the
The spectrum of presentation of cCMV varies widely, from asymptomatic infection to very
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symptomatic at birth, with important clinical sequelae for both groups[6]. While only 10-15% of infants with cCMV infection have clinical symptoms at birth, symptomatic newborns may
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develop permanent SNHL, vision loss, cerebral palsy, seizures, cognitive and/or motor impairment as well as developmental delay [7, 8]. At the same time, up to 15% of asymptomatic
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newborns develop SNHL or other permanent neurologic sequelae [9]. Treatment with oral valganciclovir for 6 months has been shown to preserve or improve hearing in symptomatic newborns, and may improve neurodevelopmental outcomes [10].
Recent expert consensus
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guidelines have classified cCMV as moderate to severely symptomatic, asymptomatic and asymptomatic with isolated SNHL, for purposes of establishing treatment criteria [11]. Treatment is only currently recommended in moderate-severe cases. However, there is no consensus on the recommended investigations in cases of cCMV infection to determine level of symptomatology in an individual patient, and practice can vary by center and treating physician.
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4 There are currently no recommendations for the use baseline CMV qPCR in the initial assessment of infant with cCMV, with limited and conflicting data on whether CMV viral load (VL) reflects disease severity among symptomatic and asymptomatic infants[4, 9, 12-15]. While treatment initiation is recommended early (within the first month of life) [10] for moderate to severely symptomatic infants, the cascade of care from time of diagnosis to completion of all investigations necessary to determine level of symptoms can be long, resulting in delays in
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treatment initiation, or unnecessary treatment in some cases. Hearing loss detected at birth may take weeks to confirm, with some failed newborn hearing assessments being subsequently
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normal after weeks of follow-up [16]. The time to complete imaging studies, including head
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ultrasound (HUS), Computerized Tomography Scan (CT) or Magnetic Resonance Imaging
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(MRI) may be considerable, with discordant results between modalities [17, 18].
OBJECTIVES:
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Given the challenges in correctly identifying level of symptoms within the first month of life among newborns with cCMV, the objective of this study was to assess whether the level of CMV
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viremia at time of diagnosis could identify moderate to severely symptomatic newborns, according to criteria defined by recent consensus guidelines [11].
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STUDY DESIGN Study Population
This was a retrospective study of children diagnosed with cCMV infection at CHU SainteJustine between 2008 and 2016.
Cases of cCMV infection were identified through the
microbiology laboratory clinical database, based on positive testing by culture or shell vial assay on urine or saliva, or qPCR in blood, urine, saliva, within the first 21 days of life [19]. If the
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5 initial test was qPCR in saliva, confirmatory urine PCR was done to confirm diagnosis. In 2007, blood qPCR for CMV was introduced at the CHU Sainte-Justine laboratory, and its use in the management of cCMV infection was at individual physician discretion. For purposes of this study, patients diagnosed with cCMV infection were included if 1) CMV PCR was done in the blood at baseline (<28 days of life and prior to initiating treatment) and 2) complete baseline information was available on all clinical findings, laboratory results, specialized investigations
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and at least one neuroimaging modality (HUS, CT or MRI) (see standard testing below).
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Approval for the study was obtained from the CHU Sainte-Justine Research Ethics Board.
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Quantitative CMV PCR
Primers and TaqMan® MGB Hydrolysis probes were designed for the UL83 gene of CMV
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AD169 strain with internal control, the Arabidopsis thaliana CSG4 gene, using Primer express 2.0. The analytical limit of detection of this method is 200 copies per ml. (See supplementary
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material for full details on the extraction methods, primers and probes).
Audiology
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Standard testing for cases of cCMV infection
As part of the Quebec universal newborn screening program (Programme québecois de
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dépistage de la surdité chez les nouveau-nés), all infants were tested using a combined protocol of automated distorsion product otoacoustic emissions (DPOAE-A) and automated auditory brainstem response (A-ABR), followed by brainstem auditory evoked potentials by three weeks of age. Although definitions of SNHL vary in the literature from 20 to 40 decibels (dB) unilateral or bilateral threshold of hearing loss [1], at our center SNHL was defined as a unilateral or bilateral hearing threshold of >40 dB for at least 2 of the frequencies tested.
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Ophthalmology At the time of diagnosis, all infants underwent formal ophthalmological examination by a pediatric ophthalmologist. In the presence of any abnormal or uncertain findings, follow-up evaluations were conducted as clinically indicated.
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Neurological and Neuroimaging Evaluation Baseline neurological evaluation included physical examination by paediatrician or neurologist.
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The presence of microcephaly, seizures or hypotonia were listed. The findings of any
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neuroimaging (Head US, MRI or CT) were recorded for all infants. Any of the following was considered abnormal neuroimaging: intracranial calcifications, lenticulostriate vasculopathy,
Laboratory Testing
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and abnormal hyperintensity signals.
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white matter abnormalities, ventriculomegaly, defects of migration, periventricular leukomalacia,
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Baseline blood tests for all infants included complete blood count (CBC) and differential, alanine aminotransferase (ALT), bilirubin (total and direct). Lumbar puncture and cerebrospinal fluid (CSF) analyses, including CMV qPCR on CSF, were performed at individual physician
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discretion, and not routinely done in the diagnostic work-up for cCMV.
Clinical Definitions Cases were classified as “moderately to severely symptomatic” according to consensus guidelines[11] if they had multiple manifestations attributable to cCMV infection from among: thrombocytopenia, petechiae, hepatomegaly, splenomegaly, raised ALT or bilirubin, systemic
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7 signs such as intrauterine growth restriction (IUGR), or central nervous system involvement such as microcephaly, radiographic abnormalities consistent with CMV central nervous system disease as described above, chorioretinitis, SNHL, abnormal CSF indices for age in the absence of another cause, or the detection of CMV DNA in CSF. Patients were classified as “mildly symptomatic” if they had up to 2 isolated manifestations of cCMV infection that were mild and transient, such as mild hepatomegaly, a single measurement of low platelet count, or raised
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levels of alanine aminotransferase. Patients were categorized as “asymptomatic cCMV infection with isolated SNHL” if they had confirmed SNHL without other clinical or laboratory or
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imaging manifestation of cCMV infection. Children were considered to have “asymptomatic
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cCMV infection” if there were no manifestations of cCMV disease including normal hearing. Finally, patients were categorized as having “neurological involvement” at baseline if they had
Statistical Analysis
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retinal scarring or optic atrophy).
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any abnormal findings on neuroimaging, neurological exam, or eye involvement (chorioretinitis,
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Infants who underwent all of the above investigations were categorized into each of the 4 groups according to criteria described above (moderate to severely symptomatic, mildly symptomatic, asymptomatic with isolated SNHL hearing loss, and asymptomatic) and median viral load (VL)
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was compared between the groups. Given that equipoise exists regarding the need to treat asymptomatic patients with isolated SNHL[11, 20], we conducted separate analyses according to 1) “Consensus criteria” (treatment for moderate to severely symptomatic vs. no treatment for mildly symptomatic, asymptomatic, or asymptomatic with isolated SNHL) 2) “Expanded criteria” that included SNHL as a manifestation of moderate to severe symptoms for treatment purposes, and 3) Comparing infants with and without neurological involvement. Baseline VL
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8 was compared between infants according to each of these criteria using Wilcoxon rank-sum tests, and results plotted. The probability of having moderate to severe symptoms according to each criteria was determined using univariate logistic regression, with baseline VL as predictor.
RESULTS Demographic and Baseline Characteristics (Table 1)
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Among 57 cases of cCMV diagnosed during the study period, 47 were included in the analysis. Ten patients were excluded because baseline VL was not available within the first 28 days of
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life. Baseline VL was done at a median of 6 days (IQR 3-12 days). Reasons for testing for
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CMV in the newborns included symptoms suggestive of CMV infection (24 cases, 51%), suspected primary infection during pregnancy in 16 cases (34%), failed newborn hearing
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screening test in 5 cases (10.6%), and targeted screening of infants of human immunodeficiency
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virus (HIV) infected mothers in 2 cases (4.3%).
All patients completed standard testing as described above. Seventy-four percent of cases had at
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least one symptom suggestive of cCMV infection (n=35). The most common clinical findings were evidence of CNS involvement (microcephaly, seizures and brain abnormalities on neuroimaging) (n=24, 52.1%), followed by IUGR (n=18, 38.3%) and SNHL (n=18, 38.3%).
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Other clinical findings included thrombocytopenia (n=17, 36.3%), neonatal jaundice (n=10, 21.3%), petechiae or purpura (n=10, 21.3%), and hepatitis (n=9, 19.1%), hepatomegaly or splenomegaly (n=6, 12.8%), and lethargy, hypotonia or poor feeding (n=5, 10.6%). Pneumonitis and eye abnormalities were less frequent affecting only 2 (4.3%) and 1 (2.1%) patient respectively.
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9 Using consensus classification criteria, 31 (66%) of newborns were categorized as moderateseverely symptomatic, none as mildly symptomatic, 4 (8.5%) asymptomatic with isolated hearing loss, and 12 (26%) completely asymptomatic. Asymptomatic infants were identified due to targeted screening of HIV exposed infants (n=2), or due to proven or suspected primary infection during pregnancy (n=10). All 47 patients had baseline VL above the level of detection (>200 copies/ml). Median baseline VL in the moderate to severely symptomatic group was
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13736 copies/ml (IQR 9917- 187999) vs. 1876 copies/ml (IQR 835-17713) in the asymptomatic or asymptomatic with isolated SNHL group, p<0.004) (Figure 1a). When infants with isolated
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SNHL were included within the expanded definition of moderate to severe symptoms, median
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VL was again significantly higher among those with moderate-severe symptoms (VL 19360, IQR 3890-83951 copies/ml) vs. those asymptomatic (1496 copies/ml, IQR 835-2812 copies/ml)
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p<0.001 (Figure 1b). Among newborns with any abnormal neurological findings, median viral load was again significantly higher than those without (17 317, IQR 3467-80372 copies/ml vs.
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2461, IQR 1148-15796 copies/m, p=0.03) (Figure 1c).
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The likelihood of having moderate-to severe symptoms according to VL are presented in Figures 2a (for consensus criteria), 2b (expanded criteria), and 2c (neurological involvement). In all 3 scenarios, baseline VL in copies/ml was a predictor of symptom severity (p<0.02 for consensus
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criteria, p<0.006 for expanded treatment criteria and p=0.005 for neurological involvement). According to consensus guidelines, a patient at our center would have a > 75% probability of presenting moderate to severe symptoms above 18 770 copies/ml according to consensus criteria, 5 378 copies/ml according to the expanded criteria, and 43 815 copies/ml when considering neurological symptoms. In all three scenarios, a threshold of 100 000 copies/ml approached a near 100% probability of meeting symptomatic criteria.
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DISCUSSION Despite the significant morbidity and public health burden associated with cCMV disease, there are limited data on the role of blood qPCR in the diagnostic workup of cCMV infection, and mixed data on whether baseline VL can predict disease severity and long-term outcomes. While we have not yet assessed long-term outcomes among these patients, our baseline assessment of
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CMV blood viral load suggests that a quantitative measure of viremia is associated with symptom severity at birth. Our results demonstrate differences in baseline qPCR level among
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those with moderate to severe symptoms (whether including those with SNHL or not), and
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among those with evidence of neurological involvement. This is consistent with a previous study which found an association between baseline viral load and neurological involvement [13],
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though differs from another study which did not find an association between viral load and the presence of hearing loss at birth [15]. We suspect that these differences are due to different
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outcome measures. Where previous studies have assessed the association between a single symptom and viral load, these have not reached statistical significance [14, 15], however, where
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studies have considered the totality of symptoms, differences in viral load have been demonstrated [9,12,13].
While it is not possible to establish a clear viral load cut-off between
symptomatic and asymptomatic infants, as this will vary depending on the CMV assay used and
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the investigations conducted to determine level of symptoms, in our series, the probability of being moderate to severely symptomatic approached 100% among those with a baseline level above 100 000 copies/ml.
Between 1000 and 10 000 copies/ml, the probability of being
moderate to severely symptomatic increased in a linear fashion such that there is no clear cut-off.
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11 These results suggest that baseline qPCR could contribute to the initial evaluation of infants with congenital CMV infection, pending results of the complete diagnostic workup.
The rapid
turnaround time for qPCR results could allow for effective triage and assessment of patients prior to the availability of the final results on full diagnostic evaluation (confirmatory hearing or neuroimaging). Initial non-specific findings on HUS such as lenticulostriate vasculopathy and cystic lesions may subsequently be normal on MRI [18], while failed newborn hearing screens
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may be normal on confirmatory testing, which may take weeks to perform. In these instances, a low level CMV PCR in the blood may help avoid unnecessary treatment of infants based on This could also have
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preliminary findings on neuroimaging and hearing assessments.
While the
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implications for universal screening programs using dried blood spots (DBS).
sensitivity and specificity of the DBS for cCMV infection is not clear (ranging from 28-100%)
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[21-26], the sensitivity increases with increasing blood viral load [27, 28]. Depending on the sensitivity of the DBS assay used in a screening program, it could potential identify only the
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most symptomatic cases of cCMV infection with highest blood viral loads.
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Our study has a number of limitations, including its retrospective design, and the lack of a standardized assay for qPCR across laboratories. The identified thresholds of 10 and 100 000 copies/ml may not be applicable across laboratories and centers [29]. However, as is the case for
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treatment criteria for immunocompromised patients with CMV disease, it is possible that each center establishes its local thresholds based upon its qPCR technology and applies this to cCMV infection [30]. Furthermore, in the absence of a systematic screening program, our patient population comprises mainly symptomatic infants and children at risk of infection and is likely not representative of all cCMV cases. None of the infants in our study were classified as mildly symptomatic, while 26% were asymptomatic. Finally, while our definition of baseline was a
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12 positive CMV PCR measured in blood within the first 28 days of life, it is unclear if time elapsed between in utero infection and blood qPCR testing affects the VL at diagnosis, or how viral load evolves over the first weeks of life in the absence of antiviral treatment.
In summary, these results demonstrate a higher baseline VL within the first month of life was present among moderate to severely symptomatic infants with cCMV infection, as compared to
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those asymptomatic. The role of viral burden in cCMV-infected infants should be further investigated in prospective studies to better understand viral pathogenicity, predictors of long-
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term outcome, and response to therapy.
CREDIT Authorship Statement
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Mina Smiljkovic: Conceptualization, Investigation, Writing-original draft; Jean- Baptiste Le Meur: Methodology, Analysis; Brigitte Malette: Resources, investigation; Isabelle Boucoiran:
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Data curation; Anne Frederique Minsart: Investigation; Valerie Lamarre: Data curation; B.T: Methodology; Christian Renaud: Resources, investigation; Fatima Kakkar:
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Conceptualization, Supervision, Writing – review and editing. All authors contributed to interpreting the data, critically revising the manuscript, and approved the final version.
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Funding: This work was supported by a clinician investigator and infrastructure grant from the Fonds de Recherche Santé Québec (FRQS) to FK and IB.
ACKNOWLEDGEMENTS/ CONFLICTS OF INTEREST We thank Dorothée Leduc, Doris G Ransy, Hugo Soudeyns, Suzanne Taillefer, Silvie Valois and Nathalie Pichette for their work with the Centre d’Infectiologie Mère-Enfant (CIME) cohort, and
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13 all members of the CIME clinical and research teams.
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All authors declare no conflicts of interest.
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22 Table 1. Characteristics of Children Diagnosed with cCMV Infection (n = 47) n (%) 38 (80.9) 7 (14.9) 2 (4.3)
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16 (34) 5 (10.6) 2 (4.3) 24 (51.1) 25 (53.2) 14 (29.8) 8 (17.0)
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Baseline characteristics Gestational age (weeks) >=37 32-37 <32 Screening indication Maternal seroconversion Failed hearing test HIV infected mother Symptomatic infant Baseline blood CMV qPCR (copies/mL) < 10 000 10 000 - 100 000 > 100 000 Degree of symptoms Moderate-severe Mild Asymptomatic with isolated hearing loss Asymptomatic Identified clinical features Intrauterine growth retardation Microcephaly, seizures, and brain abnormalities on neuroimaging Lethargy, hypotonia or poor feeding Thrombocytopenia (platelet count <100 000/mm3) Petechiae or purpura Neonatal jaundice (direct bilirubin >50 μmol/L) Hepatitis (alanine aminotranferase >80 U/L) Hepatomegaly or splenomegaly Pneumonitis Chorioretinitis, retinal scarring or optic atrophy Sensorineural hearing loss
18 (38.3) 24 (52.1) 5 (10.6) 17 (36.3) 10 (21.3) 10 (21.3) 9 (19.1) 6 (12.8) 2 (4.3) 1(2.1) 18 (38.3)
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31 (66) 0 (0) 4 (8.5) 12 (25.8)
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