Journal of Clinical Virology 60 (2014) 119–126
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Intrafamilial transmission of human cytomegalovirus (HCMV): Long-term dynamics of epitope-specific antibody response in context of avidity maturation Klaus Hamprecht a,∗,1 , Alfred Lennart Bissinger b,1,2 , Jose Arellano-Galindo a,3 , Katrin Schweinzer a , Xioajing Jiang a,4 , Katharina Göhring a , Elfriede Mikeler a , Gerhard Jahn a a b
Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tuebingen, D-72076 Tuebingen, Germany 1st Medical Department, University Hospital of Tuebingen, 72076 Tuebingen, Germany
a r t i c l e
i n f o
Article history: Received 29 December 2013 Received in revised form 8 March 2014 Accepted 10 March 2014 Keywords: CMV Primary infection Avidity Immunoblot gO sequencing Horizontal transmission
a b s t r a c t Background: The role of a special early family formation (PEKiP® ), which is popular in Germany, as a potential origin of HCMV-transmission to seronegative mothers is not documented. Objectives: To describe the clinical courses, to identify the virological origin and to evaluate a new tool for diagnosis of a cascade of intrafamilial HCMV primary infections. Study design: This prospectively analyzed long-term course of HCMV primary infection leading to hospitalization of two family members, included the evaluation of different IgG/IgM/IgG avidity-assays with an epitope-specific recombinant immunoblot-assay. Additionally, neutralization (NT) assays using fibroblast-and epithelial-target cells were performed to correlate NT50 values to avidity maturation. HCMV gN/gO/gB-RFLP-genotyping and phylogenetic analyses were performed using urine viral isolates. Results: The clinical courses of the sequentially occurring intrafamilial HCMV primary infections were unusual, leading to hospitalization. Long-term-serology of the mother revealed concordant results for an unimodal IgG-course and a rapid decrease of IgM-indices from week 7 to week 21 p.i. Interestingly, the cut-off definitions for low and high avidity ranged discordantly from 15 to 25 weeks, and from 18 to 42 weeks p.i., respectively. A good correlation was found between the increase of fibroblast-adapted NT50 values and the appearance of high avidity using the epitope-specific immunoblot (>18 weeks p.i.). RFLP-genotyping and sequencing could identify the index patient as member of PEKiP® -meetings. Conclusions: PEKiP® -meetings with naked babies may be an important source of horizontal HCMVtransmission to seronegative pregnant mothers in Germany. Using epitope-specific immunoblots, persisting HCMVp150-IgM-reactivities and good concordance between high IgG-avidity and increase of fibroblast adapted neutralization capacity were found. © 2014 Elsevier B.V. All rights reserved.
1. Background
∗ Corresponding author. Tel.: +49 7071 2984657; fax: +49 7071 5790. E-mail address: [email protected] (K. Hamprecht). 1 These authors contributed equally. 2 Present address: Labor Prof. Gisela Enders MVZ, Rosenbergstr. 85, 70193 Stuttgart, Germany. 3 Present address: Dr. Marques 162 Doctores, Delegacion Cuauhtemoc, Mexico, DF 06720, Mexico. 4 Present address: Department of Infectious Disease, Wuhan General Hospital, 627 Wu Luo Road, Wuhan, China. http://dx.doi.org/10.1016/j.jcv.2014.03.006 1386-6532/© 2014 Elsevier B.V. All rights reserved.
The overall HCMV-seroprevalence in pregnant women in Germany was estimated recently at 42% [1], while the rate of HCMV IgG-seroconversion is about 0.5% [2], similar to data from France with 0.46% [3]. No data are available on HCMV-shedding rates of infants in German day care centers. A recent meta-analysis reported on HCMV shedding-prevalence of healthy infants in US day care centers of about 23% [4]. Breastfeeding has a major impact on global HCMVepidemiology and vertical virus transmission, since nearly every seropositive mother reactivates the virus and the mother-toinfant-transmission rate is about 40% [5]. Vertical transmission of
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K. Hamprecht et al. / Journal of Clinical Virology 60 (2014) 119–126
HCMV during pregnancy in low seroprevalence-settings [6] results in rates of congenital HCMV infection of 0.7 of live births [7]. However, beside vertical transmission also horizontal transmission, notably during group day care, plays an important epidemiological role in virus spread [8–11]. A special form of interaction between babies and their parents in Germany is the ‘Prague-Programmefor-Parents-and-Children’ (PEKiP® ) [12] consisting of group-work of parents and their infants from 4 weeks to 12 months post partum, in which up to 65 000 families in Germany, Switzerland and Austria, participate weekly. 2. Objectives The primary aim of this study was to identify the origin of an unusual case series of intrafamilial HCMV-transmissions of a seronegative mother who visited a (PEKiP® )-group weekly with her daughter. Comprehensive long-term-HCMV-monitoring included avidity maturation, epitope-specific recombinant immunoblotting, and neutralization using fibroblasts- and epithelial cells was performed. 3. Study cohort and methods 3.1. Study cohort The term and healthy female infant was born in July 2007. The 9 months old healthy girl was still breastfed from her seronegative mother in 2008, and both visited a PEKiP® -group weekly until early May 2008. The mother planned her second pregnancy in summer 2008. The 53 years old healthy maternal grandmother-I, was also involved in child-care, like the 54 years old paternal grandmother-II. 3.2. Methods 3.2.1. Direct HCMV detection Virus isolation using fibroblast microculture was performed as described previously [13]. DNA was purified by spin columns using QIAmp DNA Blood Mini Kit (Qiagen, Germany) and used for qualitative nested-PCR of the IE1Ex4-target region [13]. 3.2.1.1. HCMV gB-, gN-, gO-genotyping. The variable regions from the viral glycoprotein gene sequences that encode for the proteins UL55, UL73 and UL74, were amplified and sequenced as previously reported [14–16]. Multiple alignments of the sequences using clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) were generated for the clinical isolates from the child, the mother and the grandmother I, as well as for reference strains AD169, Towne, Merlin, Toledo and Davis (Genebank access code: X17403, FJ616285, Y4468942, GU937742 and X99845, respectively) as well as for representative strains of each genotype. The pairwise identities between the isolates were calculated using the Smith Waterman algorithm using Jalview Version 2. The gO-variants were also identified using restriction-fragment-length-polymorphism (RFLP) using HpaII [16]. Additionally, the RFLP-genotyping of HCMV glycoprotein N was performed as described previously using SacI and SalI [15]. 3.2.2. HCMV serology The following commercial tests were used for diagnosis of the HCMV antibody status: “ELISA”-CMV IgG ELISA PKS/CMV IgM-ELA-Test PKS (Medac, Germany); “CLIA”-Liaison CMV IgG/IgG avidity/IgM (DiaSorin, Italy); “ECLIA” Elecsys CMV IgG/IgM/IgG avidity (Roche Diagnostics, Switzerland); “CMIA” Architect CMV IgG/IgM/IgG avidity assay (Abbott, Germany); recomblot CMV IgG/IgM/IgG avidity (Mikrogen, Germany). The recomblot assay
(recIB) detected HCMV-specific antibodies to the following HCMV recombinant proteins: IE1, p150, CM2, p65, gB1, gB2. RecIB-scoring reached from +/− weaker than IgG/IgM-control (negative), to +++, very strong reactivity.
3.2.3. HCMV neutralization Target cells were both, human foreskin-fibroblasts (HFF) and human retinal pigment-epithelial cells (ARPE-19; ATCC-CRL2302/LGC-Standards, Germany). The viral target strain originated from urine of a congenitally infected newborn. The reference sera included an HCMV-specific hyperimmunoglobulin (HIG) (Cytotect® ) and serum-pools (N = 100) from healthy seropositive and seronegative mothers at birth. Each 200 l of the cell-free target virus-passage (50×TCID50 /ml) were preincubated for 90 min at 37 ◦ C and 5%CO2 with 200 l of heat inactivated (30 min 56 ◦ C) serum dilutions from 1:50 to 1:3200 of the maternal serum. Thereafter, each 100 l of the virion-antibody mixtures were added to the different microcultures. After 18 h of incubation, monolayers were fixed and IE1 immunoperoxidase staining followed [13]. The neutralization-titers 50% (NT50 ) were calculated referring to 100% of viral infectivity in seronegative serum pool replica.
4. Results 4.1. Clinical courses and routine diagnostics 4.1.1. Index-patient In April 2008, the 9 month old girl was presented at the pediatrician with fever, flu-like symptoms and increasing hidrosis. Symptoms of an infection of the upper-respiratory tract dominated (Fig. 1a and b) and persisted for about 3 months. Serological testing was performed in July, detecting the presence of HCMV-IgG without IgM (Fig. 1a:1). Semiquantitative viruria was detectable up to November 2009, and DNAuria still 3 months longer. In total, viral DNAuria and viruria were analyzed at 8 different time points. At the end of viruria the infant was 28 months old. Viruria-levels were continuously decreasing. Peak-level of viruria (111 infected fibroblasts/ml of urine) under observation was during October 2008 at the age of 15 months. The viral isolate from observed peak-level of viruria was propagated in vitro and used for sequencing analysis (Fig. 1a:2). Viral DNAload in October 2008 was 1360 copies/ml, and in December 2008 15,000 copies/ml. Interestingly, the infant did not shed HCMV into saliva during the observation.
4.1.2. Mother of the index-patient On 24th June 2008, maternal symptoms started with fever (>39 ◦ C), flu like-symptoms with shivering, lymphadenopathy and symptoms of a pulmonary infection leading to hospitalization after ineffective empiric antibiotic treatment (doxycyclin for 6 days) (Fig. 1a and b). Computed tomography in July 2008 revealed atypical pulmonary infiltration, but no pathogen was detectable in bronchoalveolar lavage (BAL). Ultrasonography revealed hepatosplenomegaly. Laboratory findings presented elevated LDH and CRP, slightly elevated transaminases and slight lymphocytosis in the differential blood count (Fig. 1b). Routine serology revealed detection of HCMV-IgM and -IgG, but negative HCMV-PCR from blood and BAL (Fig. 1a:3–6). Interestingly, in week 25 after onset of symptoms, viral DNAuria was still detectable with >2000 copies/ml (Table 1). Since the onset of symptoms was exactly defined, it was possible to refer all later findings to that date. We assume an incubation time of about 4 weeks, thus exposition might have been in May, which fits well with the potential onset of viral shedding of the index patient.
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Fig. 1. (a) Flow chart of clinical symptoms and HCMV monitoring (gray shaded: periods of clinical symptoms). Infant: 1 – IgG pos., IgM and IgA neg., 2 – urine pos. mother: 3 – IgG and IgM pos., 4 – blood and BAL PCR neg., 5 – IgG and IgM pos., blood PCR neg., 6 – urine pos. grandmother I: 7 – IgG and IgM neg., 8 – IgG and IgM pos., 9 – urine pos. grandmother II: no CMV tests during acute disease. (b) Clinical and laboratory findings in the study cohort during acute CMV infection (n. d.: not done; n. c.: not collectable; + present; −: not present).
Table 1 Longitudinal epitope-specific HCMV rec IgG/IgM/IgG avidity immunoblotting during maternal primary infection. Full blown IgM reactivities in initial serum with persistence of p150 reactivity for 17 months p.i., while HCMV IgG avidity maturation is finished 4 months after primary infection (week 17 after onset of symptoms at 24 June 2008 or week 21 p.i.). Date
16/07/08
29/09/08
23/10/08
17/12/08
02/07/09
15/07/09
11/11/09
Weeks after symptom onset Weeks post infection
3 7
14 18
17 21
25 29
53 57
55 59
72 76
rec IgG IE1 rec IgG IE1 avidity rec IgM IE1
++ n.r. ++
++ + n.r.
+++ ++ n.r.
+++ +++ n.r.
+++ +++ n.r.
+++ +++ n.r.
++ ++ n.r.
rec IgG p150 rec IgG p150 avidity rec IgM p150
+ n.r. +++
++ ++ +
+++ +++ +
+++ +++ +
+++ +++ +
+++ +++ +
+++ +++ +
rec IgG CM2 rec IgG CM2 avidity rec IgM CM2
+++ + +++
+++ ++ +
+++ +++ +/−
+++ +++ +/−
+++ +++ +/−
+++ +++ n.r.
++ ++ n.r.
rec IgG p65 rec IgM p65
+++ +++
+++ +
+++ +
+++ +
+++ +
+++ +
++ +/−
rec IgG gB1 rec IgM gB1
n.r. +
+ n.r.
++ n.r.
++ n.r.
+++ n.r.
+++ n.r.
+++ n.r.
rec IgG gB2 rec IgM gB2
n.r. n.r.
+ n.r.
+ n.r.
+ n.r.
+++ n.r.
+++ n.r.
++ n.r.
CMV DNA blood BAL Urine
Negative Negative
Negative
Positive (38.5 ◦ C), flulike symptoms, fatigue, lymphadenopathy and increasing hidrosis accompanied with tachycardia and arterial hypertension beginning in July 2008. Early serological testing in July 2008 revealed no HCMV-specific IgG/IgM-antibodies (Fig. 1a:7). Due to persistent fever, the patient was hospitalized after ineffective empiric antibiotic treatment (ciprofloxacin for 5 days). In August 2008, X-ray examination of the chest showed no alteration, ultrasonography revealed hepatosplenomegaly. Laboratory findings presented elevated LDH and CRP, transaminases above threshold as well as a lymphocytosis in the differential blood count (Fig. 1b). Remarkably, routine serology in August 2008 detected HCMV-IgGseroconversion (Fig. 1a:8). Recombinant-immunoblotting revealed in October 2008 weak gB2-reactivity, intermediate IgG-avidity, and recIgM-reactivity (data not shown). Therefore, virus infection might have occurred at least three months before in July (Fig. 1a). In October 2008 she shed HCMV into urine, without viral DNAemia (Fig. 1a:9).
4.1.4. Grandmother-II of the index patient The paternal grandmother suffered from a febrile episode with flu-like-symptoms with distinctive fatigue accompanied with tachycardia and arterial hypertension beginning in August 2008. Laboratory-testing showed mild elevation of LDH and CRP without alteration of transaminases. In October (23/10/2013) she visited our institution as outpatient (Fig. 1a and b). She also shed HCMV into urine (21,200 copies/ml; 16 infected fibroblast nuclei/ml; Fig. 1a:9), and showed strong gB2-reactivity in context of intermediate HCMV-recIgG-avidity, and AI of 0.31 using CLIA (data not shown). 4.2. Maternal long-term HCMV serology During initial hospitalization all four IgG-assays showed a rapid increase reaching peak levels around week 18 p.i. Thereafter a decline of IgG-levels was observed at week 21 p.i. followed by four individual discordant kinetics describing slowly increasing IgGvalues. Around week 94 p.i. the IgG-values ranged from 60 U/ml
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Fig. 3. (a) Long-term course of maternal HCMV IgG avidity maturation. The time periods of switches from low to intermediate and high levels of IgG avidity depend strongly on the cut-off definitions for low and high IgG avidity. (b) The time periods of switches from low to intermediate and high levels of IgG avidity depend strongly test-dependent on the cut-off definitions for low and high IgG avidity. (c) Longitudinal course of neutralization capacity dependent from target cell and entry mode (fibroblasts versus epithelial cells). Using ARPE-19 cells it can be demonstrated strikingly, that the NT titers are at least 4-fold higher than in the HFF system. Additionally, already in week 7 high NT capacity can be detected in he ARPE-19-system. Using fibroblasts, a significant rise in NT50 value is documented in week 18 p.i.
(ELISA) to 600 AU/ml (CMIA) (Fig. 2a). In contrast, the HCMV-IgMlevels are concordantly decreasing between weeks 7 and 18 p.i. (Fig. 2b). The results from maternal recHCMV IgG/IgM/IgG avidity-testing are shown over a period of 16 months (72 weeks) after onset of clinical symptoms (Table 1). About 3 weeks after onset of clinical symptoms, specific recIgG-reactivity directed against CM2 and p65 and also IE1 is detectable with high scoring. At that time, the gB1/gB2-IgG-reactivity is not detectable, but recIgM-reactivity is detectable against all recombinant antigens with exception of gB2. Interestingly, the IgM-reactivity pattern is changing rapidly under complete or partial loss of reactivity against IE1, gB1, CM2 (about three months after infection), while reactivity against p150 and p65 is weakly detectable for about 1.5 years p.i. (Table 1: week 76). The maternal HCMV IgG-avidity-maturation was compared to the reduction of the recIB-reactivity for HCMV IE1, p150, and CM2 (Fig. 3a). Low (to intermediate) avidity is detectable using recIB up to 18 weeks p.i., while high avidity is observed after 5 months (21 weeks) p.i. (Fig. 3b; Table 1). Unfortunately, the range of test-specific “high avidity” using four different commercial IgGavidity-assays is >18–42 weeks p.i., while the range of “low avidity” includes the time period from 15 to 25 weeks p.i.
Neutralization assays with HFF-target cells fit well with avidity data of the recIgG-IB. The NT50 value increases in week 18 and remains stable over the whole observation period. The NT50 values using epithelial target cells are already at high levels in week 7 after onset of infection. NT50 RPE-values are 4-times higher than NT50 HFF during week 18–94 p.i. (Fig. 3c). Beside the mother of the index-patient also grandmother-I showed an HCMV primary infection with IgG-seroconversion and low avidity (data not shown). In contrast; grandmother-II did not show IgM-reactivity about 4 months following primary infection in the presence of strong recIgG-gB2-reactivity with intermediate IgG-avidity. 4.3. HCMV genotyping The alignment of the nucleotide sequences that encode for HCMV-gB, -gN (data files for supplemental material) and -gO region (Fig. 4) of the isolates from the child, the mother and the grandmother, showed identity of the HCMV clinical isolates of infant, mother and grandmother I (Fig. 4a–c). Genotyping by sequencing and RFLP analysis revealed a gB1/gO1/gN1-genotype. The percentage of identity of the isolates versus the reference strains Merlin,
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Fig. 4. Analysis of the N-terminal HCMV UL74 gene sequence which encodes the gO envelope protein. (A) Multiple alignment analyzed with clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) of the N-terminal variable region gB. Pairwise analysis with the reference and representative gO genotype strains was performed using Jalview V2 software (http://www.jalview.org). The identities observed were of 99.6% with the gOa1 reference strains and (C) a 100% between the clinical isolates from the child, mother and grandmother I.
Davis and Towne (gB1-genotype) were of 98%, 98% and 97.5% respectively (data not shown), whereas for gN and gO versus AD169 (gN1, gO1a genotype) was of 99.4% and 99.6%, respectively (Fig. 4b), that in conclusion all three virus isolates are identical (Fig. 4c). 5. Discussion The index-patient was breastfed at least 6 months before PEKiP® -meetings. Postnatal HCMV-transmission via breastfeeding from his initially seronegative mother can be clearly excluded. All three adults involved in home-caring of the index-patient developed a symptomatic HCMV-infection (Fig. 1a/b), leading to hospitalization of the mother and grandmother I. Fever and flulike-symptoms dominated, accompanied by pulmonary affection in the mother and cardiac symptoms in the two grandmothers. A cardiac affection due to primary HCMV-infection is a known but rare manifestation [17]. In a cohort of symptomatic HCMVprimary infections, influenza-like symptoms and fever were found mainly, but in 75% of cases pregnant women showed an asymptomatic course of HCMV-primary infection [18]. Prolonged, but spontaneous recovery was observed in all three adult patients, no specific antiviral treatment was given. The time-course suggested a transmission-cascade originating from PEKiP® -meetings (Fig. 1a). The course of maternal HCMV-IgG-longterm-serology shows a relatively good concordance with the rapid and steep initial IgG-increase with peak levels around week 18 (Fig. 2a). For interpretation of quantitative longterm HCMV-IgG-kinetics a strong
limitation exists: data from different assay systems are not comparable [19] in the absence of a WHO standard for unit-definition of HCMV-IgG. HCMV-primary infection is characterized by a steep decrease of IgM-reactivity from peak-level values early after onset of infection to week 18 p.i. All four IgM-assays used with exception of the recIgM-assay showed a completely concordant process of loss of IgM-reactivity (Fig. 2b). Interestingly, the recIB-avidity-assay detected exactly during this period of high IgM-reactivity a low IgGavidity. Recently, high IgM-antibody titer were described as strong predictor of low IgG-avidity [20]. In contrast, low IgM-indices at week 29 p.i. ( 21 weeks p.i. with a short periods of transition between low, and high avidity (Fig. 3b). This fits well with IgM-detection in all four assay systems. However, the main problem is the highly divergent definition of intermediate IgG-avidity in the three other test-systems using avidity indices
K. Hamprecht et al. / Journal of Clinical Virology 60 (2014) 119–126
(CMIA, CLIA, ECLIA). These ranges overlap from week 18 to 42 p.i., depending on the test-system used. Comparison of eight commercial HCMV-IgG-avidity assays showed that in a panel of 198 samples under evaluation none of the samples tested showed identical scoring [23]. Interestingly, the original cut-off values for the VIDAS CMV-IgG Avidity kit (bioMérieux, France) were changed recently for better analytical performance [24]. The recIgG–avidityassay seems to reflect the avidity-maturation in a more restricted time period, which correlates also to the increase of neutralization titers using HFF-target cells (Fig. 3b and c). Therefore high recIB IgG-avidity coincidences with NT50 increase from
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Intrafamilial transmission of human cytomegalovirus (HCMV): Long-term dynamics of epitope-specific antibody response in context of avidity maturation Klaus Hamprecht a,∗,1 , Alfred Lennart Bissinger b,1,2 , Jose Arellano-Galindo a,3 , Katrin Schweinzer a , Xioajing Jiang a,4 , Katharina Göhring a , Elfriede Mikeler a , Gerhard Jahn a a b
Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tuebingen, D-72076 Tuebingen, Germany 1st Medical Department, University Hospital of Tuebingen, 72076 Tuebingen, Germany
a r t i c l e
i n f o
Article history: Received 29 December 2013 Received in revised form 8 March 2014 Accepted 10 March 2014 Keywords: CMV Primary infection Avidity Immunoblot gO sequencing Horizontal transmission
a b s t r a c t Background: The role of a special early family formation (PEKiP® ), which is popular in Germany, as a potential origin of HCMV-transmission to seronegative mothers is not documented. Objectives: To describe the clinical courses, to identify the virological origin and to evaluate a new tool for diagnosis of a cascade of intrafamilial HCMV primary infections. Study design: This prospectively analyzed long-term course of HCMV primary infection leading to hospitalization of two family members, included the evaluation of different IgG/IgM/IgG avidity-assays with an epitope-specific recombinant immunoblot-assay. Additionally, neutralization (NT) assays using fibroblast-and epithelial-target cells were performed to correlate NT50 values to avidity maturation. HCMV gN/gO/gB-RFLP-genotyping and phylogenetic analyses were performed using urine viral isolates. Results: The clinical courses of the sequentially occurring intrafamilial HCMV primary infections were unusual, leading to hospitalization. Long-term-serology of the mother revealed concordant results for an unimodal IgG-course and a rapid decrease of IgM-indices from week 7 to week 21 p.i. Interestingly, the cut-off definitions for low and high avidity ranged discordantly from 15 to 25 weeks, and from 18 to 42 weeks p.i., respectively. A good correlation was found between the increase of fibroblast-adapted NT50 values and the appearance of high avidity using the epitope-specific immunoblot (>18 weeks p.i.). RFLP-genotyping and sequencing could identify the index patient as member of PEKiP® -meetings. Conclusions: PEKiP® -meetings with naked babies may be an important source of horizontal HCMVtransmission to seronegative pregnant mothers in Germany. Using epitope-specific immunoblots, persisting HCMVp150-IgM-reactivities and good concordance between high IgG-avidity and increase of fibroblast adapted neutralization capacity were found. © 2014 Elsevier B.V. All rights reserved.
1. Background
∗ Corresponding author. Tel.: +49 7071 2984657; fax: +49 7071 5790. E-mail address: [email protected] (K. Hamprecht). 1 These authors contributed equally. 2 Present address: Labor Prof. Gisela Enders MVZ, Rosenbergstr. 85, 70193 Stuttgart, Germany. 3 Present address: Dr. Marques 162 Doctores, Delegacion Cuauhtemoc, Mexico, DF 06720, Mexico. 4 Present address: Department of Infectious Disease, Wuhan General Hospital, 627 Wu Luo Road, Wuhan, China. http://dx.doi.org/10.1016/j.jcv.2014.03.006 1386-6532/© 2014 Elsevier B.V. All rights reserved.
The overall HCMV-seroprevalence in pregnant women in Germany was estimated recently at 42% [1], while the rate of HCMV IgG-seroconversion is about 0.5% [2], similar to data from France with 0.46% [3]. No data are available on HCMV-shedding rates of infants in German day care centers. A recent meta-analysis reported on HCMV shedding-prevalence of healthy infants in US day care centers of about 23% [4]. Breastfeeding has a major impact on global HCMVepidemiology and vertical virus transmission, since nearly every seropositive mother reactivates the virus and the mother-toinfant-transmission rate is about 40% [5]. Vertical transmission of
120
K. Hamprecht et al. / Journal of Clinical Virology 60 (2014) 119–126
HCMV during pregnancy in low seroprevalence-settings [6] results in rates of congenital HCMV infection of 0.7 of live births [7]. However, beside vertical transmission also horizontal transmission, notably during group day care, plays an important epidemiological role in virus spread [8–11]. A special form of interaction between babies and their parents in Germany is the ‘Prague-Programmefor-Parents-and-Children’ (PEKiP® ) [12] consisting of group-work of parents and their infants from 4 weeks to 12 months post partum, in which up to 65 000 families in Germany, Switzerland and Austria, participate weekly. 2. Objectives The primary aim of this study was to identify the origin of an unusual case series of intrafamilial HCMV-transmissions of a seronegative mother who visited a (PEKiP® )-group weekly with her daughter. Comprehensive long-term-HCMV-monitoring included avidity maturation, epitope-specific recombinant immunoblotting, and neutralization using fibroblasts- and epithelial cells was performed. 3. Study cohort and methods 3.1. Study cohort The term and healthy female infant was born in July 2007. The 9 months old healthy girl was still breastfed from her seronegative mother in 2008, and both visited a PEKiP® -group weekly until early May 2008. The mother planned her second pregnancy in summer 2008. The 53 years old healthy maternal grandmother-I, was also involved in child-care, like the 54 years old paternal grandmother-II. 3.2. Methods 3.2.1. Direct HCMV detection Virus isolation using fibroblast microculture was performed as described previously [13]. DNA was purified by spin columns using QIAmp DNA Blood Mini Kit (Qiagen, Germany) and used for qualitative nested-PCR of the IE1Ex4-target region [13]. 3.2.1.1. HCMV gB-, gN-, gO-genotyping. The variable regions from the viral glycoprotein gene sequences that encode for the proteins UL55, UL73 and UL74, were amplified and sequenced as previously reported [14–16]. Multiple alignments of the sequences using clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) were generated for the clinical isolates from the child, the mother and the grandmother I, as well as for reference strains AD169, Towne, Merlin, Toledo and Davis (Genebank access code: X17403, FJ616285, Y4468942, GU937742 and X99845, respectively) as well as for representative strains of each genotype. The pairwise identities between the isolates were calculated using the Smith Waterman algorithm using Jalview Version 2. The gO-variants were also identified using restriction-fragment-length-polymorphism (RFLP) using HpaII [16]. Additionally, the RFLP-genotyping of HCMV glycoprotein N was performed as described previously using SacI and SalI [15]. 3.2.2. HCMV serology The following commercial tests were used for diagnosis of the HCMV antibody status: “ELISA”-CMV IgG ELISA PKS/CMV IgM-ELA-Test PKS (Medac, Germany); “CLIA”-Liaison CMV IgG/IgG avidity/IgM (DiaSorin, Italy); “ECLIA” Elecsys CMV IgG/IgM/IgG avidity (Roche Diagnostics, Switzerland); “CMIA” Architect CMV IgG/IgM/IgG avidity assay (Abbott, Germany); recomblot CMV IgG/IgM/IgG avidity (Mikrogen, Germany). The recomblot assay
(recIB) detected HCMV-specific antibodies to the following HCMV recombinant proteins: IE1, p150, CM2, p65, gB1, gB2. RecIB-scoring reached from +/− weaker than IgG/IgM-control (negative), to +++, very strong reactivity.
3.2.3. HCMV neutralization Target cells were both, human foreskin-fibroblasts (HFF) and human retinal pigment-epithelial cells (ARPE-19; ATCC-CRL2302/LGC-Standards, Germany). The viral target strain originated from urine of a congenitally infected newborn. The reference sera included an HCMV-specific hyperimmunoglobulin (HIG) (Cytotect® ) and serum-pools (N = 100) from healthy seropositive and seronegative mothers at birth. Each 200 l of the cell-free target virus-passage (50×TCID50 /ml) were preincubated for 90 min at 37 ◦ C and 5%CO2 with 200 l of heat inactivated (30 min 56 ◦ C) serum dilutions from 1:50 to 1:3200 of the maternal serum. Thereafter, each 100 l of the virion-antibody mixtures were added to the different microcultures. After 18 h of incubation, monolayers were fixed and IE1 immunoperoxidase staining followed [13]. The neutralization-titers 50% (NT50 ) were calculated referring to 100% of viral infectivity in seronegative serum pool replica.
4. Results 4.1. Clinical courses and routine diagnostics 4.1.1. Index-patient In April 2008, the 9 month old girl was presented at the pediatrician with fever, flu-like symptoms and increasing hidrosis. Symptoms of an infection of the upper-respiratory tract dominated (Fig. 1a and b) and persisted for about 3 months. Serological testing was performed in July, detecting the presence of HCMV-IgG without IgM (Fig. 1a:1). Semiquantitative viruria was detectable up to November 2009, and DNAuria still 3 months longer. In total, viral DNAuria and viruria were analyzed at 8 different time points. At the end of viruria the infant was 28 months old. Viruria-levels were continuously decreasing. Peak-level of viruria (111 infected fibroblasts/ml of urine) under observation was during October 2008 at the age of 15 months. The viral isolate from observed peak-level of viruria was propagated in vitro and used for sequencing analysis (Fig. 1a:2). Viral DNAload in October 2008 was 1360 copies/ml, and in December 2008 15,000 copies/ml. Interestingly, the infant did not shed HCMV into saliva during the observation.
4.1.2. Mother of the index-patient On 24th June 2008, maternal symptoms started with fever (>39 ◦ C), flu like-symptoms with shivering, lymphadenopathy and symptoms of a pulmonary infection leading to hospitalization after ineffective empiric antibiotic treatment (doxycyclin for 6 days) (Fig. 1a and b). Computed tomography in July 2008 revealed atypical pulmonary infiltration, but no pathogen was detectable in bronchoalveolar lavage (BAL). Ultrasonography revealed hepatosplenomegaly. Laboratory findings presented elevated LDH and CRP, slightly elevated transaminases and slight lymphocytosis in the differential blood count (Fig. 1b). Routine serology revealed detection of HCMV-IgM and -IgG, but negative HCMV-PCR from blood and BAL (Fig. 1a:3–6). Interestingly, in week 25 after onset of symptoms, viral DNAuria was still detectable with >2000 copies/ml (Table 1). Since the onset of symptoms was exactly defined, it was possible to refer all later findings to that date. We assume an incubation time of about 4 weeks, thus exposition might have been in May, which fits well with the potential onset of viral shedding of the index patient.
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Fig. 1. (a) Flow chart of clinical symptoms and HCMV monitoring (gray shaded: periods of clinical symptoms). Infant: 1 – IgG pos., IgM and IgA neg., 2 – urine pos. mother: 3 – IgG and IgM pos., 4 – blood and BAL PCR neg., 5 – IgG and IgM pos., blood PCR neg., 6 – urine pos. grandmother I: 7 – IgG and IgM neg., 8 – IgG and IgM pos., 9 – urine pos. grandmother II: no CMV tests during acute disease. (b) Clinical and laboratory findings in the study cohort during acute CMV infection (n. d.: not done; n. c.: not collectable; + present; −: not present).
Table 1 Longitudinal epitope-specific HCMV rec IgG/IgM/IgG avidity immunoblotting during maternal primary infection. Full blown IgM reactivities in initial serum with persistence of p150 reactivity for 17 months p.i., while HCMV IgG avidity maturation is finished 4 months after primary infection (week 17 after onset of symptoms at 24 June 2008 or week 21 p.i.). Date
16/07/08
29/09/08
23/10/08
17/12/08
02/07/09
15/07/09
11/11/09
Weeks after symptom onset Weeks post infection
3 7
14 18
17 21
25 29
53 57
55 59
72 76
rec IgG IE1 rec IgG IE1 avidity rec IgM IE1
++ n.r. ++
++ + n.r.
+++ ++ n.r.
+++ +++ n.r.
+++ +++ n.r.
+++ +++ n.r.
++ ++ n.r.
rec IgG p150 rec IgG p150 avidity rec IgM p150
+ n.r. +++
++ ++ +
+++ +++ +
+++ +++ +
+++ +++ +
+++ +++ +
+++ +++ +
rec IgG CM2 rec IgG CM2 avidity rec IgM CM2
+++ + +++
+++ ++ +
+++ +++ +/−
+++ +++ +/−
+++ +++ +/−
+++ +++ n.r.
++ ++ n.r.
rec IgG p65 rec IgM p65
+++ +++
+++ +
+++ +
+++ +
+++ +
+++ +
++ +/−
rec IgG gB1 rec IgM gB1
n.r. +
+ n.r.
++ n.r.
++ n.r.
+++ n.r.
+++ n.r.
+++ n.r.
rec IgG gB2 rec IgM gB2
n.r. n.r.
+ n.r.
+ n.r.
+ n.r.
+++ n.r.
+++ n.r.
++ n.r.
CMV DNA blood BAL Urine
Negative Negative
Negative
Positive (38.5 ◦ C), flulike symptoms, fatigue, lymphadenopathy and increasing hidrosis accompanied with tachycardia and arterial hypertension beginning in July 2008. Early serological testing in July 2008 revealed no HCMV-specific IgG/IgM-antibodies (Fig. 1a:7). Due to persistent fever, the patient was hospitalized after ineffective empiric antibiotic treatment (ciprofloxacin for 5 days). In August 2008, X-ray examination of the chest showed no alteration, ultrasonography revealed hepatosplenomegaly. Laboratory findings presented elevated LDH and CRP, transaminases above threshold as well as a lymphocytosis in the differential blood count (Fig. 1b). Remarkably, routine serology in August 2008 detected HCMV-IgGseroconversion (Fig. 1a:8). Recombinant-immunoblotting revealed in October 2008 weak gB2-reactivity, intermediate IgG-avidity, and recIgM-reactivity (data not shown). Therefore, virus infection might have occurred at least three months before in July (Fig. 1a). In October 2008 she shed HCMV into urine, without viral DNAemia (Fig. 1a:9).
4.1.4. Grandmother-II of the index patient The paternal grandmother suffered from a febrile episode with flu-like-symptoms with distinctive fatigue accompanied with tachycardia and arterial hypertension beginning in August 2008. Laboratory-testing showed mild elevation of LDH and CRP without alteration of transaminases. In October (23/10/2013) she visited our institution as outpatient (Fig. 1a and b). She also shed HCMV into urine (21,200 copies/ml; 16 infected fibroblast nuclei/ml; Fig. 1a:9), and showed strong gB2-reactivity in context of intermediate HCMV-recIgG-avidity, and AI of 0.31 using CLIA (data not shown). 4.2. Maternal long-term HCMV serology During initial hospitalization all four IgG-assays showed a rapid increase reaching peak levels around week 18 p.i. Thereafter a decline of IgG-levels was observed at week 21 p.i. followed by four individual discordant kinetics describing slowly increasing IgGvalues. Around week 94 p.i. the IgG-values ranged from 60 U/ml
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Fig. 3. (a) Long-term course of maternal HCMV IgG avidity maturation. The time periods of switches from low to intermediate and high levels of IgG avidity depend strongly on the cut-off definitions for low and high IgG avidity. (b) The time periods of switches from low to intermediate and high levels of IgG avidity depend strongly test-dependent on the cut-off definitions for low and high IgG avidity. (c) Longitudinal course of neutralization capacity dependent from target cell and entry mode (fibroblasts versus epithelial cells). Using ARPE-19 cells it can be demonstrated strikingly, that the NT titers are at least 4-fold higher than in the HFF system. Additionally, already in week 7 high NT capacity can be detected in he ARPE-19-system. Using fibroblasts, a significant rise in NT50 value is documented in week 18 p.i.
(ELISA) to 600 AU/ml (CMIA) (Fig. 2a). In contrast, the HCMV-IgMlevels are concordantly decreasing between weeks 7 and 18 p.i. (Fig. 2b). The results from maternal recHCMV IgG/IgM/IgG avidity-testing are shown over a period of 16 months (72 weeks) after onset of clinical symptoms (Table 1). About 3 weeks after onset of clinical symptoms, specific recIgG-reactivity directed against CM2 and p65 and also IE1 is detectable with high scoring. At that time, the gB1/gB2-IgG-reactivity is not detectable, but recIgM-reactivity is detectable against all recombinant antigens with exception of gB2. Interestingly, the IgM-reactivity pattern is changing rapidly under complete or partial loss of reactivity against IE1, gB1, CM2 (about three months after infection), while reactivity against p150 and p65 is weakly detectable for about 1.5 years p.i. (Table 1: week 76). The maternal HCMV IgG-avidity-maturation was compared to the reduction of the recIB-reactivity for HCMV IE1, p150, and CM2 (Fig. 3a). Low (to intermediate) avidity is detectable using recIB up to 18 weeks p.i., while high avidity is observed after 5 months (21 weeks) p.i. (Fig. 3b; Table 1). Unfortunately, the range of test-specific “high avidity” using four different commercial IgGavidity-assays is >18–42 weeks p.i., while the range of “low avidity” includes the time period from 15 to 25 weeks p.i.
Neutralization assays with HFF-target cells fit well with avidity data of the recIgG-IB. The NT50 value increases in week 18 and remains stable over the whole observation period. The NT50 values using epithelial target cells are already at high levels in week 7 after onset of infection. NT50 RPE-values are 4-times higher than NT50 HFF during week 18–94 p.i. (Fig. 3c). Beside the mother of the index-patient also grandmother-I showed an HCMV primary infection with IgG-seroconversion and low avidity (data not shown). In contrast; grandmother-II did not show IgM-reactivity about 4 months following primary infection in the presence of strong recIgG-gB2-reactivity with intermediate IgG-avidity. 4.3. HCMV genotyping The alignment of the nucleotide sequences that encode for HCMV-gB, -gN (data files for supplemental material) and -gO region (Fig. 4) of the isolates from the child, the mother and the grandmother, showed identity of the HCMV clinical isolates of infant, mother and grandmother I (Fig. 4a–c). Genotyping by sequencing and RFLP analysis revealed a gB1/gO1/gN1-genotype. The percentage of identity of the isolates versus the reference strains Merlin,
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Fig. 4. Analysis of the N-terminal HCMV UL74 gene sequence which encodes the gO envelope protein. (A) Multiple alignment analyzed with clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) of the N-terminal variable region gB. Pairwise analysis with the reference and representative gO genotype strains was performed using Jalview V2 software (http://www.jalview.org). The identities observed were of 99.6% with the gOa1 reference strains and (C) a 100% between the clinical isolates from the child, mother and grandmother I.
Davis and Towne (gB1-genotype) were of 98%, 98% and 97.5% respectively (data not shown), whereas for gN and gO versus AD169 (gN1, gO1a genotype) was of 99.4% and 99.6%, respectively (Fig. 4b), that in conclusion all three virus isolates are identical (Fig. 4c). 5. Discussion The index-patient was breastfed at least 6 months before PEKiP® -meetings. Postnatal HCMV-transmission via breastfeeding from his initially seronegative mother can be clearly excluded. All three adults involved in home-caring of the index-patient developed a symptomatic HCMV-infection (Fig. 1a/b), leading to hospitalization of the mother and grandmother I. Fever and flulike-symptoms dominated, accompanied by pulmonary affection in the mother and cardiac symptoms in the two grandmothers. A cardiac affection due to primary HCMV-infection is a known but rare manifestation [17]. In a cohort of symptomatic HCMVprimary infections, influenza-like symptoms and fever were found mainly, but in 75% of cases pregnant women showed an asymptomatic course of HCMV-primary infection [18]. Prolonged, but spontaneous recovery was observed in all three adult patients, no specific antiviral treatment was given. The time-course suggested a transmission-cascade originating from PEKiP® -meetings (Fig. 1a). The course of maternal HCMV-IgG-longterm-serology shows a relatively good concordance with the rapid and steep initial IgG-increase with peak levels around week 18 (Fig. 2a). For interpretation of quantitative longterm HCMV-IgG-kinetics a strong
limitation exists: data from different assay systems are not comparable [19] in the absence of a WHO standard for unit-definition of HCMV-IgG. HCMV-primary infection is characterized by a steep decrease of IgM-reactivity from peak-level values early after onset of infection to week 18 p.i. All four IgM-assays used with exception of the recIgM-assay showed a completely concordant process of loss of IgM-reactivity (Fig. 2b). Interestingly, the recIB-avidity-assay detected exactly during this period of high IgM-reactivity a low IgGavidity. Recently, high IgM-antibody titer were described as strong predictor of low IgG-avidity [20]. In contrast, low IgM-indices at week 29 p.i. ( 21 weeks p.i. with a short periods of transition between low, and high avidity (Fig. 3b). This fits well with IgM-detection in all four assay systems. However, the main problem is the highly divergent definition of intermediate IgG-avidity in the three other test-systems using avidity indices
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(CMIA, CLIA, ECLIA). These ranges overlap from week 18 to 42 p.i., depending on the test-system used. Comparison of eight commercial HCMV-IgG-avidity assays showed that in a panel of 198 samples under evaluation none of the samples tested showed identical scoring [23]. Interestingly, the original cut-off values for the VIDAS CMV-IgG Avidity kit (bioMérieux, France) were changed recently for better analytical performance [24]. The recIgG–avidityassay seems to reflect the avidity-maturation in a more restricted time period, which correlates also to the increase of neutralization titers using HFF-target cells (Fig. 3b and c). Therefore high recIB IgG-avidity coincidences with NT50 increase from
