JOURNAL OF CLINICAL MICROBIOLOGY, July 2011, p. 27332739 0095-1137/11/$12.00 doi:10.1128/JCM.01358-10 Copyright 2011, American Society for Microbiology.

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Vol. 49, No. 7

Clinical and Molecular Observations of Two Fatal Cases of Rotavirus-Associated Enteritis in Children in Italy
Maria Cristina Medici,1* Laura Anna Abelli,1 Monica Martinelli,1 Domenico Corradi,2 Icilio Dodi,3 Fabio Tummolo,1 Valeria Albonetti,1 Vito Martella,4 Giuseppe Dettori,1 and Carlo Chezzi1
Section of Microbiology1 and Section of Pathology,2 Department of Pathology and Laboratory Medicine, University of Parma School of Medicine, and Unit of Paediatrics and Oncohematology, Maternal-Infantile Department,3 University Hospital of Parma, Viale A. Gramsci, 14, 43126 Parma, Italy, and Department of Public Veterinary Health, University of Bari, Bari, Italy4
Received 3 July 2010/Returned for modication 27 July 2010/Accepted 22 March 2011

Two fatal cases of infantile rotavirus enteritis occurred in northern Italy in 2005. Both children were severely dehydrated, and death was related to severe cerebral edema. Histological examination demonstrated extensive damage of the intestinal epithelium, villous atrophy or blunting, and macrophage inltration. The two rotavirus strains were of the G1P[8] type and the long electropherotype. The 2005 G1P[8] rotaviruses differed in the NSP4, VP3, VP4, and VP7 genes from G1P[8] rotaviruses circulating in 2004, suggesting the onset of a new G1P[8] strain in the local population. CASE REPORTS Patient 1 was a 2-year-old Caucasian boy who, on 25 April 2005, started developing diarrhea, vomiting, and a moderate fever (about 38.5C). Over the following 12 h, the vomiting episodes gradually decreased while the diarrhea progressively worsened, reaching 8 to 10 discharges. On this basis, the young patient was rehydrated per os at home by means of a solution (Humana Idravita) containing glucose (15.88 g/liter), sodium chloride (50 mmol/liter), maltodextrin (2.60 g/liter), potassium (20 mmol/liter), sodium (60 mmol/liter), and citrates (10 mmol/liter). At this stage, his general practitioner did not observe any sign of dehydration. Unfortunately, during the subsequent night, the patients clinical picture deteriorated further, with severe hyporeactivity and asthenia. On 27 April 2005, the child was hospitalized at our pediatric emergency department in cardiorespiratory arrest. At admission, his pupils were dilated and not photoreactive; in addition, the patient had mottled extremities and labial/nail cyanosis and respiratory movements were completely absent. The child was intubated for ventilation. However, after 30 min of cardiopulmonary resuscitation, ventilatory support was discontinued and the child was pronounced dead. Permission was given for an autopsy. Patient 2 was a 13-month-old Caucasian boy who was admitted on 29 April 2005 to our pediatric emergency department with a 24-hour history of vomiting, nonbloody diarrhea, a temperature of 40C, and decreased oral intake. On admission, the child was in good general condition despite mild dehydration. Upon laboratory analysis, the values were as follows: alanine aminotransferase (ALT), 42 IU/liter; aspartate aminotransferase (AST), 64 IU/liter; lactate dehydrogenase (LDH), 676 IU/liter; total bilirubin, 1.5 mg/dl. Serum electrolytes, blood glucose, creatinine, blood nitrogen, and complete blood
* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, Section of Microbiology, University of Parma School of Medicine, Viale Antonio Gramsci, 14, 43126 Parma, Italy. Phone: 39-0521-903499. Fax: 39-0521-993620. E-mail: mariacristina .medici@unipr.it. Published ahead of print on 27 April 2011. 2733

count were within normal ranges. C-reactive protein was 6.83 mg/liter. The child was administered intravenous rehydration. A few hours after hospital admission, the patient had a transient episode of dyspnea (saturation, 98%; pulse rate, 198/min; respiratory frequency, 96/min) and had mottled extremities. Ceftriaxone was administered intravenously after collection of blood samples. Also, the child had semiliquid loose stools that were collected for diagnostic investigation. After a couple of hours, following an abundant liquid loose stool, the child appeared poorly reactive and hypotonic and suffered respiratory and cardiac arrest. Cardiopulmonary resuscitation was initiated, and after administration of adrenaline, the breath rhythm was restored, with spontaneous eye movement (recovery of consciousness). The laboratory data collected before the respiratory arrest were as follows: ALT, 83 IU/liter; AST, 116 IU/ liter; LDH, 542 IU/liter; blood glucose, 373 mg/dl. In the blood, calcium was 7.8 mg/dl while sodium, potassium, chlorine, nitrogen, and creatine were at normal levels. Blood gas analysis revealed mixed acidosis (pH 6.813; pCO2, 119.8 mm Hg; pO2, 30.9 mm Hg; HCO3, 19 mM; blood base excess, 18.1 mM). After sedation, the child was transferred to the intensive care unit but his neurological status worsened. A computerassisted tomography scan and angiography showed diffuse cerebral edema with ischemic areas and no evidence of cerebral blood ow beyond the carotid siphon and foramen magnum from the left cerebral artery, respectively. During this time span, sodium levels were 150 to 161 mmol/liter with a chloremia of 116 to 132 mEq/liter, elevated liver enzyme levels (AST, 148 IU/liter; ALT, 152 IU/liter; LDH, 1,129 IU/liter), and a peak of blood glucose (345 mg/dl). The neurological and clinical status of the child further deteriorated, and coma de passe was established. The child was pronounced dead, and an autopsy was performed. Pathology ndings. Autopsies were performed in accordance with current Italian laws. At autopsy, the morphological ndings on the two children were similar. In both cases, death was attributed to tonsillar herniation through the foramen magnum as a consequence of severe cerebral edema. Another

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FIG. 1. (A) Low-power histopathological view of the small bowel wall with marked inammatory inltration involving the lamina propria and diffuse villous disepithelization (arrow). Hematoxylin-eosin staining is shown; original magnication, 4. (B) The villi are often blunted and sometimes fused. In addition to being disepithelized, they show a severe lymphoplasmacytic and granulocytic inltration of the lamina propria. Hematoxylin-eosin staining is shown; original magnication, 20. (C) Ileal portion with complete loss of intestinal villi. Hematoxylin-eosin staining is shown; original magnication, 4. (D) High-power histopathological view of the colonic mucosa with mild-to-moderate lymphoplasmacytic and granulocytic inltration involving the lamina propria. Hematoxylin-eosin staining is shown; original magnication, 20.

nding they had in common was a dilated bowel lumen (mainly in the ileum and jejunum) containing diffusely watery feces. In both cases, samples were collected from all organs, xed in a 10% buffered formalin solution for 24 h, and embedded in parafn tissue blocks. Five-micrometer histological sections were cut and stained with hematoxylin-eosin. The histological sections were observed through a light microscope (Olympus BX 51; Olympus, Tokyo, Japan). Upon histopathological analysis, inammation and blood stasis were observed in the central nervous system. The small bowel wall showed multiple areas of mucosal necrosis (with disepithelization) and villous blunting, as well as lymphoplasmacytic and neutrophilic inammatory inltration and edema involving the lamina propria (Fig. 1A to C), whereas the intestinal wall was not altered. In the large bowel (Fig. 1D), these changes were qualitatively similar but quantitatively less marked than in the proximal intestinal districts. The mesenteric lymph nodes displayed marked reactive hyperplastic changes. The pulmonary parenchyma showed acute blood stasis with scattered endoalveolar hemorrhages and foci of agonal emphysema. In the other organs, only signs of acute blood stasis were observed. Virological investigations. Only postmortem samples, including bowel contents and peripheral blood, from patient 1

were examined. For patient 2, stool and serum samples obtained during hospitalization were examined. Both the bowel contents from patient l and the stool sample from patient 2 revealed rotavirus-like particles upon electron microscopic observation (strain PR1598/2005/F from patient l and strain PR1609/2005/F from patient 2) performed as previously described (31). Polyacrylamide gel electrophoresis (PAGE) analysis of genomic viral double-stranded RNA (dsRNA) (32) revealed typical group A rotavirus (GARV) RNA migration patterns (4-2-3-2) with a long electropherotype (e-type). In order to determine the rotavirus VP7 and VP4 genotypes, the dsRNA extracted from 10% (wt/wt) fecal suspensions in phosphatebuffered saline (pH 7.2) using the guanidine isothiocyanateglass milk method (17), was subjected to reverse transcriptionPCR (RT-PCR) using different sets of G and P type-specic primers (4, 17, 18, 20, 29). The G1P[8] Wa strain (ATCC VR-2018) was used as a reference positive control in PCRs. Amplication products were electrophoresed at 100 V for 60 min in 2% agarose gel, stained with ethidium bromide, and visualized on a UV Transilluminator. By PCR genotyping, the two strains were characterized as G1P[8]. The RNA was extracted from peripheral blood of patient 1 and serum of patient 2 with EXTRAzol and EXTRA-

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gen kits (Nanogen Advanced Diagnostics, Turin, Italy), respectively, and screened for rotavirus using primers that amplify a 380-bp-long fragment of the rotavirus VP6 gene (19). Rotavirus RNA was not detected in the peripheral blood from patient l or in the serum from patient 2, while an amplication product of 610 bp was obtained by RT-PCR for an internal control (CPE-RNA; Nanogen Advanced Diagnostics) with specic primers (RECK; Nanogen Advanced Diagnostics) (31) used to rule out PCR inhibitors. The postmortem blood from patient 1 and the serum from patient 2 were also examined with a commercial enzyme immunoassay kit for detection of rotavirus antigen (Rotaclone; Meridian Bioscience, Inc., Cincinnati, OH). The cutoff value (optical density [OD], 0.092, i.e., 3 times the standard deviation above the mean OD) was established by testing a panel of sera collected from 16 children without gastroenteritis. Interestingly, rotaviral antigen was identied in the postmortem blood from patient 1 (OD, 0.440). These ndings are consistent with previous observations by Fischer et al. (15) documenting rotavirus antigens in the sera of children 1 or 2 days after the onset of diarrhea. Sequence and phylogenetic analyses of the VP3, VP4, VP7, and NSP4 genes. Several rotavirus genes have been associated with rotavirus virulence (VP3, VP4, VP7, NSP1, and NSP4) (3, 10, 21, 36, 43). The NSP4, VP3, VP4, and VP7 genes were sequenced in order to gather additional data on the two rotavirus strains PR1598/2005/F and PR1609/2005/F. A 680-bp and a 327-bp fragment of the NSP4 and VP3 genes, respectively, were amplied by RT-PCR as described by Lee et al. (26) and by Esona et al. (13), respectively. The VP3, VP4, VP7, and NSP4 amplicons were puried with a QIAquick gel extraction kit (Qiagen, Hilden, Germany) and sequenced on an automated sequencer (ABI-PRISM 3730 DNA analyzer; Applied Biosystems, Foster City, CA) using specic PCR primers. Reference sequences for VP3, VP4, VP7, and NSP4 gene comparisons were retrieved from the sequence databases. Sequence and phylogenetic analyses were performed using MEGA version 4.0 (25). Upon sequence analysis, strains PR1598/2005/F and PR1609/2005/F shared 100% nucleotide and amino acid identity in the NSP4, VP3, and VP4 genes and 99.9% nucleotide and 99.6% amino acid identity in the VP7 gene. In order to compare the sequences of fatal viruses with those of other G1P[8] viruses cocirculating locally in the 2005 epidemic and with strains circulating in the previous season (2004), sequences of cognate genes of G1P[8] strains detected in 2005 (12 for the NSP4 gene, 7 for the VP3 gene, 4 for the VP4 gene, and 5 for theVP7 gene) and in 2004 (6 for the NSP4 gene, 6 for the VP3 gene, 2 for the VP4 gene, and 2 for the VP7 gene) were determined. In the NSP4 gene analysis, all of the 2004 and 2005 strains belonged to NSP4 genogroup E1. However, all of the 2005 strains, including PR1598/2005/F and PR1609/ 2005/F, grouped together in a subcluster different from that of the 2004 strains (Fig. 2A). Similarly, phylogenetic analyses of the VP7 and VP4 genes revealed that the G1P[8] 2005 viruses, with a few exceptions, were distinct from the 2004 viruses. In the VP4 tree, all of the 2005 rotaviruses but strain PR1588/2005 segregated in a subcluster of the P[8]-III lineage (Fig. 2B), while in the VP7 tree, all of the 2005 G1P[8] viruses but strain PR385/2005 clustered

together within the G1-I lineage (Fig. 2C). This pattern was also observed in the VP3 analysis, with all of the 2005 strains, including PR1598/2005/F and PR1609/2005/F, segregating in a subcluster clearly separated from the 2004 G1P[8] viruses (Fig. 2D). By alignment of the deduced amino acid sequences of NSP4 genes, several mutations in the 2005 and 2004 strains were identied (data not shown) in NSP4 regions with toxigenic and functional activities. In particular, a mutation (Asn/Lys3Ser at residue 133) occurred within the amino acid (aa) 114 to 135 region, i.e., the enterotoxic peptide of NSP4 or diarrhea-inducing region (DIR) (3, 27, 42), and adjacent to the interspecies variable domain (ISVD) (residues 135 to 141), which appears to inuence NSP4-mediated pathogenicity, NSP4 cytotoxicity, diarrhea-inducing ability, and virus virulence in vivo (3, 21, 24, 33, 34, 43, 44). Detailed analysis of the deduced amino acid sequences of the VP4 gene identied several amino acid substitutions in the 2005 and 2004 G1P[8] strains (data not shown) affecting the VP8* antigenic subunit. Conversely, no hallmark was detectable within the VP3 and VP7 deduced amino acid sequences in strains PR1598/2005/F and PR1609/2005/F compared to the other strains examined (data not shown). To investigate whether the mutations observed affected the NSP4 and VP4 structures, secondary structure prediction was performed using the PSIPRED protein structure prediction server of the University College of London (8, 35) (http://bioinf.cs.ucl.ac.uk/psipred/). Three minor differences between the 2005 and 2004 viruses were mapped in the NSP4 secondary structure (Fig. 3). In the VP8* region, we mapped 6 to 11 minor structural differences (Fig. 4) between the 2005 fatal strains (PR1598/2005/F and PR1609/ 2005/F) and strains representative of 2004 G1P[8] rotaviruses within lineage P[8]-III (strain PR1533/2004) and lineage P[8]-I (strain PR2108/2004).

To date, the literature describing rotavirus-associated death is limited. Fatal cases in children aged 5 years were recorded in Toronto (21 cases) in the 1970s (11). In the same years, the rate of infant mortality associated with diarrhea in the United States averaged 12.8 deaths per 100,000 live births (23). Rotavirus-associated mortality has also been documented in Venezuela (21 cases) (37) and Nicaragua (52 cases) (1) among malnourished children from very poor socioeconomic classes. On the other hand, an 11-year-long diarrhea- and rotavirusassociated hospitalization and death surveillance in the United States among children reported a fatality rate of l death per 1,616 rotavirus-coded hospitalizations (0.06%) (14), while a Swedish 11-year surveillance of rotavirus gastroenteritis in a hospitalized pediatric population reported a fatality rate of l death per 984 children with rotavirus gastroenteritis (0.1%) (22). During a 24-year surveillance for rotavirus enteritis in children hospitalized in Parma, Italy, only the two rotavirus-associated fatal cases described in this study were documented, with an overall fatality rate of l death per 983 rotavirus enteritis cases (0.1%, data from 1987 to 2010, 2 deaths) (M. C. Medici et al., unpublished data). These deaths occurred during an exceptional 2005 epidemic season of rotavirus enteritis (Feb-

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FIG. 3. Schematic representations of NSP4 secondary structure predictions obtained with the PSIPRED protein structure prediction server. The panel depicts aa 124 to 175. (Boxes a) Beginning at aa 141 (PR1533/2004 strain) or 142 (PR2108/2004 and PR1494/2004 strains), the 2-aa -sheet of the 2004 strains has mutated into a 3-aa -sheet in the PR1598/2005/F strain. (Boxes b) Beginning at aa 146, the 8- and 5-aa -helices of the PR2108/2004 strain have mutated into a 15-aa -helix in the PR1598/2005/F strain. Conf, conrmed; Pred, predicted; AA, amino acid.

ruary to May 2005), when the prevalence of rotavirus infection reached 53.9% (132 out of 245 children examined; overall 2005 prevalence of 34.5%), compared to the 41.2% prevalence in the 2004 epidemic period (January to May), when the annual prevalence was 27.5% (median prevalence over the 11-year period of 2000 to 2010, 22.2%) (Medici et al., unpublished). In both patients, histopathological examination of the gastrointestinal tract conrmed the typical ndings associated with rotavirus enteritis (6, 12, 28, 41), dened as marked lymphoplasmacytic inammatory inltrates in the lamina propria with manifest loss of villous epithelium. The clinical courses and autopsy ndings indicated that both children were severely dehydrated. In the two children, rotavirus RNA was detected by VP6 RT-PCR, electron microscopy, and PAGE either in the bowel contents or in stool samples. Rotavirus antigen, but not rota-

virus RNA, was also revealed in the blood of patient 1. Rotavirus RNA was not detected in the tissues of the two patients. This could be accounted for by a delay in the analyses due to forensic and legal requirements. Although rotavirus dsRNA is known to be rather stable, delays in tissue formalin xation and embedding could have promoted RNA damage, hindering rotavirus RNA detection by VP6 RT-PCR in the postmortem histological sections of intestinal and extraintestinal sites. Also, formalin may easily trigger phenomena of degradation/alteration of nucleic acids. In order to rule out this hypothesis, MA104 cells infected with rotavirus strain Wa were parafn embedded as previously described (40) and therefore used as a positive extraction control using an EXTRAfn kit (Nanogen Advanced Diagnostics). In parallel, parafn-embedded postmortem histological sections of the patients were reextracted with the same protocol and tested by the VP6 RT-PCR, thus conrming that rotavirus RNA was not present in the tissues. Cerebrospinal uid samples of the two patients were not available, and therefore we could not assess if rotavirus RNA was present in the central nervous system (CNS). However, in fatal cases of rotavirus-associated disease, it is not possible to correlate CNS localization of rotavirus with neurological damage and death (29). In these two patients, it is likely that the observed neurological damage was triggered by rapid dehydration and consequent electrolyte imbalance, as suggested by the hypernatremic status of patient 2, who was not viremic. On the other hand, whether or not the presence of infectious rotavirus in the blood of patient 1 played a role in disease evolution remains unclear, as most children with rotavirus in stools can be viremic (7). Very young children do not tolerate loss of 15% of their body weight in extracellular uids or high loads of sodium because of relatively reduced glomerular ltration rates (30), and therefore a possible cause of death may be insufcient or inadequate initial uid replacement in the two children. However, patient 1 was immediately treated at home under the supervision of the family pediatrician and patient 2 was treated in a hospital under the care of expert pediatricians. The two GARVs detected in these fatal cases showed long e-type and G1P[8] specicities. G1P[8] rotaviruses appear to be predominant in Italy (2, 31) and worldwide (16, 42) and accounted for 42.4% of the strains revealed in the Parma epidemic period of February to May 2005. The G1P[8] strains circulating in the Parma area in 2005 appeared to differ in NSP4, VP3, VP4, and VP7 from the 2004 strains. A clear monophyletic pattern was observed in the NSP4 and VP3 analyses of the 2005 G1P[8] viruses, while this clustering was less strict in the VP7 and VP4 analyses, as some 2005 viruses were

FIG. 2. Neighbor-joining phylogenetic trees based on the deduced NSP4 amino acid sequences corresponding to aa 4 to 175 (corresponding to reference strain Wa, accession no. K02032) (p-distance model) (A), nucleotides 85 to 1011 (corresponding to reference strain Wa, accession no. M96825) of the VP4 gene (Kimura 2-parameter model) (B), nucleotides 127 to 995 (corresponding to reference strain Wa, accession no. K02033) (Kimura 2-parameter model) of the VP7 gene (C), and nucleotides 735 to 880 (corresponding to reference strain Wa, accession no. AY267335) (Kimura 2-parameter model) of the VP3 gene (D) of group A rotaviruses found in northern Italy in 2004 and 2005, including 2 strains detected in two fatal cases of rotavirus enteritis (PR1598/2005/F and PR1609/2005/F), and of group A rotaviruses from GenBank. The GenBank nucleotide sequence accession numbers are shown in parentheses. Percentage bootstrap values (of 1,000 replicates) above 80% are shown at the branch nodes. The scale bar in each panel indicates the number of nucleotide substitutions per position. The NSP4 genogroups (A), as well as P[8] (B) and G1 (C) lineages, are indicated by the brackets to the right of the dendrograms.

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FIG. 4. Schematic representations of VP8* secondary structure predictions obtained with the PSIPRED protein structure prediction server. Shown are aa 26 to 334. Boxes a to k highlight differences in the predicted protein secondary structure of 2005 strain PR1598/2005/F with respect to 2004 strains PR1533/2004 and PR2108/2004. Conf, conrmed; Pred, predicted; AA, amino acids.

found to segregate separately. Banyai et al. (5) suggested that genetic differentiation into lineages may contribute to the success of G1P[8] rotaviruses in causing natural infections. The mechanisms controlling rotavirus virulence have not been dissected and investigated in detail yet. Several genes have been implicated in these mechanisms, suggesting complex interactions of multiple genes yet under the inuence of the genetic context, rather than the effect/result of a single gene (10). In this study, several amino acid substitutions were identied in the 2005 G1P[8] rotaviruses with respect to 2004 GARVs and these mutations affected the toxigenic and functional regions of NSP4 (i.e., the DIR) and the VP8* antigenic region, which can inuence GARV antibody binding and the

immune response. Amino acid changes in G1 VP7 neutralization epitopes and in the signal peptide motif in various lineages and sublineages detected in different rotavirus seasons are supposed to trigger the selection of variants able to escape immunity (5), representing an important survival strategy of rotaviruses. Also, minimal differences in the conformation of NSP4 due to mutations in the diarrhea-inducing regions of NSP4, including the ISVD, can have a critical effect on enterotoxigenic activity (21). By prediction of protein secondary structure, all of the 2005 Italian G1P[8] rotaviruses were predicted to differ from the 2004 strains in the NSP4 ISVD and in the VP4 antigenic site. Accordingly, it can be speculated that these

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changes, either alone or in combination, allowed the 2005 G1P[8] strains to spread widely through the pediatric population during the 2005 epidemic period, as described and suggested in other studies (9). The spatial/temporal clustering of the two distinct fatal cases documented in the Parma area in 2005 seemed unusual and provided the rationale for the gathering of sequence data on these fatal rotavirus strains. The pathophysiology of rotavirus diarrhea is clearly multifactorial (38, 39). Hence, it remains unclear whether a host-related factor or inadequate initial uid replacement was actually responsible for the deaths of the two children, rather than possible changes in rotavirus strain properties. In conclusion, the two fatal events reported in this paper demonstrate that even nowadays in industrialized countries where children rapidly receive effective attention and therapy, fatal rotavirus enteritis can occur, highlighting the need for the adoption of rotavirus immunization programs in developed nations. Nucleotide sequence accession numbers. The GenBank accession numbers for the nucleotide sequences reported in this paper are HQ906773 to HQ906787 for the VP3 gene, HM245019 to HM245026 for the VP4 gene, HM245027 to HM245035 for the VP7 gene, and HM244999 to HM2445018 for the NSP4 gene.
This study was partly supported by grants Ricerca Scientica FIL (ex 60%) 2006, prot. 60A06-0979, and FIL 2008 to Maria Cristina Medici from the University of Parma, Parma, Italy. We have no conicts of interest to declare.
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