Journal of Medical Virology 65:6669 (2001)
Fatal Adenovirus Type 7b Infection in a Child withSmith-Lemli-Opitz Syndrome
A. Beby-Defaux,1 L. Maille,1 S. Chabot,1 A. Nassimi,2 D. Oriot,2 and G. Agius1*1Laboratory of Virology, University Hospital Center La Miletrie, Poitiers, France2Department of Pediatrics, University Hospital Center La Miletrie, Poitiers, France
Adenovirus type 7 causes worldwide respiratorytract infections, mainly in children. Severe sys-temic infections can occur, especially in immu-nocompromised patients and in patients withunderlying chronic diseases. This report de-scribes the first case of a fatal disseminatedadenovirus type 7 infection in a child with Smith-Lemli-Opitz syndrome, a rare autosomal reces-sive disorder due to a primary enzymatic defect incholesterol metabolism. Nasopharyngeal secre-tions and autopsy specimens including liver,lung, pleural fluid, and rectum were collectedfor viral culture. Adenovirus serotype 7 strainswere obtained from all anatomic sites, except theliver. All these clinical isolates were analyzedusing restriction endonuclease digestion of thegenome, identifying them as genome type 7b, avirulent type. In this case, the fatal evolutioncould have been accelerated by the presence ofan immunodeficiency although immunodefi-ciency is not included in the definition of Smith-Lemli-Opitz syndrome. The frequent recurrentbanal infections in Smith-Lemli-Opitz syndromecould be prevented by a cholesterol supplemen-tation regimen. Finally, this report emphasizesthe need for efficient therapy for dissemin-ated adenovirus infections, especially for virulentgenome types. J. Med. Virol. 65:6669,2001. 2001 Wiley-Liss, Inc.
KEY WORDS: adenovirus DNA; Smith-Lemli-Opitz syndrome; virulent gen-ome type; restriction endonu-clease digestion
The Smith-Lemli-Opitz syndrome is a rare autosomalrecessive disorder due to a primary enzymatic defectin cholesterol metabolism [Honda et al., 1995], andcharacterized by multiple congenital anomalies [Smithet al., 1964]. The enzymatic defect is responsible fordeficient plasma and tissue cholesterol levels, andaccumulation of 7-dehydrocholesterol resulting in
growth restriction and mental retardation. TheSmith-Lemli-Opitz syndrome has been subdivided into2 types on the basis of clinical severity: type I is theclassic one and type II the severe one. Recurrent banalinfections are an important part of the medicalproblems associated with the Smith-Lemli-Opitz syn-drome.
Adenovirus serotype 7 is frequently associated withsevere clinical manifestations with residual damageand sometimes fatal outcome, mainly in children[Horwitz, 1996]. Adenovirus serotype 7 may cause loca-lized diseases, such as pneumonia, acute respiratorydisease, conjunctivitis, but also severe systemic infec-tions with sepsis-like syndrome, high fever, and multi-ple-organ-system involvement, which can result indeath [Horwitz, 1996; Munoz et al., 1998]. Host factorssuch as metabolic or genetic diseases, anatomic abnor-malities, and immunologic deficiencies contribute tothe severity of adenovirus infection [Munoz et al., 1998].However, disseminated infections have also beenreported in immunocompetent children and in childrenwith underlying chronic illnesses [Munoz et al., 1998].
This report describes the first case of a fatal dis-seminated adenovirus type 7b infection in a child with atype II Smith-Lemli-Opitz syndrome.
A 5-year-old girl had severe multiple morphologicanomalies including dysmorphic facies, cleft palate,polydactyly, and pyloric stenosis requiring gastro-stomy. Since her birth, she had developed multipleear and respiratory infections, and recurrent pyelone-phritis although she had no renal tract abnormalities.The child had been living in a disabled patient centerfor two months before hospitalization. On 1 January,she was admitted to the Department of Pediatrics ofPoitiers University Hospital Center for severe pneu-monia and otitis due to respiratory syncytial virus(RSV). She was discharged a week later with anti-
*Correspondence to: Dr. G. Agius, Laboratoire de Virologie,CHU La Miletrie, BP 577, 86021 Poitiers cedex, France.E-mail: firstname.lastname@example.org
Accepted 29 January 2001
2001 WILEY-LISS, INC.
biotherapy. Nine days later, she developed fever anddiarrhea. Despite symptomatic treatment, she wasreferred to the pediatric intensive care unit on 20January for septic shock with acute pneumonia anddiarrhea. On admission, she appeared hypotrophic(weight: 13,000 g), cyanotic and lethargic, with neuro-logic hypotonic signs. Physical examination showedhigh fever, important signs of acute respiratory infec-tion, major hypotension (mean arterial pressure:25 torr), tachycardia, hepatomegaly, and massivediarrhea. Chest X-ray showed bilateral reticulomicro-nodulary infiltrates in the upper and lower lung lobes,with pleural effusion.
Arterial blood gas values with a FiO2 of 1.0 werepCO2 6.3 kPa, pO2 26.2 kPa, oxygen saturation 99%,bicarbonates 21 mmol/l and pH 7.25. Laboratory datashowed a leukocyte count of 11.4 109/l with 67%polynuclear leukocytes and 24% lymphocytes, hemo-globin at 10.8 g/dl, hematocrit at 31.3%, fibrinogen at4.9 g/l, and C-reactive protein at 125 mg/l. Lactic acidwas 5.51 mmo/l. Lumbar puncture showed clearcerebrospinal fluid (CSF), the erythrocyte count was5.1 109/l, and the leukocyte count was 0.075109/lwith 100% mononuclears.
Treatment and Evolution
Mechanical ventilation, fluid expansion, and vaso-pressive drugs were started immediately due toexcessive respiratory workload and hypotension. Thecourse worsened to episodes of cardiac arrests withrefractory shock and acute respiratory distress syn-drome. Despite intensive treatment including inotropicsupport and antibiotics, cardiovascular and respiratoryfunctions deteriorated rapidly and the child died a fewhours later.
Nasopharyngeal secretions were processed for directexamination using immunofluorescence assay withmonoclonal antibodies (Pasteur Diagnostics, Marnes-la-Coquette, France) for adenovirus, RSV, coronavirus,influenza viruses types A and B, and parainfluenzaviruses types 1, 2, and 3.
Serology for adenovirus, influenza viruses type A andB, parainfluenza viruses types 1, 2, and 3, and entero-viruses was performed using the complement fixationtest. A group-reactive antigen (Virion, Wurzburg,Germany) was used for adenovirus complement fixa-tion testing.
Nasopharyngeal secretions and autopsy specimensincluding liver, lung, pleural fluid, and rectum wereinoculated onto HeLa-229 and MRC-5 cells. Character-istic cytopathic effect was confirmed by immunofluor-escence assay using monoclonal antibodies against anadenovirus group-reactive epitope (Pasteur Diagnos-tics). Serotype determination was then performed by
neutralization with type-specific rabbit antisera (NIH,Bethesda, MD).
The adenovirus isolates were examined using restric-tion endonuclease digestion. Viral suspensions wereharvested when an extensive cytopathic effect waspresent, and adenovirus strains were kept at 808Cuntil restriction endonuclease analysis was carried out.Viral DNA was extracted and purified from infectedcells as previously described by Shinagawa et al. .Restriction endonuclease analysis was carried out withBamHI and BstEII according to a well-known pathwayused for genome type identification [Li et al., 1996;Wadell et al., 1985]. Briefly, aliquots containing 12 mgof viral DNA were digested with 1015 U/ml of eachendonuclease according to the manufacturers instruc-tions (Boehringer Mannheim, Germany). After diges-tion, nucleic acid fragments were separated byelectrophoresis at 38 V/cm for 16 hr on a 0.8% agarosegel and visualized by UV illumination following bro-mide staining. Adenovirus genotype 7c (no. 37300) andadenovirus genotype 7b (KCH4) strains circulating inEurope since the 1970s were used as reference.
Direct examination by immunofluorescence assay onnasopharyngeal secretions was positive for adenovirus.Serology for adenovirus was negative, as well asserologies for influenza viruses types A and B, parain-fluenza viruses types 1, 2, and 3, cytomegalovirus, andenterovirus. Only specific RSV antibodies were foundpositive at 1:128 by complement fixation testing.
Five days after inoculation, typical adenovirus cyto-pathic effects on heteroploid cells showing roundingand swelling of cells, refractile appearance, and clump-ing in grapelike clusters were observed in all theclinical specimens, except the liver. This preliminaryidentification was confirmed by immunofluorescenceassay, showing adenovirus serotype 7 in all anatomicsites. It should be noted that specific neutralizingantibody titer was lower than 1:4 using the adenovirusserotype 7 isolates as antigen.
Each isolate obtained from the anatomic sites yieldedcleavage patterns similar to the adenovirus genotype7b reference strain. Restriction patterns with BamHIand BstEII showed a genome type 7b (Fig. 1). The use ofBstEII allowed discriminating between genome types7b and 7b1 [Li et al., 1996].
Bacteriologic investigations including mycobacteriaresearch in blood, CSF, pleural fluid, lung, and liverwere negative as well as serologic tests for Mycoplasmapneumoniae, Chlamydia pneumoniae, and Coxiellaburneti.
In the case reported, a child with a severe geneticdefect presented a fatal adenovirus infection. The mainquestion arising is the reason why this patient wasparticularly vulnerable to disseminated adenovirusinfection. The presence of an immunodeficiency is the
Adenovirus and Smith-Lemli-Opitz Syndrome 67
major hypothesis although immunodeficiency is notrecognized as part of the Smith-Lemli-Opitz syndrome.Ostergaard et al.  showed a defective monocyteoxidative metabolism in a child with Smith-Lemli-Opitz syndrome suffering from repeated febrile infec-tions, but monocyte functions including chemotaxis,phagocytosis, and IL-1 production were normal. How-ever, other unknown immune mechanisms might leadto a fatal evolution.
Viral infections induce a temporary suppression of T-cell-mediated immunity, or impair respiratory tissueand may increase the risk of developing severesecondary infections, mainly by adenovirus [Munozet al., 1998]. In our patient, RSV detected at firstadmission might have contributed to the severity ofadenovirus serotype 7 subsequent infection.
A decrease in the number and severity of intercur-rent infections as well as improvement in developmen-tal progress have been evidenced by a cholesterolsupplementation regimen [Elias et al., 1997]. In thecase reported, the patient was not supplemented withcholesterol.
Some adenovirus infections have been suggested toresult from reactivation of latent viruses from anendogenous source [Horwitz, 1996; Munoz et al.,1998]. In this study, failure to detect adenovirusantibodies is more consistent with an acute infectionthan with a reactivation or a persistent infection.
However, some adenovirus infections that may not bedetected by complement fixation testing can yield up to50% false-negative results in children [Schmidt andLennette, 1971]. In addition, nosocomial transmissionmight be suspected, since the interval between the twohospitalizations was consistent with the incubationperiod for adenovirus infection [Horwitz, 1996].
Disseminated adenovirus infections have been char-acterized by severe pulmonary disease, multiple-organ-system involvement including the liver, and frequentfatal outcome [Munoz et al., 1998]. We demonstratednasopharynx, lung and gastrointestinal tract impair-ments but failed to find hepatic involvement althoughhepatitis has been commonly reported [Horwitz, 1996;Munoz et al., 1998]. The mortality rate is usually high,due to the type of underlying conditions and theseverity of the disease. In addition, there is no specifictherapy for adenovirus infections (ganciclovir andribavirin have been used with no significant results[McCarthy et al., 1995]). Although no convincing linkwas established between a particular type and enhan-ced virulence, genome type 7b is the most common andvirulent serotype 7 strain in Europe [Munoz et al.,1998]. However, numerous genome types and subtypescirculate and cause outbreaks with various degrees ofseverity [Horwitz, 1996].
Finally, this report emphasizes that adenovirusgenotype 7b remains a virulent genome type, and that
Fig. 1. Agarose gel electrophoresis showing BamHI (A) and BstEII(B) restriction patterns of adenovirus serotype 7 isolates. Lanes M1,M2, M3: size markers (bacteriophage ( DNA digested with HindIII,adenovirus serotype 2 digested with KpnI and VI marker, respec-
tively); lanes 13: isolates from nasopharyngeal secretions, lung, andrectum, respectively; lanes 4,5: adenovirus genotype 7b (KCH4) andadenovirus genotype 7c (no. 37300) reference strains, respectively.Numbers to the left of the gels are molecular sizes in base pairs.
68 Beby-Defaux et al.
the severity of the clinical outcome calls for an efficienttherapy for adenoviral infection, especially in patientssusceptible to threatening viral infections such as inthe Smith-Lemli-Opitz syndrome.
We are indebted to Dr. Goran Wadell, Department ofVirology, University of Umea, Sweden, for providingthe reference strains used in this study.
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