Astrovirus infection in hospitalized infants with severe combined immunodeficiency after allogeneic hematopoietic stem cell transplantation

Abstract

Infants with severe primary combined immunodeficiency (SCID) and children post-allogeneic hematopoietic stem cell transplantation (HSCT) are extremely susceptible to unusual infections. The lack of generic tools to detect disease-causing viruses among more than 200 potential human viral pathogens represents a major challenge to clinicians and virologists. We investigated retrospectively the causes of a fatal disseminated viral infection with meningoencephalitis in an infant with gamma C-SCID and of chronic gastroenteritis in 2 other infants admitted for HSCT during the same time period. Analysis was undertaken by combining cell culture, electron microscopy and sequence-independent single primer amplification (SISPA) techniques. Caco-2 cells inoculated with fecal samples developed a cytopathic effect and non-enveloped viral particles in infected cells were detected by electron microscopy. SISPA led to the identification of astrovirus as the pathogen. Both sequencing of the capsid gene and the pattern of infection suggested nosocomial transmission from a chronically excreting index case to 2 other patients leading to fatal infection in 1 and to transient disease in the others. Virus-specific, real-time reverse transcription polymerase chain reaction was then performed on different stored samples to assess the extent of infection. Infection was associated with viremia in 2 cases and contributed to death in 1. At autopsy, viral RNA was detected in the brain and different other organs, while immunochemistry confirmed infection of gastrointestinal tissues. This report illustrates the usefulness of the combined use of classical virology procedures and modern molecular tools for the diagnosis of unexpected infections. It illustrates that astrovirus has the potential to cause severe disseminated lethal infection in highly immunocompromised pediatric patients.

Figure 1. Timeline for the detection of astrovirus in samples of the 3 patients.

Sample screening listed in Tables S1,S2,S3 is summarized as follows. Grey circles indicate positive detection of astrovirus in any type of samples (stools, serum, plasma, nasopharyngeal swabs, and vesicle swabs) by real-time RT-PCR and/or cell culture confirmed by immunofluorescence. Black diamonds indicate negative astrovirus detection by real-time RT-PCR. Black star represents the end of hospitalization. T: bone marrow transplantation; †: death.

Figure 2. Electron microscopy of Caco-2 cells infected with a suspension of stool sample # 12869 from patient 1 (Table S1).

A. 25,000-fold magnification of a portion of the infected Caco-2 cell containing both nucleus and cytoplasm. Dark staining in the nucleus (white arrow) is linked to chromatin condensation and granulation, whereas staining in the cytoplasm (black arrows) is due to aggregates of viral capsids. B. 66,000-fold magnification of viral capsid aggregates (black arrow) present in the cell cytoplasm.

Figure 3. Immunofluorescence staining of non-infected (A,C) and infected (B,D) Caco-2 cells incubated with anti-astrovirus (A,B) and pan-enterovirus blend (C,D) monoclonal antibodies; magnification 63x (oil).

Figure 4. Immunohistochemistry of the small intestine autopsy tissue sections of patient 2.

Immunohistochemistry with an anti-astrovirus antibody performed on non-infected (A) and infected (B) regions of the small intestine autopsy tissue sections.

Figure 5. Phylogenetic analysis of the transmitted astrovirus strains.

A. A maximum-likelihood-based phylogenetic tree was computed using capsid gene sequences (corresponding to nt 4325 to 4867 of the astrovirus 4 complete genome [GenBank DQ344027] or nt 1 to 543 of the capsid gene) obtained from 8 samples collected from patient 1, one from patient 2, and two from patient 3 (Table 2). Corresponding sequences from representatives of the 8 human astrovirus serotypes (HAstV) and the divergent VMLB1 human astrovirus (HastVMLB1) were included to show a comparison of evolutionary distances and relationships. Bat astrovirus (BatAstV, in light gray) was used as an outgroup. All virus strains are shown with the corresponding GenBank accession number. The numbers of substitutions per site are indicated below the tree. Because of long branches, the outgroup BatAstV, as well as the strain HAstVMLB1, are shown with annotated branch lengths. B. Blow-up of a sub-tree from panel A marked with an asterisk showing relatively short branch lengths compared to the full tree.