Niraj C. Patel

Vaccine-Acquired Rotavirus in Infants with Severe Combined Immunodeficiency

Live pentavalent human-bovine reassortant rotavirus vaccine is recommended in the United States for routine immunization of infants. We describe three infants, two with failure to thrive, who had dehydration and diarrhea within 1 month after their first or second rotavirus immunization and subsequently received a diagnosis of severe combined immunodeficiency. Rotavirus was detected, by means of reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay, in stool specimens obtained from all three infants, and gene-sequence analysis revealed the presence of vaccine rotavirus. These infections raise concerns regarding the safety of rotavirus vaccine in severely immunocompromised patients.

Outcomes of patients with severe combined immunodeficiency treated with hematopoietic stem cell transplantation with and without preconditioning

BACKGROUND The effect of pretransplantation conditioning on the long-term outcomes of patients receiving hematopoietic stem cell transplantation for severe combined immunodeficiency (SCID) has not been completely determined. OBJECTIVE We sought to assess the outcomes of 23 mostly conditioned patients with SCID and compare their outcomes with those of 25 previously reported nonconditioned patients with SCID who underwent transplantation. METHODS In the present study we reviewed the medical records of these 23 consecutive, mostly conditioned patients with SCID who underwent transplantation between 1998 and 2007. RESULTS Eighteen patients (median age at transplantation, 10 months; range, 0.8-108 months) received haploidentical mismatched related donor, matched unrelated donor, or mismatched unrelated donor transplants, 17 of whom received pretransplantation conditioning (with 1 not conditioned); 13 (72%) patients engrafted with donor cells and survive at a median of 3.8 years (range, 1.8-9.8 year); 5 (38%) of 13 patients require intravenous immunoglobulin; and 6 of 6 age-eligible children attend school. Of 5 recipients (median age at transplantation, 7 months; range, 2-23 months) of matched related donor transplants, all 5 engrafted and survive at a median of 7.5 years (range, 1.5-9.5 year), 1 recipient requires intravenous immunoglobulin, and 3 of 3 age-eligible children attend school. Gene mutations were known in 16 cases: mutation in the common gamma chain of the IL-2 receptor (IL2RG) in 7 patients, mutation in the alpha chain of the IL-7 receptor (IL7RA) in 4 patients, mutation in the recombinase-activating gene (RAG1) in 2 patients, adenosine deaminase deficiency (ADA) in 2 patients, and adenylate kinase 2 (AK2) in 1 patient. Early outcomes and quality of life of the previous nonconditioned versus the present conditioned cohorts were not statistically different, but longer-term follow-up is necessary for confirmation. CONCLUSIONS Hematopoietic stem cell transplantation in patients with SCID results in engraftment, long-term survival, and a good quality of life for the majority of patients with or without pretransplantation conditioning.

Detection of polyomavirus SV40 in tonsils from immunocompetent children.

BACKGROUND BK virus (BKV), JC virus (JCV) and simian virus 40 (SV40) are nonenveloped DNA viruses, members of the family Polyomaviridae. BK and JC viruses establish persistent infections in humans, and evidence suggests that SV40 can infect humans, as well. Whether persistence occurs in the lymphoid system is unknown. METHODS Paraffin-embedded tonsils from 220 immunocompetent children (mean age 9.3 years) were examined by polymerase chain reaction (PCR) to detect viral DNA of BKV, JCV, SV40, and Epstein-Barr virus (EBV). RESULTS Polyomavirus-specific DNA sequences were detected in 8.3% (29/351) of specimens collected from 220 children. Twenty-one (9.5%) children had polyomavirus DNA present in at least one tonsil, with sequences identified as SV40 (n=20) and BKV (n=1). Polyomavirus JCV was not detected. Among patients positive for SV40, 8 of 14 (57%) contained viral DNA in both available tonsils. EBV DNA was detected in 99 (28.2%) samples from 67 (30.5%) patients. Eleven samples (3.1%) from 8 (3.6%) children were positive for both polyomavirus and EBV. SV40-positive children were significantly older than the SV40-negative subjects (P<0.001). T-antigen expression was detected in an SV40 DNA-positive tonsil by immunohistochemistry. CONCLUSIONS These results suggest that SV40 can infect tonsils, that lymphoid tissue may represent a site for polyomavirus persistence, and that immunohistochemistry is not a useful detection assay when there are very few virus-infected cells in a tissue.

Long-term outcomes of nonconditioned patients with severe combined immunodeficiency transplanted with HLA-identical or haploidentical bone marrow depleted of T cells with anti-CD6 mAb

BACKGROUND Between 1981 and 1995, 20 children with severe combined immunodeficiency (SCID; median age at transplant, 6.5 [range, 0.5-145] mo, 12 with serious infection) were treated with haploidentical T cell-depleted (anti-CD6 antibody) bone marrow (median number of 5.7 [0.8-18.8] x 10(8) nucleated cells/kg) from mismatched related donors (MMRDs), and 5 children with SCID (median age at transplant, 1.8 [0.5-5.0] mo, 1 with serious infection) were given unmanipulated bone marrow from matched related donors (MRDs). No conditioning or graft-versus-host disease (GvHD) prophylaxis was used. OBJECTIVE To assess the outcomes of patients with SCID who received bone marrow from MMRDs or MRDs. METHODS We reviewed the medical records of these 25 consecutive patients with SCID (4 with Omenn syndrome). RESULTS Of the 20 patients who received bone marrow from MMRDs, 12 engrafted, 10 survived at a median age of 15.2 [10.0-19.1] years, 4 had chronic GvHD (lung, intestine, skin), 5 required intravenous immunoglobulin, and 8 attended school or college. Two of 5 patients who died had chronic GvHD, and 2 developed lymphoproliferative disease. Of the 5 patients who received bone marrow from MRDs, 5 engrafted, 5 survived at a median age of 23.3 [18.5-26] years, 1 had chronic GvHD (lung, skin), 2 required intravenous immunoglobulin, and 4 attended school or college. CONCLUSIONS Treatment of critically ill patients with SCID with anti-CD6 antibody T cell-depleted MMRD marrow resulted in an overall 50% long-term survival of patients (83% survival of those engrafted). The principal barriers to long-term survival were delay in diagnosis, life-threatening infection, failure to engraft, and chronic GvHD. Educational goals were achieved in most of the survivors.

Viral regulatory region effects on vertical transmission of polyomavirus SV40 in hamsters

Viral strain differences influence the oncogenic potential of polyomavirus simian virus 40 (SV40). We hypothesized that viral strain differences might also affect vertical transmission of SV40 in susceptible hosts. Pregnant Syrian golden hamsters were inoculated intraperitoneally with 10(7) plaque-forming units of SV40 and offspring were sacrificed post-delivery (1-21 days, 6 months). Organ extracts were analyzed for SV40 DNA by polymerase chain reaction assay. Transmission of SV40 from mother to offspring was detected in over half of litters. Most placentas were virus-positive. Mothers inoculated with SV40 strains containing complex regulatory regions transmitted virus more frequently than those infected with simple enhancer viruses (p<0.001). Virus was detected more often in progeny brain than in spleen (p<0.05). Several progeny were virus-positive at 6 months of age, suggesting viral persistence. Maternal animals retained virus in several tissues through day 21 and developed T-antigen antibodies. These results indicate that SV40 replicates in hamsters, vertical transmission of SV40 can occur, and the viral regulatory region influences transmission.

Characterization of T and B cell repertoire diversity in patients with RAG deficiency

Differences in B and T cell repertoires in patients with RAG deficiency associate with clinical severity. Taking SCID genetics to the clinic Mutations that lead to deficiencies in the recombination-activating genes RAG1 and RAG2 result in a spectrum of immunodeficiencies ranging from loss of T and/or B cell repertoire diversity to a complete lack of T and B cells—severe combined immunodeficiency (SCID). Here, Lee et al. perform next-generation B and T cell repertoire sequencing on 12 patients with RAG mutations who have immunodeficiencies of varying severity. They found that the level of repertoire skewing was associated with the severity of disease and that specific repertoire deficiencies were associated with particular phenotypes. These data support a genotype-phenotype connection for primary immunodeficiencies. Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.

Chronic Rotavirus Infection in an Infant with Severe Combined Immunodeficiency: Successful Treatment by Hematopoietic Stem Cell Transplantation

Kobrynski et al. [1] showed gastrointestinal complaints in children can be an early sign of primary immunodeficiency disease (PIDD). Rotavirus infection associated with PIDD can be life-threatening. Rotavirus is a leading cause of childhood gastrointestinal disease worldwide [2]. Most rotavirus disease is caused by 5 G types (G1-G4 and G9) and 3 P types (P1A[8], P1B[4], and P2A[6]) [2]. Two live oral rotavirus vaccines, RotaTeq® (5 different human-bovine reassortant rotavirus strains; Merck and Co, Whitehouse Station, NJ) and Rotarix® (1 human rotavirus strain; GlaxoSmithKline, Rixensart, Belgium) are recommended for routine immunization of US infants [2]. Disease caused by emerging worldwide rotavirus type, G9P[8] may be prevented by both vaccines although this strain is not included in either vaccine. Severe combined immunodeficiency (SCID) is characterized by lack of T cells and life-threatening infections [3]. Treatment of viral infections prior to hematopoietic stem cell transplantation (HSCT) with intravenous immunoglobulin (IVIG) and antivirals has been attempted, but persistence of viral disease has been reported [3–8]. We report a 7 month-old male infant with SCID who had persistent, nonvaccine-associated rotavirus gastroenteritis and viremia despite oral and IVIG administration. T-cell engraftment following HSCT possibly helped by oral and IVIG was necessary to eliminate rotavirus infection. The full-term, formula-fed infant received RotaTeq at 2 and 4 months of age. The patient developed chronic intermittent diarrhea at 2 months of age and was hospitalized at 7 months of age with respiratory distress, diarrhea, and failure to thrive. A peripheral white blood cell count was 16,140 cells/μl with 59% neutrophils, 17% lymphocytes (absolute lymphocyte count=2743cells/μl [normal range 3,900–9,000]), 7% monocytes, and 13% eosinophils. Bronchoscopy aspirate revealed Pneumocystis jiroveci. Stool for rotavirus was positive by electron microscopy (EM). Immunoglobulins were very low including IgG 77 mg/dL (normal range 184–974 mg/dL), IgA <6 mg/dL (normal range 9–107 mg/dL), and IgM 36 mg/dL (normal range 41–197 mg/dL). The CD3+T cells were severely low (32 cells/mm3, normal range 1919–5054 cells/mm3), CD19+B cells were elevated (2715 cells/mm3, normal range 566–2535 cells/mm3), and CD3−CD56+CD16+NK cells were low (28 cells/mm3, normal range 181–901 cells/mm3). T-cell proliferation to mitogens was markedly depressed. A hemizygous mutation (nucleotide substitution A for G at position 1451 in the polyA tail region) was present in the common gamma chain of interleukin-2 receptor consistent with X-linked SCID. Multipe doses of IVIG (Gamunex®, Talecris) were given before and after transplantation, including two doses of 300 mg/kg administered orally at 8 months of age (Fig. 1). Molecular analysis of stool and serum specimens identified a non-vaccine associated human rotavirus strain G9P[8]. The patient received a 10/10 matched unrelated donor unfractionated HSCT with pretransplant myeloablative conditioning at 9.5 months of age. Rotavirus-positive diarrhea persisted until 2 months post transplant (age 11.5 months), coincident with T-cell engraftment (Fig. 1). The patient, last tested at 14.5 months of age, had no detectable rotavirus. Figure 1 The detection of rotavirus in relation to the presence of CD3+,CD4+, and CD8+T-cell quantification before and after bone marrow transplantation. *=CD3+T cells were 100% donor origin calculated by short tandem repeat studies; ¥=Several serum samples ... Reverse transcriptase polymerase chain reaction (RT-PCR) using rotavirus gene 9 and gene 4 primer sets resulted in cDNA products from stool and serum samples. Homology of gene 9 and gene 4 amplicon sequences to GenBank database sequences confirmed the patient’s stools contained rotavirus strain G9P[8]. There was 98% nucleotide homology between the stool rotavirus gene 4 sequence, which comprised 51% of the 2328 nt ORF, and two fully-sequenced P[8] rotaviruses but no significant homology with a partial RotaTeq vaccine gene 4 sequence. There was 98% nucleotide homology between the stool rotavirus gene 9 sequence, which comprised 85% of the 978 nt ORF, and two fully-sequenced G9 rotaviruses. There was a single nucleotide change in gene 9 (residue 595 C→T, resulting in a silent mutation) between two stools obtained 74 days apart. There was no change in gene 4 sequence between stools obtained 54 days apart. Neutralizing antibodies to rotavirus G9 were present in the orally administered immunoglobulin product at a concentration of 1:1600. Neutralizing antibodies to serotypes G1(WA, 1:800; K8, 1:1600) and G3 (SA11, 1:3200) were present at similar concentrations. CD3+T cells were very low (32 cells/ml, normal range 2500–6500 cells/ml) prior to transplantation (Fig. 1). Rotavirus became undetectable by EM two months post transplantation with CD3+, CD4+, and CD8+T-cell engraftment as shown by return of lymphocytes by 65 days post transplantation (CD3+T cells=138/mm3 at 2 months post transplantation). T-cell proliferation, as assessed by response to mitogens, was <3% of normal range and became present at five months post transplantation (data not shown). Chimerism analysis showed presence of donor T cells (100%) and absence of donor B cells (0%) at two and seven months post transplantation.

Scurvy Mimicking Osteomyelitis: Case Report and Review of the Literature

A 9-year-old African American male with a history of autism and nonverbal communication presented to a children’s hospital with refusal to bear weight and a draining mouth sore. The patient had jumped off of a bed and hit his left knee 6 weeks earlier. He initially favored his left knee; however, a plain radiograph at an urgent care facility was negative. The following week, he developed pain in his right leg and a mouth sore. A dental exam during this time revealed gingival bleeding that was attributed to poor dental hygiene. The gingival bleeding progressed and leg swelling developed 2 weeks prior to admission and was accompanied by refusal to bear weight. Plain radiographs of his left leg were unremarkable 1 week prior to admission. Swelling and warmth of the left knee and drainage of yellow-green fluid from a mouth sore prompted evaluation in the emergency department. At presentation, he was afebrile, had a pulse of 124, blood pressure 114/62 mm Hg, and respiratory rate of 22. His weight was at the 37th percentile, height at the 56th percentile, and body mass index at the 20th percentile. Physical examination was remarkable for severe gingivitis with purulent fluid at the gingival line as well as warmth, erythema, tenderness, and swelling of the proximal anterior left tibia with limited range of motion of the left knee and no appreciable effusion. Laboratory evaluation revealed a normal white blood cell count, normocytic anemia, erythrocyte sedimentation rate >140 mm/h, and C-reactive protein 1.3 mg/dL (normal <1.0 mg/dL). Repeat plain radiographs of the left leg were normal. Orthopedic surgery was consulted for concern of osteomyelitis. He was admitted to the hospital for further imaging and intravenous antibiotics. A detailed history showed a diet devoid of everything except oatmeal and soy milk due to sensory aversions. Magnetic resonance imaging showed multifocal areas of increased signal intensity of the distal femurs, left proximal femur, and distal and proximal tibias bilaterally as well as a subperiosteal fluid collection that was suspicious for an abscess (Figure 1A and B). A needle was inserted into the fluid collection yielding a gelatinous red material. Intravenous clindamycin and cefazolin were started empirically for presumed osteomyelitis. Because of the multifocality of the bony lesions and the restricted diet, a vitamin C level was obtained that was undetectable. Bacterial cultures from the needle aspirate and subsequent open left distal femur biopsy were negative and antibiotics were discontinued. The bone biopsy showed changes in the periosteum suggestive of a recent subperiosteal hemorrhage (Figure 1C), which was suggestive of scurvy. Oral medicine evaluated the patient and diagnosed an oral pyogenic granuloma. The patient was started on vitamin C 500 mg once daily and, in conjunction with speech therapy, his diet was expanded to include fruit and vegetable purees. His gingivitis, energy level, and mobility quickly improved. Four weeks after dietary changes and vitamin C supplementation were started, a repeat vitamin C level was normal.