Masako Shimamura

Saliva Polymerase-Chain-Reaction Assay for Cytomegalovirus Screening in Newborns

BACKGROUND Congenital cytomegalovirus (CMV) infection is an important cause of hearing loss, and most infants at risk for CMV-associated hearing loss are not identified early in life because of failure to test for the infection. The standard assay for newborn CMV screening is rapid culture performed on saliva specimens obtained at birth, but this assay cannot be automated. Two alternatives--real-time polymerase-chain-reaction (PCR)-based testing of a liquid-saliva or dried-saliva specimen obtained at birth--have been developed. METHODS In our prospective, multicenter screening study of newborns, we compared real-time PCR assays of liquid-saliva and dried-saliva specimens with rapid culture of saliva specimens obtained at birth. RESULTS A total of 177 of 34,989 infants (0.5%; 95% confidence interval [CI], 0.4 to 0.6) were positive for CMV, according to at least one of the three methods. Of 17,662 newborns screened with the use of the liquid-saliva PCR assay, 17,569 were negative for CMV, and the remaining 85 infants (0.5%; 95% CI, 0.4 to 0.6) had positive results on both culture and PCR assay. The sensitivity and specificity of the liquid-saliva PCR assay were 100% (95% CI, 95.8 to 100) and 99.9% (95% CI, 99.9 to 100), respectively, and the positive and negative predictive values were 91.4% (95% CI, 83.8 to 96.2) and 100% (95% CI, 99.9 to 100), respectively. Of 17,327 newborns screened by means of the dried-saliva PCR assay, 74 were positive for CMV, whereas 76 (0.4%; 95% CI, 0.3 to 0.5) were found to be CMV-positive on rapid culture. Sensitivity and specificity of the dried-saliva PCR assay were 97.4% (95% CI, 90.8 to 99.7) and 99.9% (95% CI, 99.9 to 100), respectively. The positive and negative predictive values were 90.2% (95% CI, 81.7 to 95.7) and 99.9% (95% CI, 99.9 to 100), respectively. CONCLUSIONS Real-time PCR assays of both liquid- and dried-saliva specimens showed high sensitivity and specificity for detecting CMV infection and should be considered potential screening tools for CMV in newborns. (Funded by the National Institute on Deafness and Other Communication Disorders.).

DRIED BLOOD SPOT REAL-TIME POLYMERASE CHAIN REACTION ASSAYS TO SCREEN NEWBORNS FOR CONGENITAL CYTOMEGALOVIRUS INFECTION

CONTEXT Reliable methods to screen newborns for congenital cytomegalovirus (CMV) infection are needed for identification of infants at increased risk of hearing loss. Since dried blood spots (DBS) are routinely collected for metabolic screening from all newborns in the United States, there has been interest in using DBS polymerase chain reaction (PCR)-based methods for newborn CMV screening. OBJECTIVE To determine the diagnostic accuracy of DBS real-time PCR assays for newborn CMV screening. DESIGN, SETTING, AND PARTICIPANTS Between March 2007 and May 2008, infants born at 7 US medical centers had saliva specimens tested by rapid culture for early antigen fluorescent foci. Results of saliva rapid culture were compared with a single-primer (March 2007-December 2007) and a 2-primer DBS real-time PCR (January 2008-May 2008). Infants whose specimens screened positive on rapid culture or PCR had congenital infection confirmed by the reference standard method with rapid culture testing on saliva or urine. MAIN OUTCOME MEASURES Sensitivity, specificity, and positive and negative likelihood ratios (LRs) of single-primer and 2-primer DBS real-time PCR assays for identifying infants with confirmed congenital CMV infection. RESULTS Congenital CMV infection was confirmed in 92 of 20,448 (0.45%; 95% confidence interval [CI], 0.36%-0.55%) infants. Ninety-one of 92 infants had positive results on saliva rapid culture. Of the 11,422 infants screened using the single-primer DBS PCR, 17 of 60 (28%) infants had positive results with this assay, whereas, among the 9026 infants screened using the 2-primer DBS PCR, 11 of 32 (34%) screened positive. The single-primer DBS PCR identified congenital CMV infection with a sensitivity of 28.3% (95% CI, 17.4%-41.4%), specificity of 99.9% (95% CI, 99.9%-100%), positive LR of 803.7 (95% CI, 278.7-2317.9), and negative LR of 0.7 (95% CI, 0.6-0.8). The positive and negative predictive values of the single-primer DBS PCR were 80.9% (95% CI, 58.1%-94.5%) and 99.6% (95% CI, 99.5%-99.7%), respectively. The 2-primer DBS PCR assay identified infants with congenital CMV infection with a sensitivity of 34.4% (95% CI, 18.6%-53.2%), specificity of 99.9% (95% CI, 99.9%-100.0%), positive LR of 3088.9 (95% CI, 410.8-23 226.7), and negative LR of 0.7 (95% CI, 0.5-0.8). The positive and negative predictive values of the 2-primer DBS PCR were 91.7% (95% CI, 61.5%-99.8%) and 99.8% (95% CI, 99.6%-99.9%), respectively. CONCLUSION Among newborns, CMV testing with DBS real-time PCR compared with saliva rapid culture had low sensitivity, limiting its value as a screening test.

Human Cytomegalovirus Infection Elicits a Glycoprotein M (gM)/gN-Specific Virus-Neutralizing Antibody Response

ABSTRACT Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that infects 40 to 90% of adult human populations. HCMV infections are often asymptomatic in healthy individuals but can cause severe organ and life-threatening disease in immunocompromised patients. The antiviral antibody response to HCMV infection is complex and is known to include virus-neutralizing antibody production against surface glycoproteins encoded by HCMV. We have investigated the human antibody response to a complex of HCMV surface glycoproteins composed of glycoprotein M (gM)/gN, the gene products of the UL100 and UL73 open reading frames. Mouse monoclonal antibodies generated against gM/gN have previously been shown to neutralize HCMV infection of human fibroblasts in vitro. To determine whether human antibodies reactive with the gM/gN complex possess virus-neutralizing properties, we isolated human antibodies reactive with gM/gN from pooled human HCMV hyperimmune globulin by affinity purification using recombinant gM/gN. The affinity-purified human anti-gM/gN antibodies reacted specifically by immunofluorescence with HCMV-infected human fibroblasts and with cells transiently expressing gM/gN, but not with cells transfected with plasmids encoding other immunogenic HCMV proteins. The anti-gM/gN antibodies also reacted specifically only with gM/gN in immunoblot assays using lysates of transfected cells expressing specific HCMV proteins. Last, human anti-gM/gN antibodies efficiently neutralized infectious HCMV in vitro with a capacity comparable to that of human anti-gB antibodies. These data indicated that gM/gN can elicit a virus-neutralizing antibody response in humans infected with HCMV and therefore should be considered a potential candidate for inclusion in prophylactic CMV vaccines.

The lysosomal targeting and intracellular metabolism of trypanosome lytic factor by Trypanosoma brucei brucei.

Trypanosome lytic factor (TLF) provides innate protection for humans against infection by the animal pathogen Trypanosoma brucei brucei but not against the agent of human African sleeping sickness, Trypanosoma brucei rhodesiense. TLF exists in two forms, TLF-1 and TLF-2. Prior studies suggested that TLF-1 causes lysosomal disruption and subsequent cell death in T. b. brucei. Here we confirm the lysosomal targeting of TLF-1 by immunolocalization with the trypanosome lysosomal membrane protein p67, and by co-fractionation of radiolabelled TLF-1 with lysosomal enzymes. In addition, pulse-chase studies indicate that TLF-1 is not degraded within the lysosome as compared to the host protein transferrin. In TLF-1 treated cells, transferrin is degraded normally, indicating that lysosomal proteases remain active during the early phase of TLF-1 treatment but fail to degrade TLF-1. Following endocytosis a TLF lipoprotein appears to undergo disulfide bond reduction prior to entering the lysosome. Results presented here indicate that TLF-1 lipoproteins are targeted to the lysosome but are resistant to trypanosome lysosomal proteases.

Insight into the mechanism of trypanosome lytic factor-1 killing of Trypanosoma brucei brucei.

It has been known for almost a century that normal human serum can lyse the extracellular blood parasite Trypanosoma brucei brucei. This process is a result of a non-immune killing factor in human sera known as trypanosome lytic factor (TLF). In this work, we demonstrate that killing of T. b. brucei by trypanosome lytic factor-1 (TLF-1) in vitro is inhibited by the lipophyllic iron chelator, LI, the lipophyllic antioxidant DPPD, and the protease inhibitors antipain and E64. Thus TLF-1 killing likely requires iron, oxidants, and serine and cysteine proteases. Furthermore, we demonstrate that TLF-1 mediated lysis causes measurable peroxidation in T. brucei lipids via a reaction that is inhibited by DPPD, weak bases, and human haptoglobin. We hypothesize that TLF-1 lysis requires intracellular factors within the trypanosome including high intracellular H2O2 and high polyenoic lipid concentrations, lysosomal acidification and proteases, and intracellular iron sources. The data presented supports the hypothesis that the combination of these factors with TLF-1 inside the lysosome results in lysosomal membrane breakdown, release of the lysosomal contents, and subsequent autodigestion of the cell.

Human cytomegalovirus induces TGF-β1 activation in renal tubular epithelial cells after epithelial-to-mesenchymal transition.

Human cytomegalovirus (HCMV) infection is associated epidemiologically with poor outcome of renal allografts due to mechanisms which remain largely undefined. Transforming growth factor-β1 (TGF-β1), a potent fibrogenic cytokine, is more abundant in rejecting renal allografts that are infected with either HCMV or rat CMV as compared to uninfected, rejecting grafts. TGF-β1 induces renal fibrosis via epithelial-to-mesenchymal transition (EMT) of renal epithelial cells, a process by which epithelial cells acquire mesenchymal characteristics and a migratory phenotype, and secrete molecules associated with extracellular matrix deposition and remodeling. We report that human renal tubular epithelial cells infected in vitro with HCMV and exposed to TGF-β1 underwent morphologic and transcriptional changes of EMT, similar to uninfected cells. HCMV infected cells after EMT also activated extracellular latent TGF-β1 via induction of MMP-2. Renal epithelial cells transiently transfected with only the HCMV IE1 or IE2 open reading frames and stimulated to undergo EMT also induced TGF-β1 activation associated with MMP-2 production, suggesting a role for these viral gene products in MMP-2 production. Consistent with the function of these immediate early gene products, the antiviral agents ganciclovir and foscarnet did not inhibit TGF-β1 production after EMT by HCMV infected cells. These results indicate that HCMV infected renal tubular epithelial cells can undergo EMT after exposure to TGF-β1, similar to uninfected renal epithelial cells, but that HCMV infection by inducing active TGF-β1 may potentiate renal fibrosis. Our findings provide in vitro evidence for a pathogenic mechanism that could explain the clinical association between HCMV infection, TGF-β1, and adverse renal allograft outcome.

Acyclovir-resistant chronic verrucous vaccine strain varicella in a patient with neuroblastoma.

A 21-month-old girl with neuroblastoma developed chronic verrucous Oka strain varicella-zoster infection during chemotherapy. Virus isolated from the patient demonstrated high-level acyclovir resistance, and its thymidine kinase had no in vitro enzymatic activity. After foscarnet therapy, she underwent stem cell transplantation without varicella reactivation. This is only the second reported case of resistant varicella zoster virus caused by Oka strain virus.

Cytomegalovirus Enhances Macrophage TLR Expression and MyD88-Mediated Signal Transduction To Potentiate Inducible Inflammatory Responses

Circulating monocytes carrying human CMV (HCMV) migrate into tissues, where they differentiate into HCMV-infected resident macrophages that upon interaction with bacterial products may potentiate tissue inflammation. In this study, we investigated the mechanism by which HCMV promotes macrophage-orchestrated inflammation using a clinical isolate of HCMV (TR) and macrophages derived from primary human monocytes. HCMV infection of the macrophages, which was associated with viral DNA replication, significantly enhanced TNF-α, IL-6, and IL-8 gene expression and protein production in response to TLR4 ligand (LPS) stimulation compared with mock-infected LPS-stimulated macrophages during a 6-d in vitro infection. HCMV infection also potentiated TLR5 ligand–stimulated cytokine production. To elucidate the mechanism by which HCMV infection potentiated inducible macrophage responses, we show that infection by HCMV promoted the maintenance of surface CD14 and TLR4 and TLR5, which declined over time in mock-infected macrophages, and enhanced both the intracellular expression of adaptor protein MyD88 and the inducible phosphorylation of IκBα and NF-κB. These findings provide additional information toward elucidating the mechanism by which HCMV potentiates bacteria-induced NF-κB–mediated macrophage inflammatory responses, thereby enhancing organ inflammation in HCMV-infected tissues.

Comparison of saliva PCR assay versus rapid culture for detection of congenital cytomegalovirus infection.

As part of the CMV and Hearing Multicenter Screening (CHIMES) study, 72,239 newborns were screened for cytomegalovirus by rapid culture and real-time PCR of saliva samples. Of the 266 infants with congenital cytomegalovirus infection, discordance between rapid culture and PCR was observed in 14 children, and 13 were identified only by PCR, demonstrating the superiority of the PCR assay.

Δγ134.5 Herpes Simplex Viruses Encoding Human Cytomegalovirus IRS1 or TRS1 Induce Interferon Regulatory Factor 3 Phosphorylation and an Interferon-Stimulated Gene Response

ABSTRACT The chimeric herpes simplex viruses (HSV) are Δγ134.5 vectors encoding the human cytomegalovirus (HCMV) IRS1 or TRS1 genes. They are capable of late viral protein synthesis and are superior to Δγ134.5 HSVs in oncolytic activity. The interferon (IFN) response limits efficient HSV gene expression and replication. HCMV TRS1 and IRS1 restore one γ134.5 gene function: evasion of IFN-inducible protein kinase R, allowing late viral protein synthesis. Here we show that, unlike wild-type HSV, the chimeric HSV do not restore another γ134.5 function, the suppression of early IFN signaling mediated by IFN regulatory factor 3 (IRF3).