Kumud K. Singh

Human Bocavirus: Prevalence and Clinical Spectrum at a Children's Hospital

Abstract Background. Molecular methods of pathogen discovery have recently led to the description of several new respiratory viruses. Human bocavirus (HBoV), a proposed member of the family Parvoviridae, is one of the most recently described respiratory viruses. Initial reports indicate that HBoV is a common cause of respiratory tract infection in children. Methods. A total of 1474 nasal scraping specimens collected over a 20-month period were screened by polymerase chain reaction for the presence of HBoV nucleic acid. Positive results were confirmed with a second polymerase chain reaction assay from a different genomic region. The medical records of patients with positive results were reviewed for demographic and clinical data. Results. HBoV DNA was identified in 82 samples (5.6%). The peak rate of HBoV infection occurred during the period of March through May in both 2004 and 2005. Sixty-three percent of infected patients were <12 months of age. The most common symptoms were cough, rhinorrhea, and fever. Other symptoms of interest included diarrhea and a “paroxysmal” cough that was clinically suspected to be caused by Bordetella pertussis. Conclusions. HBoV DNA is commonly present in children with upper and lower respiratory tract infections. The presence of a pertussis-like cough and diarrhea in association with HBoV infection merits further investigation.

An MDR1-3435 variant is associated with higher plasma nelfinavir levels and more rapid virologic response in HIV-1 infected children.

Objective:The multidrug-resistance transporter gene (MDR1) encoding for P-glycoprotein (P-gp) and genes encoding for isoenzymes of cytochrome P450 (CYP) have an important role in transport and metabolism of antiretroviral agents. This research examined the impact of single nucleotide polymorphisms (SNP) of MDR1 and CYP genes on nelfinavir and efavirenz pharmacokinetics and the response to highly active antiretroviral therapy (HAART) in HIV-1 infected children. Methods:Seventy-one HIV-1-infected children from PACTG 382 receiving nelfinavir, efavirenz and one or two nucleoside reverse transcriptase inhibitors had genomic DNA from PBMC evaluated for MDR1 and CYP SNP by real-time PCR. Plasma drug concentrations, CD4 lymphocyte counts and HIV-1 RNA were measured during HAART. Results:The frequencies of C/C, C/T and T/T genotypes in the MDR1-3435-C→T polymorphisms were 44% (n = 31), 46% (n = 33) and 10% (n = 7), respectively. Ninety-one percent of children with the C/T genotype reached plasma HIV-1 RNA < 400 copies/ml by week 8 compared to 59% of children with the C/C genotype (P = 0.01). Children with the C/T genotypes had higher 8 h postdose concentration (P = 0.02) and lower clearance rate (P = 0.04) for nelfinavir compared to those with the C/C genotype. The seven children with the T/T genotype had nelfinavir pharmacokinetics and virologic response similar to those with the C/C genotype. No compensatory polymorphisms were observed between MDR1 and CYP genotypes. Conclusions:HIV-1 infected children with the MDR1-3435-C/T genotype had more rapid virologic responses to HAART at week 8 with higher plasma nelfinavir concentrations compared to those with the C/C genotype. These findings suggest that P-gp may play an important role in the pharmacokinetics and virologic response to HAART containing nelfinavir.

Longevity of lobsters is linked to ubiquitous telomerase expression

Mammals have high growth rates in embryonic and juvenile phases and no growth in adult and senescent phases. We analyzed telomerase activity in a fundamentally different animal which grows indeterminately. Lobsters (Homarus americanus) grow throughout their life and the occurrence of senescence is slow. A modified TRAP assay was developed and the lobster telomeric repeat sequence TTAGG was determined. We detected telomerase activities which were dependent on RNA and protein components, required dGTP, dATP and dTTP, but not dCTP. Telomerase products with a five nucleotide periodicity were generated. High telomerase activities were detected in all lobster organs. We conclude that telomerase activation is a conserved mechanism for maintaining long‐term cell proliferation capacity and preventing senescence, not only in cellular models or embryonic life stages but also in adult multicellular organisms.

Human immunodeficiency virus type 1 subtype C tat fails to induce intracellular calcium flux and induces reduced tumor necrosis factor production from monocytes

ABSTRACT Over 50% of all human immunodeficiency virus type 1 (HIV-1) infections worldwide are caused by subtype C strains, yet most research to date focuses on subtype B, the subtype most commonly found in North America and Europe. The HIV-1 trans-acting regulatory protein (Tat) is essential for regulating productive replication of HIV-1. Tat is secreted by HIV-infected cells and alters several functions of uninfected bystander cells. One such function is that, by acting at the cell membrane, subtype B Tat stimulates the production of tumor necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2) from human monocytes and can act as a chemoattractant. In this study, we show that the mutation of a cysteine to a serine at residue 31 of Tat commonly found in subtype C variants significantly inhibits the abilities of the protein to bind to chemokine (C-C motif) receptor 2 (CCR2), induce intracellular calcium flux, stimulate TNF and CCL2 production, and inhibit its chemoattractant properties. We also show that TNF is important in mediating some effects of extracellular Tat. This report therefore demonstrates the important functional differences between subtype C and subtype B Tat and highlights the need for further investigation into the different strains of HIV-1.

Undiagnosed Respiratory Viruses in Children

OBJECTIVE. An estimated 12 to 32 million upper respiratory infections occur in young children each year. In addition, 20% to 53% of infants will have ≥1 episode of lower respiratory infection in the first year of life. The current methods of diagnosing respiratory viruses are limited in scope and sensitivity. Polymerase chain reaction is a more sensitive method than antigen detection and is often used for newly discovered viruses. Using polymerase chain reaction, we sought to diagnose adenoviruses, human bocavirus, and human metapneumovirus at our childrens hospital. METHODS. Nasal-swab specimens submitted for antigen detection of respiratory viruses were collected and processed by using polymerase chain reaction to detect adenoviruses, human bocavirus, and human metapneumovirus. Inpatient and emergency department records were reviewed for clinical and demographic data. RESULTS. Approximately 1500 specimens were collected over 21 months; they contained adenoviruses, human bocavirus, and human metapneumovirus in 5.9%, 5.6%, and 5.2% of children, respectively. Using polymerase chain reaction and antigen detection, a viral agent was identified in as many as 62% of the specimens. Lower respiratory tract disease was present most frequently in patients infected with human metapneumovirus (63%) and least frequently in those infected with adenoviruses (45%). We detected adenoviruses by polymerase chain reaction in 59 patients for whom the antigen-detection test results were negative. A paroxysmal cough led to clinical suspicion of Bordetella pertussis infection in 19% of patients infected with human bocavirus. CONCLUSIONS. Adenoviruses, human bocavirus, and human metapneumovirus were each present in ∼5% of specimens submitted for respiratory virus rapid testing. The lower respiratory tract was more commonly affected in patients with human bocavirus and human metapneumovirus infections. Adenovirus was often undiagnosed by antigen detection. Other findings included the presence of a pertussis-like illness associated with human bocavirus.

Rapid kinetic characterization of hammerhead ribozymes by real-time monitoring of fluorescence resonance energy transfer (FRET).

In established methods for analyzing ribozyme kinetics, radiolabeled RNA substrates are primarily used. Each data point requires the cumbersome sampling, gel electrophoretic separation, and quantitation of reaction products, apart from the continuous loss of substrate by radioactive decay. We have used stable, double fluorescent end-labeled RNA substrates. Fluorescence of one fluorophore is quenched by intramolecular energy transfer (FRET). Upon substrate cleavage, both dyes become separated in two RNA products and fluorescence is restored. This can be followed in real time and ribozyme reactions can be analyzed under multiple (substrate excess) and under single (ribozyme excess) turnover conditions. A detailed comparison of unlabeled, single, and double fluorescent-labeled RNAs revealed moderate kinetic differences. Results with two systems, hammerhead ribozymes in I/II (small ribozyme, large substrate) and in I/III format (large ribozyme, small substrate), are reported.

Genetic polymorphisms in CX3CR1 predict HIV-1 disease progression in children independently of CD4+ lymphocyte count and HIV-1 RNA load

BACKGROUND The impact of CX3CR1 polymorphisms on human immunodeficiency virus type 1 (HIV-1) pathogenesis is controversial, with conflicting reports of their role in disease progression in HIV-1-infected adults. METHODS A cohort of 1055 HIV-1-infected children were genotyped for 2 CX3CR1 polymorphisms, V/I249 and T/M280, and their impact on HIV-1-related disease progression, including central nervous system (CNS) impairment, was evaluated. RESULTS Children with the CX3CR1 I/I249 genotype experienced more-rapid disease progression (I/I249 vs. V/V249: relative hazard [RH], 2.19 [95% confidence interval {CI}, 1.30-3.68], P=.003; I/I249 vs. V/I249: RH, 1.77 [95% CI, 1.00-3.14], P=.05) and a trend toward more CNS impairment (I/I249 vs. V/V249: RH, 2.19 [95% CI, 1.00-4.78], P=.049; I/I249 vs. V/I249: RH, 2.02 [95% CI, 0.85-4.83], P=.11). Children with the V249-T280 haplotype experienced significantly less disease progression (RH, 0.42 [95% CI, 0.24-0.73]; P=.002) and CNS impairment (RH, 0.39 [95% CI, 0.39-0.22]; P=.001). Of note, these effects remained significant after CD4+ lymphocyte count and plasma HIV-1 RNA load at baseline were adjusted for and in a longitudinal, multivariate analysis. CONCLUSIONS CX3CR1 genotypes and haplotypes impact HIV-1 disease progression independently of CD4+ lymphocyte count and plasma HIV-1 RNA load, suggesting that the fundamental role of CX3CR1 in the alteration of disease progression might be the recruitment of immunomodulatory cells responsible for the control of HIV-1.

APOE ε4 and MBL-2 O/O genotypes are associated with neurocognitive impairment in HIV-infected plasma donors

Background:Host genetic factors are important determinants for risk of HIV-1 infection and disease progression. This study examined associations of host genetic variants and neurocognitive impairment in Chinese individuals infected through contaminated blood products. Methods:Two hundred and one HIV-infected patients from Anhui, China, had neuropsychological tests at baseline and 12 months. DNA was genotyped for APOE ϵ2, ϵ3 and ϵ4 alleles; MBL2-A/O; CCR5-wt/Δ32; CCR5-59029-G/A; CCR2-180-G/A; SDF-1-G/A; IL4-589-C/T; MCP-1-2518-A/G; CX3CR1-745-G/A; −849-C/T polymorphisms and CCL3L1 copy number variants using real-time PCR. Univariate and multivariate analyses were performed. Results:The cohort included 61% men, with mean education 5.5 years, AIDS diagnosis 113 (55%), on antiretrovirals 114 (56%), mean baseline CD4+ cell count 349 cells/μl and mean log10 RNA 4.09. At baseline, 37% had global neuropsychological impairment increasing to 44% after 12 months. Of 43 patients with the APOE ϵ4 allele, 58% were cognitively impaired compared with 31% without the ϵ4 allele (P = 0.001, odds ratio 3.09, 95% confidence interval 1.54–6.18). The mean global deficit score (GDS) for ϵ4-positive participants on antiretrovirals for 12 months was 0.88 (0.55) compared with 0.63 (0.54) for ϵ4-negative participants (P = 0.053, 95% confidence interval −0.004 to 0.51). For MBL2, 52% of patients with the O/O genotype declined in cognitive function over 12 months compared with 23% with A/A (odds ratio 3.62, 95% confidence interval 1.46–9.03, P = 0.004). No associations were observed for the other genetic variants. Conclusion:The APOE ϵ4 allele was associated with increased risk for cognitive deficits, whereas the MBL2 O/O genotype was associated with increased risk for progressive cognitive decline in Chinese individuals infected with HIV through contaminated blood products.

Host Genetic Determinants of Human Immunodeficiency Virus Infection and Disease Progression in Children

Increasing data support host genetic factors as an important determinants of human immunodeficiency virus type-1 (HIV-1) susceptibility, mother-to-child transmission (MTCT), and disease progression. Of these genetic mediators, those impacting innate and adaptive immune responses seem to play a critical role in viral infectivity and pathogenesis. During primary infection, CCR5 using virus is predominantly transmitted and polymorphisms that affect the expression of CCR5 alter the risk for MTCT and rate of disease. Chemokines that naturally bind to coreceptors alter infectivity and viral pathogenesis. Additional genes that affect innate immunity including those encoding for MBL2 and those modulating the adaptive immune response including CX3CR1 and human leukocyte antigen types can significantly modify susceptibility and response to HIV-1 infection. As young children develop, the dependence on certain arms of the immune system varies and can alter the effect of genetic variants. Additionally, host genetic factors may alter the response to antiretrovirals. Finally, because HIV-infected children progress more rapidly than adults and have fewer background cofactors, such as drug use and coinfections, the effects of host factors on HIV-1 disease may be more clearly identified. In this review, we summarize available data on the impact of host genetics on MTCT and disease progression of HIV-infected children.

Human metapneumovirus associated with central nervous system infection in children.

Background: Human metapneumovirus (hMPV) is an established pathogen of the respiratory tract of children and adults. hMPV is related to other paramyxoviruses known to cause encephalitis. Reports suggest that hMPV may cause disease of the central nervous system (CNS). Methods: Two groups of patients were studied. The first group consisted of children between birth and 18 years from whom nasal scrapings were obtained between January 2004 and October 2005. hMPV RNA amplification by PCR was done and records were reviewed for clinical and demographic data. The second group consisted of patients with encephalitis referred to the California Encephalitis Project (CEP) for comprehensive diagnostic testing between November 2004 and June 2006. Results: In group 1, 1474 specimens were examined for hMPV RNA. Sixty-three evaluable patients were infected with hMPV of whom 4 (6.3%) had seizures, compared with 145 patients infected with RSV of whom 1 had seizures (0.7%, P = 0.031). Comparing respiratory syncytial virus (RSV) and hMPV infections, there was no significant difference in the occurrence of fever. All children with hMPV infections and seizures were hospitalized and 3 were intubated because of status epilepticus. Group 2 consisted of 205 pediatric cases referred to CEP between November 2004 and June 2006 who had hMPV testing done. hMPV was detected in nasopharyngeal swabs of 5 patients. Neither hMPV RNA nor antihMPV specific IgM were detectable in the CSF from the 5 patients for whom CSF was available. Conclusion: Nine cases of CNS illness temporally associated with the presence of hMPV nucleic acid in the upper airway are described. Compared with children infected with RSV, children with hMPV were significantly more likely to have had a seizure. Our data, in conjunction with previously reported cases suggest that hMPV may be associated with a spectrum of CNS disease ranging from febrile seizures to severe, fatal encephalitis.