Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

Influenza hospitalizations in Australian children.

Australia's National Immunisation Program (NIP) provides free influenza vaccination for children at high risk of severe influenza; a pilot-funded programme for vaccine in all children aged 6 months to <5 years in one of eight states, has seen poor vaccine impact, related to recent vaccine safety concerns. This retrospective review examined influenza hospitalizations in children aged <16 years from three seasons (2011-2013) at two paediatric hospitals on opposite sides of the country. Comparisons of this cohort were made with state-based data on influenza-coded hospitalizations and national immunization register data on population-level immunization coverage. Of 740 hospitalizations, the majority were aged <5 years (476/740, 64%), and a substantial proportion (57%) involved healthy children, not currently funded for influenza vaccine. Intensive care unit admission occurred in 8·5%, and 1·5% of all children developed encephalitis. Use of antiviral therapy was uncommon (20·5%) and decreasing. Of those hospitalized, only 5·0% of at-risk children, who are currently eligible for free vaccine, and 0·7% of healthy children were vaccinated prior to hospitalization. This was consistent with low population-wide estimates of influenza vaccine uptake. It highlights the need to examine alternative strategies, such as universally funded paediatric influenza vaccination, to address disease burden in Australian children.

A Rare Case of Q Fever Osteomyelitis in a Child From Regional Australia.

Q fever osteomyelitis is a rare disease. We report an eighth pediatric case from regional Australia. Serology is the first-line diagnostic test, with confirmation by PCR on tissue specimens. In endemic settings, Q fever should be considered in the differential diagnosis of chronic osteomyelitis; in particular, presumed chronic-recurrent multifocal osteomyelitis should be considered a possible presentation of Q fever osteo-articular disease in children.

Frequency of oseltamivir resistance in Sydney, during the Newcastle outbreak of community transmitted oseltamivir-resistant influenza A(H1N1)pdm09 virus, Australia, June to August 2011.

Although oseltamivir-resistant pandemic influenza A(H1N1)pdm09 is uncommon in immunocompetent individuals, a recent report from Newcastle, Australia, showed the first sustained community spread, from June to August 2011, of oseltamivir-resistant influenza A(H1N1)pdm09 virus carrying the H275Y neuraminidase (NA) mutation. To determine the frequency and the extent of this viral variant spread in the nearest major city to Newcastle, we performed a sequencebased genotypic assessment on samples from 143 oseltamivir untreated and 23 oseltamivir post-treatment individuals with influenza collected contemporaneously in Sydney, 120 km southwest of Newcastle. The detection of two of 143 (1.4%) community-derived samples containing H275Y suggests a low prevalence of oseltamivir-resistant influenza A(H1N1)pdm09 virus in the general community and no convincing evidence of spread of the NA H275Y-bearing influenza A(H1N1)pdm09 virus. In oseltamivir treated patients, oseltamivir-resistant influenza A(H1N1)pdm09 virus continue to emerge with three of 23 (13%) post-treatment samples containing the H275Y mutation. The observation of signature mutations and distinct phylogenetic relationship in full-length sequences of haemagglutinin and neuraminidase genes derived from 2011 strains against 2009 strains indicates continued genetic evolution and antigenic drift of the influenza A(H1N1)pdm09 viruses circulating in Australia.

The burden of rotavirus gastroenteritis in children presenting to a paediatric hospital.

The aim of this study was to determine the burden, management and outcomes of rotavirus infection in young children presenting to a tertiary paediatric hospital in Sydney, Australia. All laboratory-confirmed cases of rotavirus in children aged <5 years were identified and medical records reviewed. In 2004, 80 children aged <5 years presented to the hospital with rotavirus gastroenteritis confirmed by stool testing. Infants aged <24 months comprised 75% of cases, with more males than females affected. Most children (86%) acquired rotavirus infection in the community, with a mean length of hospital admission of 2.3 days. There were eight cases of nosocomial infection at a rate of 3/10 000 admissions. The rates of intravenous fluid management (46%) and antibiotic use (28%) were high, reflecting the severity of disease presenting in a hospital setting. These data will help inform the assessment of the recently introduced rotavirus vaccination programme in Australia.

Pancreatitis and adenoviral infection in children after blood and marrow transplantation.

Pancreatitis is a well-recognized consequence of blood and marrow transplantation (BMT). In a 4-year period, between January 2001 and December 2004, five children who received a BMT in our institution were diagnosed as having pancreatitis. Four of these five children also had adenoviral infection. We report these four cases and highlight the importance of investigating for pancreatitis patients who have any abdominal symptoms post BMT, and include specific stool culture for viral isolation, if it is not already known.

Mycoplasma pneumoniae infections in Australian children.

OBJECTIVES: To describe the epidemiology, clinical features and outcome of Mycoplasma pneumoniae infection in children presenting to a tertiary children's hospital. METHODS: Sixty-three month retrospective review of serologically diagnosed M. pneumoniae infections. RESULTS: There were 76 children, 42 boys and 34 girls, mean age 6.3 +/- 3.5 years. The age group most commonly affected was 5-9 years, followed by children 1-5 years. More than half of the patients had failed to respond to antibiotics before referral. The commonest presentation was with cough and fever. Coryza, diarrhoea, vomiting, tachypnoea and recession were significantly more common in children less than 5 years than in children 5-15 years. Hospitalized patients were more likely than non-hospitalized patients to have respiratory distress with recession and wheeze. Radiographic findings were non-specific. Thrombocytosis was found in 29 (41.4%) of 70 children studied. CONCLUSION: The clinical features of M. pneumoniae infection were different in children less than 5 years than in children aged 5-9 years. The presence of thrombocytosis in 40% of the cases has not previously been reported in children.

Cytomegalovirus antiviral resistance associated with treatment induced UL97 (protein kinase) and UL54 (DNA polymerase) mutations.

HCMV-related illness due to infections with antiviral resistant virus was verified by phenotypic and genotypic assays in 17% (8/47) of high-risk immunocompromised Australian patients. Selective PCR-sequencing of UL97 (protein kinase; PK) and UL54 (DNA polymerase; DNApol) regions important for antiviral sensitivity, identified the majority (6/8) of resistant strains through detection of mutations known to confer antiviral resistance. Additionally, eight UL54 (DNApol) mutations (N408K, T691S, A692V, S695T, L737M, A834P, V955I, and A972V) of unknown phenotype were identified in six specimens from patients with clinical evidence of antiviral resistant infections. One isolate was resistant to ganciclovir (GCV) and another resistant to PFA on phenotypic testing where mutations in UL97 (PK) or UL54 (DNApol) were not detected, suggesting a loss of correlation between phenotype and genotype. Selective PCR-sequencing of UL97 (PK) and UL54 (DNApol) provided rapid and comprehensive results, but missed some resistance detected by phenotypic assays. A combination of phenotypic and genotypic assays is recommended for complete analysis of CMV antiviral resistance, as well as further definition of the clinical relationship between novel UL54 (DNApol) mutations and antiviral resistance.

Bacteraemia and antibiotic use in respiratory syncytial virus infections.

AIMS: To examine the frequency of and risk factors for bacteraemia in children hospitalised with respiratory syncytial virus (RSV) infection; and to determine current use of antibiotics in hospitalised children with RSV infection. METHODS: Retrospective study of all children, aged 0-14 years, admitted to a tertiary children's hospital with proven RSV infection over a four year period. Children with concurrent bacteraemia and RSV infection were identified, and risk factors examined for bacteraemia. The case notes of a randomly selected comparison sample of 100 of these RSV infected children were examined to assess antibiotic use and population incidence of risk factors for severe RSV infection. RESULTS: A total of 1795 children had proven RSV infection, and blood cultures were sent on 861 (48%). Eleven (0.6%) of the 1795 RSV positive children had bacteraemia. RSV positive children had a significantly higher incidence of bacteraemia if they had nosocomial RSV infection (6.5%), cyanotic congenital heart disease (6.6%), or were admitted to the paediatric intensive care unit (2.9%). Forty five (45%) of the random comparison sample of RSV infected children received antibiotics. CONCLUSIONS: Bacteraemia is rare in RSV infection. Children with RSV infection are more likely to be bacteraemic, however, if they have nosocomial RSV infection, cyanotic congenital heart disease, or require intensive care unit admission.

Diagnosis and management of paediatric hepatitis C virus infection.

Hepatitis C virus (HCV) infection in children is uncommon and there are few guidelines indicating optimal management. It is estimated that 125-250 children are infected vertically with HCV in Australia each year and very few of these children are diagnosed and followed medically. Without accurate diagnosis and follow up, these children cannot be offered optimal care, and are at risk of presenting in adult life with significant liver pathology and long-term sequelae.

Transcriptional regulation of major histocompatibility complex class I by flavivirus West Nile is dependent on NF-kappaB activation.

Infection by the flavivirus West Nile (WNV) is associated with a virus-specific increase of major histocompatibility complex class I (MHC-I) molecules on the cell surface of diploid vertebrate cells. The increased MHC-I cell surface expression is functional and is associated with increased susceptibility to secondary WNV-immune and alloimmune cytotoxic T cells. WNV-induced up-regulation of cell surface MHC-I expression is associated with NF-kappaB activation and increased transcription of MHC-I mRNA. WNV infection increases luciferase activity of RAWa4 long terminal repeat (LTR) cells, which are transfected stably with a plasmid containing 2 NF-kappaB binding sites, the human immunodeficiency virus LTR linked to a luciferase reporter gene. The NF-kappaB-induced complexes are a p50/p65 heterodimer and another faster migrating species containing p50 homodimers. WNV-induced activation of NF-kappaB and the up-regulation of MHC-I were blocked by the protein kinase C inhibitor H-7 and salicylate, both of which block phosphorylation of inhibitor kappaB.

Management of neonatal herpes simplex virus infection.

Herpes simplex viruses (HSV) are ubiquitous pathogens which can be transmitted vertically causing significant morbidity and mortality in neonates. Neonatal HSV infection is infrequent with an incidence ranging from 1 in 3,500 to 1 in 20,000, depending on the population. Neonatal HSV infection is much more frequent in infants born to mothers experiencing a primary HSV infection with an incidence approaching 50%, while infants born to mothers experiencing recurrent HSV infection have an incidence of less than 3%. Neonatal infections are clinically categorised according to the extent of the disease. They are: (i) skin, eye and mouth (SEM) infections; (ii) central nervous system infection (encephalitis)--neonatal encephalitis can include SEM infections; and (iii) disseminated infection involving several organs, including the liver, lung, skin and/or adrenals. The central nervous system may also be involved in disseminated infections. Caesarean section, where the amniotic membranes are intact or have been ruptured for less than 4 hours, is recommended for those women who have clinical evidence of active herpes lesions on the cervix or vulva at the time of labour. This procedure significantly decreases the risk of transmission to the infant. Diagnosis of neonatal infection requires a very high level of clinical awareness as only a minority of mothers will have a history of genital HSV infection even though they are infected. Careful physical examination and appropriate investigations of the infant should accurately identify the infection in the majority of cases. Treatment is recommended where diagnosis is confirmed or there is a high level of suspicion. The current recommendation for treatment is aciclovir 20 mg/kg 3 times daily by intravenous infusion. Careful monitoring of hydration and renal function as well as meticulous supportive care of a very sick infant is also required. The newer anti-herpes agents, valaciclovir and famciclovir, offer no advantage over aciclovir and are not recommended for neonatal HSV infection. Prognosis is dependent upon the extent of disease and the efficacy of treatment, with highest rates of morbidity and mortality in disseminated infections, followed by central nervous system infection and the least in SEM infection. However, SEM infection is associated with poor developmental outcome even in infants who do not have encephalitis. Studies to improve the outcome of SEM infection are in progress. Neonatal HSV infections, although being relatively uncommon, are associated with significant morbidity and mortality if unrecognised and specific treatment is delayed. Diagnosis relies on a high level of clinical suspicion and appropriate investigation. With early therapy, the prognosis for this infection is considerably improved.

The use of flow cytometry to detect antiviral resistance in human cytomegalovirus.

A method for detecting the antiviral susceptibility of human cytomegalovirus (HCMV) isolates to antiviral agents using flow cytometry was developed. This method has been used to detect the resistance phenotype of HCMV isolates to ganciclovir (GCV). The procedure involves infecting MRC-5 cells with 10(4) pfu HCMV for 120 h, then fixing and permeabilising the cells to allow intracellular labelling of the HCMV early and late antigens. The percentage reduction in the fluorescence positive population of HCMV-infected MRC-5 cells treated with GCV at concentrations of 20 or 50 microM compared with control cultures without GCV was determined. The IC50 defined as a < 50% reduction in the fluorescence positive population in cells infected in the presence of 20 microM GCV or an IC90 defined as a < 90% reduction in the fluorescence-positive population in cells infected in the presence of 50 microM GCV, correlated with resistance determined by a plaque reduction assay. The FACS assay is a rapid and reproducible method for detecting antiviral resistance of HCMV.

Use of aciclovir in herpes simplex virus infections.

Herpes simplex virus type 1 and type 2 cause a wide range of illnesses ranging from minor cold sores to severe necrotising encephalitis or disseminated systemic infections seen in immunocompromised patients including neonates. Following primary infection, the virus is not eradicated from the body but is latent in sensory nerve ganglia where it can reactivate and cause recurrent disease. Aciclovir is the most studied and used antiviral agent with activity against herpes simplex virus infections. In most situations the use of aciclovir shortens the duration of clinical illness and viral shedding and reduces morbidity and mortality. All life- or sight-threatening infections should be managed in an inpatient hospital setting with intravenous therapy. The use of oral aciclovir is recommended in patients with non-life-threatening illness who may still have significant symptoms.

Differential tropism and chemokine receptor expression of human immunodeficiency virus type 1 in neonatal monocytes, monocyte-derived macrophages, and placental macrophages.

Laboratory-adapted (LA) macrophage-tropic (M-tropic) human immunodeficiency virus type 1 (HIV-1) isolates (e.g., HIV-1(Ba-L)) and low-passage primary (PR) isolates differed markedly in tropism for syngeneic neonatal monocytes, monocyte-derived macrophages (MDMs), and placental macrophages (PMs). Newly adherent neonatal monocytes and cultured PMs were highly refractory to infection with PR HIV-1 isolates yet were permissive for LA M-tropic isolates. Day 4 MDMs were also permissive for LA M-tropic isolates and additionally, were permissive for over half the PR isolates tested. Qualitative differences in PR HIV-1 infection of monocytes/MDMs could not be correlated with CD4 levels alone, and in all three cell types the block to PR HIV-1 strain replication preceded reverse transcription. Neonatal monocyte susceptibility to PR HIV-1 strains correlated with increasing CCR-5 expression during maturation. CCR-5 could not be detected on newly adherent (day 1) neonatal monocytes, in contrast to adult monocytes (H. Naif et al., J. Virol. 72:830-836, 1998), but was readily detectable after 4 to 7 days of culture. However, moderate CCR-5 mRNA levels were present in day 1 neonatal monocytes and remained constant during monocyte maturation. CCR-5 was not detectable on the surface of PMs, yet the receptor was present within permeabilized cells. Notably, two brain-derived PR HIV-1 isolates from a single patient, differing in their V3 loops, were discordant in their abilities to infect neonatal monocytes/MDMs and PMs, yet both isolates could infect newly adherent adult monocytes. Together these data strongly suggest that LA HIV-1 isolates are able to infect neonatal monocytes at earlier stages of maturation and lower-level expression of CCR-5 than PR isolates. The differences between neonatal and adult monocytes in susceptibility to PR isolates may also be related to the level of CCR-5 expression.

Progressive vaccinia treated with ribavirin and vaccinia immune globulin.

A 67-year-old man with metastatic melanoma and chronic lymphocytic leukemia was inadvertently given a vaccinia melanoma oncolysate vaccination. He developed progressive vaccinia at the site of inoculation. The lesion started to heal only when he was treated with ribavirin. Vaccinia immune globulin was administered and appeared to help control the initial lesion and limit the development of satellite lesions.

Advances in antiviral therapy.

For many years, acyclovir has been used to treat herpes simplex and varicella zoster infections in adults and children, although new drugs with improved bioavailability and dosage regimens (ie, famciclovir, valaciclovir) are replacing it for the outpatient management of these conditions in adults. Acyclovir remains the treatment of choice for severe herpes infections in both immunocompetent and immunosuppressed patients. Data on the newer antiherpes drugs in children are not available. Treatment of severe cytomegalovirus infections with ganciclovir and foscarnet is difficult because of toxicity; whether improved formulations of these drugs or newer agents prove clinically useful remains to be seen. For the most part, treatment of other herpesviruses is not indicated. The major advance in pediatric HIV treatment is the reduction in vertical transmission with peripartum zidovudine, although the optimal use of antiretrovirals in this situation remains to be determined. The nucleoside analogues zidovudine, zalcitabine, didanosine, and stavudine have been assessed in HIV-infected children; pediatric data about appropriate combinations (eg, with the protease inhibitors and the nonnucleoside reverse transcriptase inhibitors) and dosage regimens lag well behind the adult literature. The effectiveness of ribavirin in respiratory syncytial virus disease is uncertain. Preliminary data suggest that interferons may have a role in the management of chronic hepatitis B and C.