A comparative evaluation of the sensitivity of two automated and two manual nucleic acid extraction methods for the detection of norovirus by RT-PCR.

The aim of the study was to compare the sensitivity of a norovirus RT-PCR method using two manual RNA extraction methods (Qiagen and Roche) and two automated RNA extraction methods (Qiagen and Corbett). All four RNA extraction methods gave similar sensitivities although the automated methods, especially the Corbett, required significantly less labour than the manual methods. The automated methods also enabled RNA extraction of approximately two to three times the number of specimens in a given time period compared to manual methods.

Multiple outbreaks of Norwalk-like virus gastro-enteritis associated with a Mediterranean-style restaurant.

The role of diverse infectious agents, particularly Norwalk-like viruses (NLV), in three successive gastro-enteritis outbreaks in one setting (a restaurant) was evaluated. Methods included standard bacteriological tests, specific tests for Escherichia coli, tests for verocytotoxins, electron microscopy (EM) for viruses and reverse transcription-PCR (RT-PCR) methodology for NLV. No pathogenic bacteria were detected. Verocytotoxin genes, although detected by PCR in the first outbreak, could not be confirmed in the E. coli isolated, so they did not appear to be of significance. NLV was the main agent detected in each of the three outbreaks. DNA sequencing and phylogenetic analysis of the amplified products obtained from the RT-PCR positive specimens indicated that only one NLV strain was involved in each outbreak, but the NLV strains responsible for the three outbreaks were different from each other. PCR technology for detection of NLV proved highly sensitive, but failed to detect one specimen which was positive by EM. The restaurant associated with the outbreaks is a Mediterranean-style restaurant where food from a common platter is typically eaten with fingers. The findings indicate that NLV was introduced by guests or staff and was not due to a long-term reservoir within the setting.

High level excretion of Norwalk-like virus following resolution of clinical illness.

We report the case of an elderly woman excreting high levels (about 5 x 10(5) virions per gram of faeces) of Norwalk-like virus (NLV) in the absence of any clinical symptoms of gastroenteritis. Analysis by reverse transcription, polymerase chain reaction and DNA sequencing was carried out on a 342-nucleotide region of open reading frame 1. This indicated that the NLV belonged to genogroup 2 and was more closely related to the Camberwell subgroup, the most common circulating in southeast Australia at present, than to the Norwalk and Mexico viruses.

Virological aspects of human immunodeficiency virus infection.

The virology of human immunodeficiency virus (HIV) infection is reviewed. The transmission of HIV is restricted to direct contact with the blood or other body fluids of infected human beings. Ordinary social contact with infected individuals holds no risk but in the health care setting all patients must be considered to be potentially infectious and universal precautions taken. The replication of HIV in cells of the immune system carrying the CD4 receptor creates a complex relationship between the virus infection and the host immune response. The pathogenesis and the principles of the laboratory diagnosis of HIV infection are reviewed. Since its discovery HIV has quickly become one of the most studied and best characterized of human pathogens. The diagnosis of HIV infection, because of its implications, has been made more accurate than for any other infection. This understanding has significantly improved treatment but has yet to provide curative therapy, and prevention of infection is still the basis of the fight against AIDS.

PIP: HIV can be acquired only through direct contact with the blood or other body fluids of infected individuals. There is therefore no risk of contracting or transmitting HIV through normal social contact with HIV-infected individuals. However, in health care settings, universal precautions must be applied with all patients under the assumption that they could be infected with HIV and are therefore infectious. The virology of HIV infection is reviewed in sections on virology, transmission, life cycle, pathogenesis, and laboratory diagnosis. The diagnosis of HIV infection is more accurate than for any other infection, reflecting the importance of HIV as an infectious agent and the implications of infection. While treatment against HIV and AIDS has improved, curative therapy remains to be developed. Preventing HIV infection therefore remains the central strategy against AIDS.

What is the cause of a rash after measles-mumps-rubella vaccination?

Surveillance and laboratory confirmation of measles will increase in importance as Australia implements enhanced measles control. We describe a 17-month-old child with fever and rash after measles-mumps-rubella vaccination. Detection of vaccine-strain measles virus in his urine by polymerase chain reaction confirmed the diagnosis of a vaccine reaction rather than wild-type measles. We propose that measles virus should be sought and identified as vaccine or wild-type virus when the relationship between vaccination and measles-like illness is uncertain.

Investigation of optimal specimen type and sampling time for detection of measles virus RNA during a measles epidemic.

At various times postonset of rash, 74 patients positive for measles virus-specific immunoglobulin M provided samples for detection of measles virus RNA by a reverse transcriptase PCR. Of lymphocytes, urine, throat swab, and serum specimens, throat swab specimens were optimal for detection of measles virus RNA during the first 2 weeks after the rash.

Viral haemorrhagic fevers: current status, future threats.

In developing countries, the major outbreaks of viral haemorrhagic fevers such as Marburg, Ebola and Lassa fever viruses have been nosocomially spread. The high mortality and absence of specific treatment have had a devastating effect. Epidemics of this highly contagious disease remain a constant threat to Australia and, as a result, carefully planned laboratory and public health strategies and clinical infection control measures have been instituted for the management of suspected cases.

Measles immunity among young adults in Victoria.

Measles outbreaks in Victoria in 1999 and 2001 have suggested that a substantial proportion of young Victorian adults may be susceptible to measles infection. We performed a serosurvey of 300 18-30-year-old healthy blood donors and 312 sera retrieved after diagnostic testing for a non-rash illness in patients of the same age group, with the aim of estimating the proportion of young adults in Victoria immune to measles. We also aimed to define more precisely the birth cohorts at risk of measles infection, with cohorts reflecting the measles immunisation policies of previous years. There was no significant difference in measles immunity between the 300 blood donors (79.0%, 95% confidence interval 73.9-83.5) and the 312 patients whose sera had been stored (84.0%, 95% CI 79.4-87.9, p=0.11). There was, however, a significant difference in immunity by birth cohort. In the combined results from both samples, the proportion of people born between 1968 and 1974 who were immune to measles was 88.4 per cent (95% CI 84.1-91.6) while the proportion of those born between 1975 and 1981 was 74.1 per cent (95% CI 68.7-79.1). This study confirms that a substantial proportion of young Victorian adults are susceptible to measles, but also demonstrates that those born between 1975 and 1981 are more likely to be non-immune than those born before 1975. A review of published Australian data supports this conclusion and confirms the need for a measles control program aimed at young adults.

Heminested multiplex reverse transcription-PCR for detection and differentiation of Norwalk-like virus genogroups 1 and 2 in fecal samples.

The present study describes a heminested multiplex reverse transcription (RT)-PCR assay which enables simultaneous detection and differentiation of Norwalk-like virus (NLV) genogroups from clinical fecal samples without the need to perform sequencing or hybridization. The assay developed was able to detect concentrations of fewer than 100 viral particles per 5 microl of clarified fecal extract and could differentiate the two genogroups with a specificity of 100%. Although the multiplex RT-PCR assay failed to detect NLV in about 3% of the fecal samples which were NLV positive by electron microscopy (EM), the assay was approximately six times more sensitive than EM for NLV detection.

Suppression of the humoral immune response by mycophenolate mofetil.

BACKGROUND: No conventional immunosuppressive agent preferentially inhibits antibody production. Studies in experimental animals and in human cells in vitro suggested mycophenolate mofetil (MMF) might have such an effect. If this was the case in vivo it could have significant implications in terms of both MMF toxicity and the rational design of immunotherapeutic regimens. METHODS: Subjects were renal transplant recipients (25 patients treated with prednisolone, cyclosporine and azathioprine, and 13 treated with prednisolone, cyclosporine and MMF) and 20 normal controls. The three groups received influenza vaccination, and the antibody response to it was measured 4-6 weeks later using a standard haemagglutination assay. RESULTS: MMF profoundly suppressed the humoral immune response to influenza vaccination when added to prednisolone and cyclosporine. This effect could be seen when comparing the rise in the mean titre of antibody after vaccination. It was also reflected in the number of patients mounting responses deemed to be clinically protective by either demonstrating a 4-fold rise in titre or an increase in titre to > or = 40. CONCLUSIONS: Suppression of the humoral immune response by MMF has implications for the design of immunization protocols to protect the immunosuppressed, and raises the possibility that MMF use may be accompanied by more or different infections than complicate more conventional immunosuppression. More importantly, consideration should be given to harnessing the relatively specific effect of MMF on antibody production to treat antibody-mediated diseases.

Failure to detect norovirus in a large group of asymptomatic individuals.

Noroviruses are a major cause of both sporadic and epidemic gastroenteritis in humans, but the mechanisms by which norovirus circulates within the community are poorly understood. In this study, we examined the hypothesis that asymptomatic people act as a reservoir for norovirus. Faecal specimens from 399 asymptomatic individuals were tested for norovirus by reverse transcription polymerase chain reaction (RT-PCR) methodology, and no norovirus was detected. The failure to detect norovirus was not apparently due to the test sample being resistant to norovirus infection, nor to the presence of PCR inhibitors in the test sample. The findings suggest that, if norovirus is carried by asymptomatic people, the carriage rate is very low; the upper bound (95% confidence interval, binomial distribution) of the carriage rate was only 0.8%. Thus, it is unlikely that asymptomatic people are an important reservoir for norovirus.

Viral haemorrhagic disease of rabbits and human health.

Viral haemorrhagic disease of rabbits (VHD), a potential biological control for wild rabbits in Australia and New Zealand, escaped from quarantined field trials on Wardang Island and spread to the mainland of Australia in October 1995. This study looked for any evidence of infection or illness in people occupationally exposed to the virus. Two hundred and sixty-nine people were interviewed and 259 blood samples were collected. Exposures to VHD-infected rabbits ranged from nil to very high. No VHD antibodies were detected in any of the 259 sera when tested by VHD competitive enzyme immunoassay, which had been validated with 1013 VHDV-specific antibody negative sera. A questionnaire designed to elicit symptoms of disease in a range of organ systems found no significant differences between illness in those exposed and those not exposed to VHD, nor could an association be found between exposure and subsequent episodes of illness. The findings are consistent with the view that exposure to VHD is not associated with infection or disease in humans.

Mumps and rubella: a year of enhanced surveillance and laboratory testing.

In Victoria (Australia) surveillance for mumps and rubella has historically been passive, with most notified cases clinically diagnosed. In July 2001, the Victorian Department of Human Services implemented an enhanced surveillance system focusing on improved laboratory testing. We tested 85% of notifications and only 9% of all mumps and 27% of rubella notifications were laboratory confirmed. While most notified cases were children who had been clinically diagnosed, we found most laboratory-confirmed cases were in adults. The positive predictive value of the clinical case definition was low: mumps (10%); rubella (22%). These results highlight the value of laboratory confirmation of the diagnosis when mumps and rubella are rare, failure to do so is likely to overestimate disease incidence.

Cytomegalovirus myocarditis in a heart transplant recipient: sensitive monitoring of viral DNA by the polymerase chain reaction.

A 54-year-old female heart transplant recipient had an acute episode of graft rejection that was treated with high-dose immunosuppression therapy. During this therapy a second febrile illness developed, which was accompanied by hypotension, anemia, and rash. Findings for subsequent myocardial biopsy specimens were negative for cytomegalovirus by culture but were strongly positive for cytomegalovirus deoxyribonucleic acid by the polymerase chain reaction. Histologic observation of viral inclusions in the myocardial fibers supported the diagnosis of cytomegalovirus myocarditis. The polymerase chain reaction therefore can provide a rapid and highly sensitive method for heart transplant patients with suspected cytomegalovirus myocarditis.

Evidence of patient-to-patient transmission of hepatitis C virus through contaminated intravenous anaesthetic ampoules.

Two separate cases of acute hepatitis C virus (HCV) infection following medical procedures, arthroscopy and colonoscopy, are reported. In both episodes, patient risk factors were reviewed, and staff and other patients' sera were tested for HCV antibodies and RNA. HCV RNA positive samples were genotyped, sequenced, and subjected to phylogenetic analysis. No risk factors for HCV infection were identified for either case except for medical procedures. HCV RNA positive patients were identified preceding both cases on the respective theatre lists. HCV infection in a second low risk patient was also identified. Nucleic acid sequencing and phylogenetic analysis of HCV from the two putative source patients and the three recipient patients demonstrated a high degree of relatedness respectively. The results suggest that patient-to-patient transmission occurred in both episodes via contamination of intravenous anaesthetic ampoules with HCV used on multiple patients. Injectable medication ampoules should not be used for more than one patient.

Combined infection of Norwalk-like virus and verotoxin-producing bacteria associated with a gastroenteritis outbreak.

Detection of multiple pathogens, particularly a combination of viruses and bacteria, is infrequently documented in outbreaks of gastroenteritis. This paper reports the presence of Norwalk-like virus (NLV) and enterohaemorrhagic verotoxin-producing Escherichia coli in one individual, and NLV and verotoxin-producing Aeromonas sobria in another individual, both part of a large gastroenteritis outbreak. The causes of gastroenteritis in such outbreaks may be more complex than previously thought.

Molecular analysis of human immunodeficiency virus strains associated with a case of criminal transmission of the virus.

An investigation was done of the evidence for transmission of human immunodeficiency virus (HIV) from an HIV-positive man to several male and female sex contacts. Phylogenetic analysis of sequences from the gag and env genes showed a close relationship between the predominant virus strains from the source and 2 contacts. However, the likelihood that a female contact was infected by the source could not be determined, despite contact tracing indicating that this may have occurred. One male, shown by contact tracing and molecular evidence to have been infected by the source, subsequently transmitted HIV to his female sex partner. HIV sequence from a plasma sample used as a control in the phylogenetic analysis contained env and gag sequences that were closely related to those from the source. An epidemiologic link between these 2 individuals was subsequently confirmed by contact tracing.