Low prevalence of Babesia hongkongensis infection in community and privately-owned cats in Hong Kong.

Domestic cats are susceptible to infection with at least 11 species of Babesia. In Hong Kong, where dogs are commonly infected with B. gibsoni, a single infection in a cat by a novel species, B. hongkongensis, was reported previously. The aim of this study was to investigate the frequency of Babesia spp. detection in cats in Hong Kong. Residual blood-derived DNA from healthy free-roaming community cats (n = 239), and privately-owned cats with and without anaemia undergoing diagnostic investigations (n = 125) was tested for Babesia spp. DNA using a pan-Babesia PCR targeting mitochondrial Cytochrome B, and a B. hongkongensis specific PCR targeting 18S rRNA. Positive samples were confirmed by sequencing and comparative sequence analysis against the GenBank nucleotide database. Babesia hongkongensis was detected in 4/239 (1.7 %) community cats, and 0/125 (0.0 %) privately-owned cats. Babesia gibsoni was detected in 0/239 community cats and 1/125 (0.8 %) privately-owned cats. Cats infected with B. hongkongensis were clinically healthy at the time of sampling. The B. gibsoni-infected cat was anaemic and thrombocytopenic. Cats in Hong Kong can be infected with B. hongkongensis and B. gibsoni, albeit at low frequency. The tick vector for B. hongkongensis is yet to be identified.

Anti-VP6 IgG antibodies against group A and group C rotaviruses in South India.

In an epidemiological survey from South India, 936 serum samples were tested for IgG against recombinant baculovirus-expressed VP6 proteins from human group A and group C rotaviruses. The overall seroprevalence for group A was 100% and for group C was 25.32% (95% CI 22.64-28.21). The lowest seroprevalence for group C was in children aged <10 years (16.79%). An age-related rise in seroprevalence in group C, but not group A, suggests different patterns of exposure. Seroprevalence was similar in rural and urban subjects, unlike the higher prevalence in rural subjects in studies elsewhere.

Allogeneic hematopoietic stem cell transplantation and norovirus gastroenteritis: a previously unrecognized cause of morbidity.

BACKGROUND: A retrospective study of the clinical, epidemiologic, and virologic features of norovirus gastroenteritis in 12 adult allogeneic hematopoietic stem cell transplant (HSCT) recipients. METHODS: Norovirus infection was diagnosed by reverse-transcriptase polymerase chain reaction. Strains were genotyped by nucleic acid sequence of the most highly conserved region of the norovirus gene encoding the capsid S (shell) domain. RESULTS: Ten of 12 patients presented with vomiting of short duration, but diarrhea was present in all. The median time from onset to norovirus diagnosis was 1 month (range, 0.25-6.0 months). Eleven patients were receiving immunosuppression when norovirus infection was diagnosed: 8 for graft-versus-host disease (GVHD) in an organ other than gut, 1 for previous gut GVHD, and 2 for presumed gut GVHD that proved to be norovirus gastroenteritis. Six patients required enteral or parenteral nutrition for severe weight loss. In 10 patients, diarrhea lasted a median of 3 months (range, 0.5-14 months) and virus was shed at a high level throughout. The remaining 2 patients died after 4 months of diarrhea (one died of unrelated complications, and the other died of malnutrition). The noroviruses found were GII (untyped), GII-3, GII-4, and GII-7 in 1, 1, 9, and 1 patients, respectively. Eleven of the 12 patients had acquired their infection in the community. Phylogenetic analysis of the GII-4 strains demonstrated that all differed. CONCLUSIONS: Noroviruses are a hitherto unsuspected cause of prolonged morbidity and mortality in adults after allogeneic HSCT. The use of reverse-transcriptase polymerase chain reaction to detect high viral load levels in feces distinguishes norovirus gastroenteritis from gut GVHD.

Temperature inactivation of Feline calicivirus vaccine strain FCV F-9 in comparison with human noroviruses using an RNA exposure assay and reverse transcribed quantitative real-time polymerase chain reaction-A novel method for predicting virus infectivity.

A one-step reverse transcription quantitative real-time polymerase chain reaction (RT-QPCR) method in combination with RNase treatment and low copy number samples was developed in order to examine the effect of temperature on the ability of virus capsids to protect their RNA content. The method was applied to a non-cultivable virus (GII.4 norovirus) and Feline calicivirus vaccine strain F-9 (FCV) which is often used as a norovirus surrogate. Results demonstrated that FCV RNA is exposed maximally after 2min at 63.3 degrees C and this correlated with a greater than 4.5log reduction in infectivity as assessed by plaque assay. In contrast human GII.4 norovirus RNA present in diluted clinical specimens was not exposed maximally until 76.6 degrees C, at least 13.3 degrees C greater than that for FCV. These data suggest that norovirus possesses greater thermostability than this commonly used surrogate. Further, these studies indicate that current food processing regimes for pasteurisation are insufficient to achieve inactivation of GII.4 NoVs. The method provides a novel molecular method for predicting virus infectivity.

Clinical laboratory practices for the detection of rotavirus in England and Wales: can surveillance based on routine laboratory testing data be used to evaluate the impact of vaccination?

Two rotavirus vaccines have recently been licensed in Europe. Rotavirus surveillance data in many European countries are based on reports of laboratory-confirmed rotavirus infections. If surveillance data based on routine laboratory testing data are to be used to evaluate the impact of vaccination programmes, it is important to determine how the data are influenced by differences in testing practices, and how these practices are likely to affect the ability of the surveillance data to represent trends in rotavirus disease in the community. We conducted a survey of laboratory testing policies for rotavirus gastroenteritis in England and Wales in 2008. 60% (94/156) of laboratories responded to the survey. 91% of reporting laboratories offered routine testing for rotavirus all year round and 89% of laboratories offered routine rotavirus testing of all stool specimens from children under the age of five years. In 96% of laboratories, rotavirus detection was presently done either by rapid immunochromatographic tests or by enzyme-linked immunosorbent assay. Currently, rotavirus testing policies among laboratories in England and Wales are relatively homogenous. Therefore, surveillance based on laboratory testing data is likely to be representative of rotavirus disease trends in the community in the most frequently affected age groups (children under the age of five years) and could be used to help determine the impact of a rotavirus vaccine.

Viral gastroenteritis outbreaks in deployed British troops during 2002-7.

OBJECTIVES: The aim of this study was to see what lessons could be learnt from the suspected viral gastroenteritis outbreaks that have occurred in deployed British troops during 2002-7. METHOD: Epidemiological and laboratory data from identifiable outbreaks were reviewed, including epidemic curves and the results of PCR testing for enteropathic viruses. RESULTS: The epidemic curves of outbreaks varied predictably in accordance with the size of the population at risk and whether this population was constant or expanding. Of 11 outbreaks identified, 10 (91%) had a proven viral cause and 10 (91%) occurred in Iraq. Of 84 enteropathic viruses identified, 61 (73%) were noroviruses and these included both unknown strains and those that were common in the UK and Europe. Of the 10 viral outbreaks, 3 (30%) occurred in medical units, 5 (50%) were associated with large-scale relief in place (RiP) deployments and 5 (50%) involved >3 different viruses, which is strongly suggestive of food or water contamination. CONCLUSION: These findings can help to predict future viral gastroenteritis outbreaks and target improved prevention strategies appropriately. However, more systematic studies are now required.

Infection and reactivation with cytomegalovirus strains in lung transplant recipients.

Cytomegalovirus pneumonia is a major cause of morbidity and death following lung transplantation (LT) (1). The case fatality rate is highest in the CMV-seronegative recipients (R-) of organs from seropositive donors (D+), which suggests that transmission of CMV may occur with the graft (1), but in seropositive recipients (R+) the comparative importance of reactivation of endogenous virus and reinfection with donor virus is poorly understood.

Rotavirus epidemiology and surveillance.

There is extensive antigenic and genomic diversity among co-circulating human rotaviruses. They are differentiated into groups, subgroups and types. There are at least 7 groups (A-G) and 4 subgroups within group A. To distinguish types within group A, a dual classification system has been established with the glycoprotein VP7 defining G types, and the protease-sensitive protein VP4 defining P types. At least 14 G types and more than 20 P types have been distinguished, of which at least 10 G types and at least 11 P types have been found in humans. Using the typing system, the complex molecular epidemiology of rotaviruses was investigated. Rotaviruses of different G and P types co-circulate. The main types found are G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8]; their relative incidence rates change over time in any one location and are different at the same time between different locations. Viruses with G/P constellations such as G1P1B[4] and G2P1A[8] are mostly natural reassortants of the co-circulating main virus types emerging after double infection of hosts. Viruses carrying G and or P types not represented in the four most common types, e.g. G8P[8], G1P[6] or G9P[6], could be introduced into the population by reassortment with animal viruses, or directly from animals or exotic human sources. Naturally circulating rotaviruses constantly undergo point mutations which can be used to classify lineages and sublineages within types. The full significance of human infections with group B and C rotaviruses remains to be established. Surveillance of rotavirus types in different parts of the world is essential to monitor the emergence of new types or of new G/P constellations which may predominate over time. The efficacy and effectiveness of any future rotavirus vaccine may differ depending on the predominant natural strain types. Detailed epidemiological and molecular surveillance data should be utilized to study the transmission dynamics of rotaviruses.

Evaluation of a broadly reactive nucleic acid sequence based amplification assay for the detection of noroviruses in faecal material.

A recently described nucleic acid sequence based amplification (NASBA) assay for the detection of genogroup I (GI) and genogroup II (GII) norovirus RNA in faecal samples was evaluated against a reverse transcription polymerase chain reaction (RT-PCR). Both assays were used to screen a panel of 38 faecal samples known to contain 17 different norovirus strains and 131 clinical samples collected from 60 gastroenteritis outbreaks of unknown aetiology. The NASBA assay detected 13 out of the 17 strains of norovirus in the characterised panel, failing to detect a single GII strain and three GI strains. There was 90% agreement between the two assays used to detect norovirus in clinical samples from outbreaks. NASBA detected norovirus RNA in all 64 samples positive by RT-PCR and also detected norovirus RNA in additional 13 samples that were negative by RT-PCR. The sensitivity and specificity of NASBA was 100% and 80%, respectively, compared to RT-PCR results. The norovirus NASBA assay was shown to be highly sensitive and specific, and its ease of use and rapid turnaround time makes it a favourable alternative to RT-PCR for the investigation of norovirus outbreaks.

Evaluation of a commercial ELISA for detecting Norwalk-like virus antigen in faeces.

A commercially available enzyme immunoassay, the IDEIA Norwalk-like virus (NLV) enzyme linked immunosorbent assay (ELISA; Dako Cytomation, Ely, UK) for detecting NLV antigen in faecal samples and determining the NLV genogroup was evaluated. The performance of the ELISA was compared with that of electron microscopy and the reverse transcription polymerase chain reaction by testing a panel of faecal samples collected from patients involved in outbreaks of gastroenteritis. When compared with reverse transcription-polymerase chain reaction (RT-PCR), the ELISA had a sensitivity and specificity of 55.5 and 98.3%, respectively. This compares with a sensitivity and specificity for EM of 23.9 and 99.2%, respectively. The sensitivity and specificity of the ELISA for determining the aetiology of a Norwalk virus-like outbreak, based on two or more positive samples within an outbreak, were 52.2 and 100% when two samples were collected from an outbreak and 71.4 and 100% when six or more samples were collected. The ELISA correctly identified the NLV genogroups of viruses previously characterised by partial DNA sequencing. The ELISA is a suitable alternative to the preliminary screening by EM for investigating outbreaks of gastroenteritis. Outbreaks, negative by ELISA should be examined by RT-PCR in order to detect strains non-reactive in the assay and virus strains from representative ELISA positive outbreaks should be characterised fully to allow the genetic diversity of NLVs co-circulating in the population to be described.

Haemorrhagic colitis and haemolytic-uraemic syndrome: false positive reaction with a rotavirus latex agglutination test.

A stool sample from a child with haemorrhagic colitis and haemolytic-uraemic syndrome gave a positive reaction with the RotaScreen latex agglutination test in the absence of other evidence of rotavirus infection. When this test is performed on bloody specimens, positive reactions should be interpreted with caution and confirmed by other means.

Problems with serological diagnosis of Toxoplasma gondii infections in heart transplant recipients.

Of the first 128 patients to receive either heart or heart and lung transplants at Papworth Hospital, four developed Toxoplasma gondii infections acquired from the donor heart and two died. Six patients had passively acquired T gondii antibody as a result of blood transfusions around the time of transplantation. Eight patients developed antibodies against T gondii, which were detectable by changes in the latex agglutination test titres but not by those of the dye test. These false positive latex agglutination reactions occurred simultaneously with cytomegalovirus infection and were associated with the IgM serum fraction in the patients' sera. These reactions were not associated with cytomegalovirus specific IgM, Paul-Bunnell antibody, nor detectable rheumatoid factor. These findings emphasise the need for T gondii dye test confirmation of latex agglutination test titre rises in heart transplant recipients.

Cytomegalovirus infections in heart and heart and lung transplant recipients.

Of the first 166 heart and 15 heart and lung transplant recipients at Papworth Hospital, Cambridge, who survived for more than one month after transplantation, 162 were investigated for cytomegalovirus (CMV) infection by serological methods. Altogether, 73 (45%) developed CMV infection after transplantation: 30 (18.5%) had acquired primary infection and 43 (26.5%) reactivation or reinfection. Six patients died of primary infection, probably acquired from the donor organ. Recipients negative for CMV antibody who received an organ from an antibody positive donor had the most severe disease. Heart and lung transplant recipients experienced more severe primary CMV infection than those in whom the heart alone was transplanted. The most sensitive and rapid serological method was a mu-capture enzyme linked immunosorbent assay (ELISA) for detecting CMV specific IgM, the amount of which was often of prognostic value and influenced the management of patients.

Antibiotic resistant fever associated with herpes simplex virus infection in neutropenic patients with haematological malignancy.

The incidence of mucocutaneous herpes simplex virus infection confirmed by culture and occurring during febrile neutropenic episodes was determined in 43 patients with haematological malignancy. The outcome of 72 episodes of neutropenic fever was determined and correlated with the presence or absence of herpes simplex virus (HSV) infection. Twenty four patients had mucocutaneous HSV infection during at least one episode. In 24 episodes in which HSV was isolated only 12.5% of fevers responded to antibiotics and 75% of fevers were otherwise unexplained. Conversely, in 48 episodes of neutropenic fever in which HSV was not isolated 67% of fevers responded to antibiotics and only 8.3% were unexplained. The difference in incidence of antibiotic resistant fever in the two groups was significant. There was, therefore, a strong association between mucocutaneous HSV infection and antibiotic resistant fever in immunosuppressed neutropenic patients. As most HSV infections are the result of virus reactivation, establishing the HSV serological state of patients would identify those at risk of infection and hence those in whom the prophylactic use of acyclovir would be indicated.

Internal quality assurance in a clinical virology laboratory. II. Internal quality control.

AIMS: In April 1991 additional quality control procedures were introduced into the virology section of the Clinical Microbiology and Public Health Laboratory, Cambridge. Internal quality control (IQC) samples were gradually included in the serological assays performed in the laboratory and supplemented kit controls and standard sera. METHODS: From April 1991 to December 1993, 2421 IQC procedures were carried out with reference sera. RESULTS: The IQC samples were evaluated according to the Westgard rules. Violations were recorded in 60 of 1808 (3.3%) controls and were highest in the IQC samples of complement fixation tests (25/312 (8%) of controls submitted for complement fixation tests). CONCLUSIONS: The inclusion of IQC samples in the serological assays performed in the laboratory has highlighted batch to batch variation in commercial assays. The setting of acceptable limits for the IQC samples has increased confidence in the validity of assay results.

Serological reactivity against cyst and tachyzoite antigens of Toxoplasma gondii determined by FAST-ELISA.

AIMS: To obtain quantitative data on the human serological response to Toxoplasma gondii tachyzoite and bradyzoite antigens. METHODS: Serum samples from 30 patients who had positive antibody titres against T gondii and from 14 who were seronegative, together with sequential serum samples from four infected individuals, were screened by FAST-ELISA. RESULTS: Serum samples from the 30 seropositive patients showed high IgG and IgM titres against the T gondii tachyzoite antigen but very low responses to cyst antigen. This result was borne out in sequential serum samples from patients with toxoplasmosis. CONCLUSION: Antibody recognition of the cystic stage of T gondii is low, implying that either this stage is poorly immunogenic or that the antigen load is low.

Toxoplasmosis in heart and heart and lung transplant recipients.

Of the first 250 heart and 35 heart and lung transplant recipients at Papworth Hospital, Cambridge, who survived for more than one month after transplantation, 217 heart and 33 heart and lung patients were investigated serologically for evidence of Toxoplasma gondii infection. Six patients acquired primary T gondii infection, most probably from the donor organ. Five patients experienced T gondii recrudescence, two of whom had recovered from primary infection a few years earlier. Two patients died from primary T gondii infection and the severity of symptoms in the other patients with primary infection was related to the amount of immunosuppressive treatment. Prophylaxis with pyrimethamine (25 mg a day for six weeks) was introduced for T gondii antibody negative transplant recipients who received a heart from a T gondii antibody positive donor after the first four cases of primary toxoplasmosis. Of the seven patients not given pyrimethamine, four (57%) acquired primary T gondii infection. This compared with two of the 14 patients (14%) given prophylaxis.

Internal quality assurance in a clinical virology laboratory. I. Internal quality assessment.

AIMS: In April 1991 an internal quality assessment scheme (IQAS) was introduced into the virology section of the Clinical Microbiology and Public Health Laboratory, Cambridge. The IQAS was established to identify recurring technical and procedural problems, to check the adequacy of current techniques, and to calculate the frequency of errors. METHODS: Between April 1991 and December 1993, 715 anonymous clinical serum samples were submitted to the laboratory to test 3245 individual procedures of diagnostic viral serology. RESULTS: A total of 485 (14.9%) procedural and 61 (1.9%) technical discrepancies were observed, the technical discrepancies mainly being recorded in complement fixation tests. Twenty two (0.7% of total procedures) of the technical discrepancies were diagnostically significant. CONCLUSIONS: Evaluation criteria developed with the introduction of IQAS to viral serology, and technical and procedural discrepancies are assessed. As yet, IQAS has not been introduced to other sections of the diagnostic virology laboratory (virus isolation, electron microscopy, immunofluorescence, and enzyme linked immunosorbent assays for viral and chlamydial antigens).

Accuracy of serology for the diagnosis of Helicobacter pylori infection--a comparison of eight kits.

AIMS: To determine the accuracy of eight commercially available kits for the serological diagnosis of Helicobacter pylori infection, and hence whether a serology service could be introduced to reduce endoscopy workload. METHODS: Eighty four patients newly presenting to their general practitioners with dyspepsia were recruited. Gold standard diagnosis of H pylori infection was obtained both by a histological examination of gastroduodenal biopsy specimens and by the 14C-urea breath test (UBT). The performance of six quantitative and two qualitative enzyme linked immunosorbent assays for H pylori IgG, used according to the manufacturers' instructions, with serum samples obtained during the endoscopy visit, were compared. RESULTS: The study population had a median age of 45 years, and the prevalence of H pylori infection was 35%. With one exception, where the patient had received a course of anti-H pylori treatment between endoscopy and UBT, there was 100% concordance in the results of the two gold standard techniques. Discordant serology results were more common in patients aged > 50 years (42% of the total) than in younger patients (21%), and this was most noticeable in uninfected patients. The sensitivity of the kits was good (90-100%), but specificity was more variable (76-96%), and the rate of equivocal results was unacceptably high in some cases (0-12%). The overall accuracy of the kits ranged from 83 to 98%. Two kits in particular performed well (Pylori-Elisa II, Bio-Whitaker and Premier, Launch; qualitative) with 98% and 100% accuracy, respectively. CONCLUSIONS: In a symptomatic population with a prevalence of H pylori infection of 35%, particularly in patients aged < 50 years, some but not all serology kits may be used as a highly accurate and inexpensive alternative to the gold standard techniques.

Single-tube real-time nested polymerase chain reaction for detecting human papillomavirus DNA.

A single-tube real-time nested polymerase chain reaction (PCR) was developed to detect human Papillomavirus (HPV) DNA in a closed tube system. The oligonucleotide primers MY09/MY11 and GP5+/GP6+ were included in contiguous reactions, thus eliminating the need to transfer first round PCR product into a second tube. The sensitivity and specificity of the optimized single-tube nested PCR were comparable with that achieved by two separate reactions on a conventional thermal block system using serial dilutions derived from plasmids containing DNA of 20 HPV types. A minimum of 10 copies of HPV types 11 and 16 DNA could be detected by both systems. In clinical samples, HPV types 1A, 2, 3, 5, 6-8, 10, 11, 14, 16, 17, 18, 20, 31, 33, 35, 39, 45, 49, 50, 52-54, 57, 62, 66, 70, CP8304 and LVX82/MM7 could be detected by both PCR methods. A total of 145 samples collected from patients were tested for the presence of HPV DNA with the two PCR systems; 124 (86.1%) of 144 samples gave concordant results in both assays. The HPV DNA positive PCR amplicons were typed and concordant results were obtained in 47 of 67 positive samples tested in both amplicons. In samples containing multiple HPV types at least one type was common to both amplicons.