Human herpes virus 6 and endogenous biotin in salivary glands.

AIMS: To detect the presence of human herpes virus 6 (HHV6) and endogenous biotin in paraffin wax embedded and frozen salivary glands. METHODS: Two stage indirect and streptavidin-biotin immunoperoxidase techniques were used to visualise the antigens. RESULTS: HHV6 could not be shown in any of the tissues. However, considerable endogenous biotin antigenicity was detected in the glandular elements of the paraffin wax embedded material. CONCLUSIONS: Results obtained with avidin-biotin detection systems should be interpreted with caution, especially when glandular epithelium is being stained. This may apply to both immunoperoxidase and in situ hybridisation techniques. The use of an anti-biotin antibody as a standard control should be considered.     

Type II heat-labile enterotoxin-producing Escherichia coli isolated from animals and humans.

Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs. Of 236 LT-producing E. coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay. These isolates presumably lost plasmids coding for LT-I during storage. A total of 75% of LT-producing E. coli isolates (27 of 36) from cows, 64% of LT-producing E. coli isolates (7 of 11) from buffalo, 31% of LT-producing E. coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E. coli isolates (3 of 168) from humans contained genes coding for LT-II. Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E. coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E. coli isolates from 12 other children with diarrhea. A total of 9% of LT-II-producing E. coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E. coli (EHEC) adhesin fimbriae. E. coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae. Five VT-producing, LT-II-producing E. coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2. LT-II-producing E. coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans.     

First external quality assurance of antibody diagnostic for SARS-new coronavirus.

To confirm an infection with the new coronavirus (SARS-CoV) causing the severe acute respiratory syndrome (SARS) diagnostic assays for detection of SARS-CoV specific antibody are necessary. To evaluate the diagnostic performance of laboratories an external quality assurance (EQA) study was performed in 2004. Participating laboratories (9/20) correctly detected anti-SARS antibodies in serum samples without false positive results in an immunofluorescence assay. In contrast, only 4/13 laboratories detected most of the anti-SARS antibody positive samples without false positive results using enzyme immunoassays (EIA) and/or immunoblot. The overall results clearly demonstrate that serological diagnosis of SARS-CoV remains at an early stage of development, with further technical improvements required, particularly with respect to the use of SARS specific EIAs.     

Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children.

BACKGROUND: The quality of clinical specimens is a crucial determinant for virological diagnosis. OBJECTIVES: We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1-3 and adenovirus. RESULTS: Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. CONCLUSION: NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard.     

Evaluation and Validation of an Enzyme-Linked Immunosorbent Assay and an Immunochromatographic Test for Serological Diagnosis of Severe Acute Respiratory Syndrome

A newly developed severe acute respiratory syndrome (SARS)-specific enzyme-linked immunosorbent assay (ELISA) was further validated to confirm cutoff values and evaluate its diagnostic performance with clinical samples. In parallel, an immunochromatographic test was also evaluated. A total of 227 clinical serum specimens collected from SARS patients were used in the study, together with 385 samples from healthy donors. By use of an immunofluorescent (IF) test as the “gold standard, ” both the ELISA and the immunochromatographic test were able to detect immunoglobulin G antibodies to SARS not only from late-convalescent-stage samples (>21 days from the onset of clinical symptoms), as previously established, but also from early-acute-phase samples (1 to 10 days from onset). The ELISA, using an optical density (OD) of 0.25 as its cutoff value, produced the best sensitivity while maintaining high specificity. It detected SARS-specific antibodies in 58, 70, 75, and 95%, respectively, of the four groups of samples collected from patients 1 to 10 days, 11 to 20 days, 21 to 30 days, and more than 30 days after the onset of clinical symptoms. Similarly, the immunochromatographic test detected SARS-specific antibodies in 55, 68, 81, and 79% of the four groups, respectively. The overall specificities for the ELISA and the rapid test were 99.5 and 97.7%, respectively. Although the positive correlation observed between the ELISA OD values and the IF titers was moderate (r = 0.6915; P < 0.001), the detection rates of both the ELISA and the rapid test were found well in agreement with the IF titers.     

Longitudinal Profile of Immunoglobulin G (IgG), IgM, and IgA Antibodies against the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in Patients with Pneumonia Due to the SARS Coronavirus

By using a recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein-based enzyme-linked immunosorbent assay (ELISA) and serum specimens serially collected (from day 0 to day 240 after symptom onset) from patients with pneumonia due to SARS-CoV, we analyzed the longitudinal profiles of immunoglobulin G (IgG), IgM, and IgA antibodies against the SARS-CoV nucleocapsid protein in patients with pneumonia due to SARS-CoV. For IgG, the median optical density at 450 nm (OD₄₅₀) turned positive at day 17 and a biphasic response was observed. At day 240, all patients were still positive for anti-nucleocapsid protein IgG antibody. For IgM, the median OD₄₅₀ turned positive at day 20.5, peaked at about day 80, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgM antibody. For IgA, the median OD₄₅₀ turned positive at day 17, peaked at about day 50, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgA antibody. The time of seroconversion detected by the recombinant SARS-CoV nucleocapsid protein-based ELISA and that detected by indirect immunofluorescence assay were similar. The median times of seroconversion for IgG, IgM, and IgA detected by the indirect immunofluorescence assay were 17 days (17 days by ELISA), 16.5 days (20.5 days by ELISA), and 17.5 days (17 days by ELISA), respectively, after disease onset. One, four, and one of the six patients who died did not produce any IgG, IgM, and IgA antibodies against the nucleocapsid protein of SARS-CoV, respectively, although these antibodies were detected in all six patients by the indirect immunofluorescence assay. Further studies should be performed to see whether SARS-CoV nucleocapsid protein antibody positivity has any prognostic significance.     

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.     

Pathogenesis of severe acute respiratory syndrome

Severe acute respiratory syndrome (SARS) is a zoonotic infectious disease caused by a novel coronavirus (CoV). The tissue tropism of SARS-CoV includes not only the lung, but also the gastrointestinal tract, kidney and liver. Angiotensin-converting enzyme 2 (ACE2), the C-type lectin CD209L (also known L-SIGN), and DC-SIGN bind SARS-CoV, but ACE2 appears to be the key functional receptor for the virus. There is a prominent innate immune response to SARS-CoV infection, including acute-phase proteins, chemokines, inflammatory cytokines and C-type lectins such as mannose-binding lectin, which plays a protective role against SARS. By contrast there may be a lack of type 1 interferon response. Moreover, lymphopenia with decreased numbers of CD4+ and CD8+ T cells is common during the acute phase. Convalescent patients have IgG-class neutralizing antibodies that recognize amino acids 441-700 of the spike protein (S protein) as the major epitope.     

Molecular Epidemiology of Hospital Outbreak of Middle East Respiratory Syndrome,Riyadh, Saudi Arabia, 2014

We investigated an outbreak of Middle East respiratory syndrome (MERS) at King Fahad Medical City (KFMC), Riyadh, Saudi Arabia, during March 29–May 21, 2014. This outbreak involved 45 patients: 8 infected outside KFMC, 13 long-term patients at KFMC, 23 health care workers, and 1 who had an indeterminate source of infection. Sequences of full-length MERS coronavirus (MERS-CoV) from 10 patients and a partial sequence of MERS-CoV from another patient, when compared with other MERS-CoV sequences, demonstrated that this outbreak was part of a larger outbreak that affected multiple health care facilities in Riyadh and possibly arose from a single zoonotic transmission event that occurred in December 2013 (95% highest posterior density interval November 8, 2013–February 10, 2014). This finding suggested continued health care–associated transmission for 5 months. Molecular epidemiology documented multiple external introductions in a seemingly contiguous outbreak and helped support or refute transmission pathways suspected through epidemiologic investigation.