Laboratory containment of SARS virus.

Following the severe acute respiratory syndrome (SARS) outbreak in 2003, a large number of clinical and environmental samples containing/potentially containing SARS coronavirus (SARSCoV) as well as SARS-CoV stocks were retained in clinical and research laboratories. The importance of laboratory biosafety was demonstrated by the occurrence of laboratory incidents in Singapore, Taiwan and Beijing. It is imperative that safe practice and techniques, safety equipment and appropriate facility design should be in place to reduce or eliminate exposure of laboratory workers, other persons and the outside environment to SARS-CoV containing materials. Discussion on laboratory containment of SARS-CoV was initiated in Hong Kong in August 2003. It was agreed that an inventory of all specimens with the potential presence of SARS-CoV collected for any diagnostic or research purposes from November 2002 to July 2003 should be established in each laboratory. They should be stored in a secure place at the appropriate biosafety level with access control. Un-needed samples collected during the period should be destroyed. These laboratories should be audited to ensure inventories are updated. The audit should include safety and security measures to detect irregularities. Any laboratory accidents involving materials suspected of containing SARS-CoV should be reported to the authorities and all personnel exposed closely followed medically. A contingency plan should be in place in the laboratory and a drill conducted regularly to test its efficacy. By January 2004, all clinical laboratories performing SARS-CoV testing in Hong Kong set up inventories to document location and types of SARS-CoV containing materials retained in their laboratory. Audits of these laboratories in 2004 showed that laboratory safety and containment requirements as recommended were generally met.

Laboratory diagnosis of SARS.

The virologic test results of 415 patients with severe acute respiratory syndrome (SARS) were examined. The peak detection rate for SARS-associated coronavirus occurred at week 2 after illness onset for respiratory specimens, at weeks 2 to 3 for stool or rectal swab specimens, and at week 4 for urine specimens. The latest stool sample that was positive by reverse transcription-polymerase chain reaction (RT-PCR) was collected on day 75 while the patient was receiving intensive care. Tracheal aspirate and stool samples had a higher diagnostic yield (RT-PCR average positive rate for first 2 weeks: 66.7% and 56.5%, respectively). Pooled throat and nasal swabs, rectal swab, nasal swab, throat swab, and nasopharyngeal aspirate specimens provided a moderate yield (29.7%-40.0%), whereas throat washing and urine specimens showed a lower yield (17.3% and 4.5%). The collection procedures for stool and pooled nasal and throat swab specimens were the least likely to transmit infection, and the combination gave the highest yield for coronavirus detection by RT-PCR. Positive virologic test results in patient groups were associated with mechanical ventilation or death (p < 0.001), suggesting a correlation between viral load and disease severity.

Immunofluorescence assay for serologic diagnosis of SARS.

We evaluated an indirect immunofluorescence assay based on virus-infected cells for detecting anti-severe acute respiratory syndrome-associated coronavirus (SARS-CoV) immunoglobulin (Ig) G antibody. All confirmed SARS cases demonstrated seroconversion or fourfold rise in IgG antibody titer; no control was positive. Sensitivity and specificity of this assay were both 100%. Immunofluorescence assay can ascertain the status of SARS-CoV infection.

Human metapneumovirus detection in patients with severe acute respiratory syndrome.

We used a combination approach of conventional virus isolation and molecular techniques to detect human metapneumovirus (HMPV) in patients with severe acute respiratory syndrome (SARS). Of the 48 study patients, 25 (52.1%) were infected with HMPV; 6 of these 25 patients were also infected with coronavirus, and another 5 patients (10.4%) were infected with coronavirus alone. Using this combination approach, we found that human laryngeal carcinoma (HEp-2) cells were superior to rhesus monkey kidney (LLC-MK2) cells commonly used in previous studies for isolation of HMPV. These widely available HEp-2 cells should be included in conjunction with a molecular method for cell culture followup to detect HMPV, particularly in patients with SARS.

Genetic variation of glycoproteins B and H of human herpesvirus 7 in Hong Kong.

Glycoprotein B (gB) and glycoprotein H (gH) of human herpesvirus 7 (HHV-7) are believed to play an important role in virus entry and as targets for host immune response. This study examined the genetic diversity of these glycoproteins among 90 HHV-7 isolates collected from different individuals in Hong Kong. Overall, both the gB and gH genes were found to be highly conserved. Nucleotide polymorphism was detected only at four positions of the gB-encoding region, and all of these were synonymous substitutions. Most (97.8%) Hong Kong isolates were of gB allele group C. Two isolates collected from a Pakistani family showed a novel sequence pattern that did not match known gB allele groups. This sequence pattern was detected consistently from serial samples collected from the same individual, indicating a stable genetic entity. The gH-encoding region exhibited nucleotide polymorphism at six positions. Three of these were nonsynonymous substitutions (codon 271 Lys --> Gln, codon 308 Gly --> Glu, codon 397 Asn --> Tyr). Most (84.4%) Hong Kong isolates were of the gH allele group B, and all others were of the gH allele group C. These data indicate the possibility of using gB or gH alleles as markers for studying world-wide population movements and genetics.

Predictors of positive stool culture in adult patients with acute infectious diarrhea.

Stool cultures for bacterial pathogens are often requested for investigation of patients with infectious diarrhea, but the literature reports low yield for this diagnostic test. The identification of clinical predictors of positive stool culture will help the physician in determining the necessity for stool requests. A retrospective study was performed in the setting of an Emergency Department (ED) in Hong Kong, to compare presenting features of adult patients with positive stool culture against those with negative culture. We compared 130 consecutive cases with positive stool culture, over a 12-month period, against 119 control cases obtained from a random sampling of 524 consecutive negative cases over the same period. In multivariate analysis, the independent variables found to be associated with positive stool culture were: the month of presentation (summer season), fever, duration of abdominal pain, and requirement of IV fluid therapy. Neither bloody diarrhea nor persistent diarrhea was associated with positive stool culture.

Case report: human herpesvirus 7 associated fatal encephalitis in a peripheral blood stem cell transplant recipient.

Previous studies have suggested a neuroinvasive and neuropersistent potential of human herpesvirus 7 (HHV-7). In this report, a case of fatal encephalitis is described and its association with HHV-7 infection is discussed. An 8-year-old girl received a peripheral blood stem cell transplant for relapsed acute lymphoblastic leukaemia. The post-transplant period was uneventful and a course of intrathecal chemotherapy was given on Day-30. On Day-41, she developed acute encephalopathy with diplopia and nystagmus. She ran a rapid downhill course and succumbed despite antiviral treatment. The only positive pathological finding was the multiple microscopic foci of haemorrhage associated with neuronal degeneration detected in the brain stem. All microbiological investigations were negative, except for the presence of HHV-7 DNA in cerebrospinal fluid and brain stem tissue samples.