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1.
Gastroenterology ; 159(1): 81-95, 2020 07.
Article in English | MEDLINE | ID: mdl-32251668

ABSTRACT

BACKGROUND & AIMS: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which has been characterized by fever, respiratory, and gastrointestinal symptoms as well as shedding of virus RNA into feces. We performed a systematic review and meta-analysis of published gastrointestinal symptoms and detection of virus in stool and also summarized data from a cohort of patients with COVID-19 in Hong Kong. METHODS: We collected data from the cohort of patients with COVID-19 in Hong Kong (N = 59; diagnosis from February 2 through February 29, 2020),and searched PubMed, Embase, Cochrane, and 3 Chinese databases through March 11, 2020, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We analyzed pooled data on the prevalence of overall and individual gastrointestinal symptoms (loss of appetite, nausea, vomiting, diarrhea, and abdominal pain or discomfort) using a random effects model. RESULTS: Among the 59 patients with COVID-19 in Hong Kong, 15 patients (25.4%) had gastrointestinal symptoms, and 9 patients (15.3%) had stool that tested positive for virus RNA. Stool viral RNA was detected in 38.5% and 8.7% among those with and without diarrhea, respectively (P = .02). The median fecal viral load was 5.1 log10 copies per milliliter in patients with diarrhea vs 3.9 log10 copies per milliliter in patients without diarrhea (P = .06). In a meta-analysis of 60 studies comprising 4243 patients, the pooled prevalence of all gastrointestinal symptoms was 17.6% (95% confidence interval [CI], 12.3-24.5); 11.8% of patients with nonsevere COVID-19 had gastrointestinal symptoms (95% CI, 4.1-29.1), and 17.1% of patients with severe COVID-19 had gastrointestinal symptoms (95% CI, 6.9-36.7). In the meta-analysis, the pooled prevalence of stool samples that were positive for virus RNA was 48.1% (95% CI, 38.3-57.9); of these samples, 70.3% of those collected after loss of virus from respiratory specimens tested positive for the virus (95% CI, 49.6-85.1). CONCLUSIONS: In an analysis of data from the Hong Kong cohort of patients with COVID-19 and a meta-analysis of findings from publications, we found that 17.6% of patients with COVID-19 had gastrointestinal symptoms. Virus RNA was detected in stool samples from 48.1% patients, even in stool collected after respiratory samples had negative test results. Health care workers should therefore exercise caution in collecting fecal samples or performing endoscopic procedures in patients with COVID-19, even during patient recovery.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/prevention & control , Diarrhea/virology , Feces/virology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Load , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Diarrhea/diagnosis , Diarrhea/epidemiology , Endoscopy, Gastrointestinal/standards , Gastrointestinal Tract/diagnostic imaging , Gastrointestinal Tract/virology , Hong Kong/epidemiology , Humans , Infection Control/standards , Infectious Disease Transmission, Patient-to-Professional/prevention & control , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Prevalence , RNA, Viral/isolation & purification , SARS-CoV-2
2.
Influenza Other Respir Viruses ; 7(4): 531-9, 2013 Jul.
Article in English | MEDLINE | ID: mdl-22813463

ABSTRACT

BACKGROUND: The effects of individual lifestyle factors on the mortality risk after influenza infection have not been explored. OBJECTIVES: In this study, we assessed the modifying effects of cigarette smoking on mortality risks associated with influenza in a cohort of Hong Kong elders with a follow-up period of 1998-2009. METHODS: We used the Cox proportional hazards model with time-dependent covariates of weekly proportions of specimens positive for influenza (termed as influenza virus activity), to calculate the hazard ratio of mortality associated with a 10% increase in influenza virus activity for never, ex- and current smokers. Other individual lifestyle and socioeconomic factors as well as seasonal confounders were also added into the models. RESULTS: The overall hazard ratio associated with influenza was 1·028 (95% confidence interval, 1·006, 1·051) for all natural cause mortality and 1·035 (1·003, 1·068) for cardiovascular and respiratory mortality. We found that influenza-associated hazard ratio was greater in current and ex-smokers than in never smokers for mortality of all natural causes, cardiovascular and respiratory diseases. CONCLUSIONS: The findings suggest that smoking might increase influenza-associated mortality risks among elders.


Subject(s)
Influenza, Human/mortality , Smoking/adverse effects , Aged , Aged, 80 and over , Cohort Studies , Female , Hong Kong , Humans , Male , Risk Factors , Survival Analysis
3.
J Clin Virol ; 55(3): 209-13, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22824231

ABSTRACT

BACKGROUND: Sequence-independent amplification of clinical specimens can lead to the identification of novel pathogens. OBJECTIVES: To identify novel viruses in human stool specimens from patients with diarrhea and to investigate the ecology and clinical significance of such viruses. STUDY DESIGN: Nucleic acid extracted from stool specimens from patients with diarrhea with no known etiology were subjected to random PCR amplification and Roche/454 pyrosequencing. Novel viruses identified were genetically and epidemiologically characterized. RESULTS: Four gyroviruses, chicken anemia virus (CAV), human gyrovirus (HGV)/avian gyrovirus 2 (AGV2), gyrovirus 3 (GyV3) and a novel gyrovirus (tentatively designated as gyrovirus 4 (GyV4)) were identified in human stool specimens. GyV4, as well as CAV and AGV2/HGV were also detected in chicken skin and meat used for human consumption. CONCLUSIONS: A novel gyrovirus (GyV4) was identified in human stool and in chicken meat sold for human consumption. This virus was phylogenetically distinct from previously reported gyroviruses in chicken and humans (chicken anemia virus, human gyrovirus, avian gyrovirus 2 and recently reported gyrovirus 3). The epidemiology and pathogenesis of this virus in humans and in chicken needs to be further investigated.


Subject(s)
Circoviridae Infections/virology , Feces/virology , Gyrovirus/classification , Gyrovirus/isolation & purification , Meat/virology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Chickens , Child , Child, Preschool , Cluster Analysis , DNA, Viral/chemistry , DNA, Viral/genetics , Diarrhea/virology , Female , Gyrovirus/genetics , Humans , Infant , Male , Middle Aged , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Young Adult
4.
Cancer Res ; 72(5): 1116-25, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22282657

ABSTRACT

Nasopharyngeal carcinoma (NPC) is endemic in China and Southeast Asia where it is tightly associated with infections by Epstein-Barr virus (EBV). The role of tumor-associated viral antigens in NPC renders it an appealing candidate for cellular immunotherapy. In earlier preclinical studies, a novel adenoviral vector-based vaccine termed AdE1-LMPpoly has been generated that encodes EBV nuclear antigen-1 (EBNA1) fused to multiple CD8(+) T-cell epitopes from the EBV latent membrane proteins, LMP1 and LMP2. Here, we report the findings of a formal clinical assessment of AdE1-LMPpoly as an immunotherapeutic tool for EBV-associated recurrent and metastatic NPC. From a total of 24 patients with NPC, EBV-specific T cells were successfully expanded from 16 patients with NPC (72.7%), whereas six patients with NPC (27.3%) showed minimal or no expansion of virus-specific T cells. Transient increase in the frequencies of LMP1&2- and EBNA1-specific T-cell responses was observed after adoptive transfer to be associated with grade I flu-like symptoms and malaise. The time to progression in these patients ranged from 38 to 420 days with a mean time to progression of 136 days. Compared with patients who did not receive T cells, the median overall survival increased from 220 to 523 days. Taken together, our findings show that adoptive immunotherapy with AdE1-LMPpoly vaccine is safe and well tolerated and may offer clinical benefit to patients with NPC.


Subject(s)
Epstein-Barr Virus Infections/therapy , Herpesvirus 4, Human , Immunization, Passive , Nasopharyngeal Neoplasms/therapy , Nasopharyngeal Neoplasms/virology , Adenoviridae/immunology , Adult , Carcinoma , Disease Progression , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Nuclear Antigens/immunology , Herpesvirus 4, Human/immunology , Humans , Middle Aged , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/immunology , Nasopharyngeal Neoplasms/pathology , Neoplasm Metastasis , T-Lymphocytes, Cytotoxic/immunology , Viral Matrix Proteins/immunology
5.
Proc Natl Acad Sci U S A ; 108(48): 19359-64, 2011 Nov 29.
Article in English | MEDLINE | ID: mdl-22084096

ABSTRACT

Populations of seasonal influenza virus experience strong annual bottlenecks that pose a considerable extinction risk. It has been suggested that an influenza source population located in tropical Southeast or East Asia seeds annual temperate epidemics. Here we investigate the seasonal dynamics and migration patterns of influenza A H3N2 virus by analysis of virus samples obtained from 2003 to 2006 from Australia, Europe, Japan, New York, New Zealand, Southeast Asia, and newly sequenced viruses from Hong Kong. In contrast to annual temperate epidemics, relatively low levels of relative genetic diversity and no seasonal fluctuations characterized virus populations in tropical Southeast Asia and Hong Kong. Bayesian phylogeographic analysis using discrete temporal and spatial characters reveal high rates of viral migration between urban centers tested. Although the virus population that migrated between Southeast Asia and Hong Kong persisted through time, this was dependent on virus input from temperate regions and these tropical regions did not maintain a source for annual H3N2 influenza epidemics. We further show that multiple lineages may seed annual influenza epidemics, and that each region may function as a potential source population. We therefore propose that the global persistence of H3N2 influenza A virus is the result of a migrating metapopulation in which multiple different localities may seed seasonal epidemics in temperate regions in a given year. Such complex global migration dynamics may confound control efforts and contribute to the emergence and spread of antigenic variants and drug-resistant viruses.


Subject(s)
Demography , Disease Outbreaks , Evolution, Molecular , Genetic Variation , Influenza A Virus, H3N2 Subtype/genetics , Influenza, Human/epidemiology , Seasons , Asia/epidemiology , Australasia/epidemiology , Base Sequence , Bayes Theorem , Europe/epidemiology , Humans , Influenza A Virus, H3N2 Subtype/physiology , Models, Biological , Models, Genetic , Molecular Sequence Data , New York/epidemiology , Phylogeography , Population Dynamics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA
6.
Immunology ; 134(2): 161-71, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21896011

ABSTRACT

T cells simultaneously producing multiple cytokines and possessing cytotoxic capacity termed polyfunctional cells (PFCs) are increasingly recognized as the immune correlate of protection against pathogenic viruses. We investigated co-expression of four cytokines (interferon-γ, macrophage inflammatory protein 1-α, tumour necrosis factor-α and interleukin-2) and degranulation capacity (CD107a surface expression) of Epstein-Barr virus (EBV) -specific CD4(+) and CD8(+) T cells upon stimulation by overlapping peptides of EBV lytic (BZLF1) and latent (EBNA1, EBNA3 and LMP2) proteins, in 20 healthy Chinese long-term carriers. Two patients with post-transplant lymphoproliferative disorder (PTLD), who had impaired T-cell immunity, were studied for comparison. Both EBV-specific CD4(+) and CD8(+) PFCs were readily generated in long-term carriers and showed immunodominance hierarchies of latent proteins (EBNA1 > EBNA3/LMP2 and EBNA3 > LMP2 > EBNA1 for CD4(+) and CD8(+) T cells, respectively), as evidenced by a higher proportion of PFCs generated by immunodominant EBV proteins than by subdominant viral proteins. In contrast, the proportion of EBV-specific PFCs was markedly decreased in patients with PTLD. The EBV-specific PFCs produced more cytokine per cell than single-functional T cells and comprised different subsets. Five-functional CD4(+) and CD8(+) T cells were detected and four-functional CD4(+) T cells were mainly CD107a negative and expressed all four cytokines whereas four-functional CD8(+) T cells were mainly CD107a positive and expressed three of the four cytokines (interleukin-2-negative). We conclude that EBV-specific PFCs are generated in much higher proportions in the long-term carriers than in the patients with PTLD and maintain the immunodominant characteristics of the virus.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Carrier State/immunology , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Nuclear Antigens/immunology , Herpesvirus 4, Human/immunology , Adult , Asian People , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/virology , Carrier State/virology , Chemokine CCL3/biosynthesis , Chemokine CCL3/immunology , Child , Female , Humans , Immunodominant Epitopes/immunology , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin-2/biosynthesis , Interleukin-2/immunology , Liver Transplantation/immunology , Lymphoproliferative Disorders/immunology , Lymphoproliferative Disorders/metabolism , Lymphoproliferative Disorders/virology , Lysosomal-Associated Membrane Protein 1/immunology , Male , Middle Aged , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunology , Young Adult
7.
J Virol Methods ; 165(2): 302-4, 2010 May.
Article in English | MEDLINE | ID: mdl-20138917

ABSTRACT

The recent emergence of a novel H1N1 influenza A virus in humans caused the first influenza pandemic of this century. Many clinical diagnostic laboratories are overwhelmed by the testing demands related to the infection. Three novel H1N1-specific primer-probe sets reported during the early phase of the pandemic were tested in three commercial real-time RT-PCR mixtures. The amplification efficiencies and detection limits of these assays were determined. A ready-to-use premixed RT-PCR stored in a lyophilized format was developed. The detection limits of the studied assays were highly variable, ranging from 1.68E-01 to 1.68E-05 TCID(50) per reaction. The detection limit of the lyophilized reaction mixture was found to be 1.68E-05 TCID(50) per reaction, but the amplification efficiency of the assay was lower than those deduced from the other assays. All respiratory samples from infected patients and all control nasopharyngeal aspirates were positive and negative, respectively, in the newly developed assay. The results highlighted that, to enhance the sensitivity of an assay, it is essential to evaluate a primer-probe set with different commercial RT-PCR assays. This study also demonstrated the feasibility of using lyophilized reaction mixtures for the molecular diagnosis of novel H1N1.


Subject(s)
Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , DNA Primers , Freeze Drying , Humans , Reagent Kits, Diagnostic
8.
PLoS One ; 4(11): e7918, 2009 Nov 20.
Article in English | MEDLINE | ID: mdl-19936250

ABSTRACT

BACKGROUND: It is increasingly clear that influenza A infection induces cross-subtype neutralizing antibodies that may potentially confer protection against zoonotic infections. It is unclear whether this is mediated by antibodies to the neuraminidase (NA) or haemagglutinin (HA). We use pseudoviral particles (H5pp) coated with H5 haemagglutinin but not N1 neuraminidase to address this question. In this study, we investigate whether cross-neutralizing antibodies in persons unexposed to H5N1 is reactive to the H5 haemagglutinin. METHODOLOGY/PRINCIPAL FINDINGS: We measured H5-neutralization antibody titers pre- and post-vaccination using the H5N1 micro-neutralization test (MN) and H5pp tests in subjects given seasonal vaccines and in selected sera from European elderly volunteers in a H5N1 vaccine trial who had detectable pre-vaccination H5N1 MN antibody titers. We found detectable (titer > or = 20) H5N1 neutralizing antibodies in a minority of pre-seasonal vaccine sera and evidence of a serological response to H5N1 in others after seasonal influenza vaccination. There was excellent correlation in the antibody titers between the H5N1 MN and H5pp tests. Similar correlations were found between MN and H5pp in the pre-vaccine sera from the cohort of H5N1 vaccine trial recipients. CONCLUSIONS/SIGNIFICANCE: Heterosubtype neutralizing antibody to H5N1 in healthy volunteers unexposed to H5N1 is mediated by cross-reaction to the H5 haemagglutinin.


Subject(s)
Hemagglutinins/chemistry , Influenza A Virus, H5N1 Subtype/immunology , Influenza, Human/immunology , Influenza, Human/virology , Aged , Animals , Antibodies, Viral/chemistry , Cell Line , Child , Dogs , Europe , Humans , Influenza Vaccines/immunology , Influenza, Human/blood , Middle Aged , Neuraminidase/metabolism , Neutralization Tests
9.
Pediatrics ; 119(6): 1076-82, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17545373

ABSTRACT

OBJECTIVES: We conducted this study to test the hypothesis that intradermal influenza vaccination at one fifth of a standard dose elicits comparable immunogenicity to full-dose intramuscular vaccination in children. PATIENTS AND METHODS: We conducted a randomized, open-label study in 112 healthy children aged 3 to <18 years to compare the immunogenicity and safety of intradermal vaccination at one fifth of a dose with standard intramuscular vaccination. Analyses of hemagglutination inhibition antibody titers to each antigen in each group included geometric mean titers before and 21 days after vaccination, fold increase in geometric mean titers after vaccination, seroprotection rate, and seroconversion rate. RESULTS: The mean age of the subjects was 10.11 +/- 4.04 years in the intradermal vaccination group and 10.57 +/- 3.91 years in the intramuscular group. Intradermal vaccination was safe. Induration and mild erythema at the injection site were reported at 25% and 57%, respectively, in the intradermal group. Fold increase of geometric mean titers against influenza A/Caledonia was robust in both groups (11.1-fold and 12.9-fold increase in the intramuscular and intradermal groups, respectively), whereas that for B/Shandong was more modest (4.3-4.4). Both approaches elicited very high geometric mean titers against influenza A/Panama: 1360.5 and 893.9 for the intramuscular and intradermal groups, respectively, but because the prevaccination antibody titers were high, the fold increase of geometric mean titers was only 4.5 and 2.6, respectively. CONCLUSION: The immunogenicity of one fifth of a dose of influenza vaccine delivered by the intradermal route is comparable to the standard-dose intramuscular vaccination in children as young as 3 years of age.


Subject(s)
Antibodies, Viral/biosynthesis , Influenza Vaccines/administration & dosage , Influenza Vaccines/immunology , Vaccination/adverse effects , Adolescent , Antibodies, Viral/blood , Child , Child, Preschool , Erythema/epidemiology , Erythema/immunology , Female , Humans , Influenza Vaccines/adverse effects , Injections, Intradermal , Injections, Intramuscular , Male , Pain/epidemiology , Pain/immunology
10.
Emerg Infect Dis ; 13(6): 899-901, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17553231

ABSTRACT

We describe a 1-step reverse-transcription loop-mediated isothermal amplification assay for detection of highly pathogenic avian influenza A (H5N1) viruses. The assay was tested by using a panel of highly pathogenic H5N1 subtypes isolated over the past 10 years and clinical specimens. The assay produced negative results for all non-H5N1 subtypes.


Subject(s)
DNA, Viral/classification , Influenza A Virus, H5N1 Subtype/classification , Influenza in Birds/diagnosis , Influenza, Human/diagnosis , Molecular Diagnostic Techniques/methods , Poultry/virology , Virology/methods , Animals , DNA, Viral/genetics , Humans , Influenza A Virus, H5N1 Subtype/genetics , Influenza in Birds/genetics , Influenza in Birds/virology , Influenza, Human/genetics , Influenza, Human/virology , Molecular Sequence Data
11.
PLoS Med ; 3(4): e121, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16515368

ABSTRACT

BACKGROUND: The impact of influenza on morbidity and hospitalization in the tropics and subtropics is poorly quantified. Uniquely, the Hong Kong Special Administrative Region has computerized hospital discharge diagnoses on 95% of total bed days, allowing disease burden for a well-defined population to be accurately assessed. METHODS AND FINDINGS: Influenza-associated morbidity and hospitalization was assessed by Poisson regression models for weekly counts of hospitalizations in Hong Kong during 1996 to 2000, using proportions of positive influenza types A (H1N1 and H3N2) and B isolations in specimens sent for laboratory diagnosis as measures of influenza virus circulation. We adjusted for annual trend, seasonality, temperature, and relative humidity, as well as respiratory syncytial virus circulation. We found that influenza was significantly associated with hospitalization for acute respiratory disease (International Classification of Diseases version 9 codes [ICD9] 460-466 and 480-487) and its subcategory pneumonia and influenza (ICD9 480-487) for all age groups. The annual rates of excess hospitalization per 100,000 population for acute respiratory diseases for the age groups 0-14, 15-39, 40-64, 65-74, and 75+ were 163.3 (95% confidence interval [CI], 135-190), 6.0 (95% CI, 2.7-8.9), 14.9 (95% CI, 10.7-18.8), 83.8 (95% CI, 61.2-104.2), and 266 (95% CI, 198.7-330.2), respectively. Influenza was also associated with hospitalization for cerebrovascular disease (ICD9 430-438) for those aged over 75 y (55.4; 95% CI, 23.1-87.8); ischemic heart disease (ICD9 410-414) for the age group 40-64 y (5.3; 95% CI, 0.5-9.5) and over 75 y (56.4; 95% CI, 21.1-93.4); and diabetes mellitus (ICD9 250) for all age groups older than 40 y. CONCLUSIONS: Influenza has a major impact on hospitalization due to cardio-respiratory diseases as well as on cerebrovascular disease, ischemic heart disease, and diabetes mellitus in the tropics and subtropics. Better utilization of influenza vaccine during annual epidemics in the tropics will enhance global vaccine production capacity and allow for better preparedness to meet the surge in demand that is inevitable in confronting a pandemic.


Subject(s)
Hospitalization/statistics & numerical data , Influenza, Human/complications , Influenza, Human/epidemiology , Adult , Age Factors , Aged , Female , Hong Kong , Humans , Male , Middle Aged , Morbidity , Regression Analysis , Seasons , Tropical Climate
12.
PLoS Med ; 3(2): e27, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16379499

ABSTRACT

BACKGROUND: Cellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) in the lungs of patients with SARS is important in confirming the etiological association of the virus with disease as well as in understanding the pathogenesis of the disease. To our knowledge, there have been no comprehensive studies investigating viral infection at the cellular level in humans. METHODS AND FINDINGS: We collected the largest series of fatal cases of SARS with autopsy material to date by merging the pathological material from two regions involved in the 2003 worldwide SARS outbreak in Hong Kong, China, and Toronto, Canada. We developed a monoclonal antibody against the SARS-CoV nucleoprotein and used it together with in situ hybridization (ISH) to analyze the autopsy lung tissues of 32 patients with SARS from Hong Kong and Toronto. We compared the results of these assays with the pulmonary pathologies and the clinical course of illness for each patient. SARS-CoV nucleoprotein and RNA were detected by immunohistochemistry and ISH, respectively, primarily in alveolar pneumocytes and, less frequently, in macrophages. Such localization was detected in four of the seven patients who died within two weeks of illness onset, and in none of the 25 patients who died later than two weeks after symptom onset. CONCLUSIONS: The pulmonary alveolar epithelium is the chief target of SARS-CoV, with macrophages infected subsequently. Viral replication appears to be limited to the first two weeks after symptom onset, with little evidence of continued widespread replication after this period. If antiviral therapy is considered for future treatment, it should be focused on this two-week period of acute clinical disease.


Subject(s)
Nucleocapsid Proteins/analysis , Severe Acute Respiratory Syndrome/virology , Severe acute respiratory syndrome-related coronavirus/physiology , Virus Replication , Antibodies, Monoclonal , Autopsy , Coronavirus Nucleocapsid Proteins , Humans , Immunohistochemistry , In Situ Hybridization , Pulmonary Alveoli/virology , Respiratory Mucosa/virology , Reverse Transcriptase Polymerase Chain Reaction , Severe Acute Respiratory Syndrome/mortality , Severe Acute Respiratory Syndrome/physiopathology , Time Factors
13.
Clin Chem ; 52(2): 303-6, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16339303

ABSTRACT

BACKGROUND: Malaria is one of the most important parasitic infections in humans. A sensitive diagnostic test for malaria that could be applied at the community level could be useful in programs to control the disease. The aim of the present work was to develop a simple, inexpensive molecular test for Plasmodium falciparum. METHODS: Blood was collected from controls (n = 100) and from patients diagnosed with falciparum malaria infection (n = 102), who were recruited to the study. Heat-treated blood samples were tested by a loop-mediated isothermal amplification (LAMP) assay for P. falciparum. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. To evaluate the assay, DNA from these samples was purified and tested by PCR. Results from the LAMP and PCR assays were compared. RESULTS: The LAMP assay detected P. falciparum directly from heat-treated blood. The quantitative data from the assay correlated to the parasite counts obtained by blood-film microscopic analyses. When we used the PCR assay as the comparison method, the sensitivity and specificity of the LAMP assay were 95% and 99%, respectively. CONCLUSIONS: Unlike PCR, the LAMP assay does not require purified DNA for efficient DNA amplification, thereby reducing the cost and turnaround time for P. falciparum diagnosis. The assay requires only basic instruments, and assay positivity can be verified by visual inspection.


Subject(s)
DNA, Protozoan/blood , Hot Temperature , Malaria, Falciparum/diagnosis , Plasmodium falciparum/isolation & purification , Polymerase Chain Reaction , Specimen Handling/methods , Animals , Base Sequence , Calibration , DNA, Protozoan/genetics , Humans , Malaria, Falciparum/blood , Molecular Sequence Data , Plasmodium falciparum/genetics , Polymerase Chain Reaction/economics , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/standards , Sensitivity and Specificity
14.
J Clin Microbiol ; 43(7): 3457-9, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16000477

ABSTRACT

We compared the performance of a recently established real-time loop-mediated amplification (LAMP) assay with the one from a highly sensitive quantitative PCR assay. None of these assays produced false-positive results in this study. For samples isolated from patients within the first 3 days of disease onset, the detection rate of the quantitative PCR assay was higher (14 of 15 were positive) than the LAMP assay (9 of 15 were positive). By contrast, the detection rates of these assays toward specimens sampled from patients with more than 3 days of illness were similar (32 of 44 for PCR and 33 of 44 for LAMP were positive). The simpler operation of LAMP might be a possible solution for on-site diagnosis.


Subject(s)
Nucleic Acid Amplification Techniques/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Severe Acute Respiratory Syndrome/diagnosis , Severe Acute Respiratory Syndrome/virology , Severe acute respiratory syndrome-related coronavirus/isolation & purification , Humans , Nasopharynx/virology , Reproducibility of Results , Severe acute respiratory syndrome-related coronavirus/genetics , Sensitivity and Specificity
15.
J Med Virol ; 76(4): 435-40, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15977248

ABSTRACT

Four clinical isolates of SARS coronavirus were serially passaged in two primate cell lines (FRhK4 and Vero E6). Viral genetic sequences encoding for structural proteins and open reading frames 6--8 were determined in the original clinical specimen, the initial virus isolate (passage 0) and at passages 5, 10, and 15. After 15 passages, a total of 15 different mutations were identified and 12 of them were non-synonymous mutations. Seven of these mutations were recurrent mutation and all located at the spike, membrane, and Orf 8a protein encoding sequences. Mutations in the membrane protein and a deletion in ORF 6--8 were already observed in passage 0, suggesting these amino acid substitutions are important in the adaptation of the virus isolate in primate cell culture. A mutation in the spike gene (residue 24079) appeared to be unique to adaptation in FRhK4 cells. It is important to be aware of cell culture associated mutations when interpreting data on molecular evolution of SARS coronavirus.


Subject(s)
Mutation , Severe acute respiratory syndrome-related coronavirus/genetics , Adaptation, Biological , Amino Acid Substitution , Animals , Cell Line , Coronavirus M Proteins , Coronavirus Nucleocapsid Proteins , DNA, Viral/chemistry , DNA, Viral/genetics , Haplorhini , Membrane Glycoproteins/genetics , Nucleocapsid Proteins/genetics , Open Reading Frames , Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus/growth & development , Severe acute respiratory syndrome-related coronavirus/isolation & purification , Sequence Analysis, DNA , Serial Passage , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus , Viral Envelope Proteins/genetics , Viral Matrix Proteins/genetics , Viroporin Proteins
16.
J Clin Microbiol ; 43(1): 427-30, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15635005

ABSTRACT

Here we describe the use of the loop-mediated isothermal amplification (LAMP) method to detect human influenza viruses (H1 to H3). Our results were correlated 100% with results deduced from routine clinical diagnostic tests. In addition, we also developed a LAMP assay specific for human beta-actin cDNA as a quality control test.


Subject(s)
Influenza A virus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Actins/genetics , DNA Primers , DNA, Complementary/analysis , DNA, Viral/analysis , Humans , Influenza A virus/genetics , Influenza, Human/virology , Sensitivity and Specificity
17.
Emerg Infect Dis ; 10(2): 294-9, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15030700

ABSTRACT

Cases of severe acute respiratory syndrome (SARS) were investigated for SARS coronavirus (SARS-CoV) through RNA tests, serologic response, and viral culture. Of 537 specimens from patients in whom SARS was clinically diagnosed, 332 (60%) had SARS-CoV RNA in one or more clinical specimens, compared with 1 (0.3%) of 332 samples from controls. Of 417 patients with clinical SARS from whom paired serum samples were available, 92% had an antibody response. Rates of viral RNA positivity increased progressively and peaked at day 11 after onset of illness. Although viral RNA remained detectable in respiratory secretions and stool and urine specimens for >30 days in some patients, virus could not be cultured after week 3 of illness. Nasopharyngeal aspirates, throat swabs, or sputum samples were the most useful clinical specimens in the first 5 days of illness, but later in the illness viral RNA could be detected more readily in stool specimens.


Subject(s)
Severe Acute Respiratory Syndrome/diagnosis , Severe Acute Respiratory Syndrome/virology , Severe acute respiratory syndrome-related coronavirus/isolation & purification , Antibodies, Viral/blood , Humans , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus/genetics , Severe Acute Respiratory Syndrome/immunology , Time Factors
18.
Lancet ; 361(9371): 1773-8, 2003 May 24.
Article in English | MEDLINE | ID: mdl-12781536

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome (SARS) is a novel infectious disease with global impact. A virus from the family Coronaviridae has been identified as the cause, but the pathogenesis is still unclear. METHODS: Post-mortem tissue samples from six patients who died from SARS in February and March, 2003, and an open lung biopsy from one of these patients were studied by histology and virology. Only one full autopsy was done. Evidence of infection with the SARS-associated coronavirus (SARS-CoV) and human metapneumovirus was sought by reverse-transcriptase PCR and serology. Pathological samples were examined by light and electron microscopy and immunohistochemistry. FINDINGS: All six patients had serological evidence of recent infection with SARS-CoV. Diffuse alveolar damage was common but not universal. Morphological changes identified were bronchial epithelial denudation, loss of cilia, and squamous metaplasia. Secondary bacterial pneumonia was present in one case. A giant-cell infiltrate was seen in four patients, with a pronounced increase in macrophages in the alveoli and the interstitium of the lung. Haemophagocytosis was present in two patients. The alveolar pneumocytes also showed cytomegaly with granular amphophilic cytoplasm. The patient for whom full autopsy was done had atrophy of the white pulp of the spleen. Electron microscopy revealed viral particles in the cytoplasm of epithelial cells corresponding to coronavirus. INTERPRETATION: SARS is associated with epithelial-cell proliferation and an increase in macrophages in the lung. The presence of haemophagocytosis supports the contention that cytokine dysregulation may account, at least partly, for the severity of the clinical disease. The case definition of SARS should acknowledge the range of lung pathology associated with this disease.


Subject(s)
Lung/pathology , Severe Acute Respiratory Syndrome/pathology , Adult , Biopsy , Bronchi/pathology , Cell Nucleus/ultrastructure , Fatal Outcome , Female , Giant Cells/ultrastructure , Humans , Lung/virology , Male , Metaplasia , Middle Aged , Organ Size , Severe acute respiratory syndrome-related coronavirus/isolation & purification , Severe Acute Respiratory Syndrome/complications , Severe Acute Respiratory Syndrome/virology
19.
Int J Cancer ; 105(5): 706-9, 2003 Jul 10.
Article in English | MEDLINE | ID: mdl-12740922

ABSTRACT

We assessed 5 EBV specific assays for their capacity to effect serologic diagnosis of suspected NPC. The assays were the immunofluorescent assays, VCA IgA and EA IgA, the enzyme-linked immunosorbent assays specific for EBNA 1 IgA or zta IgG and an EBV DNA assay. Serum samples were taken from 218 symptomatic NPC patients presenting consecutively at a public hospital in Hong Kong, 51 of whom were subsequently diagnosed as having NPC; 4 had EBV-associated lung cancer with similar serology as NPC. The remaining patients included 23 who had other cancers and 140 who had other diseases. Objectives of serodiagnosis under such clinical settings, therefore, are to both exclude and predict a diagnosis of NPC. None of the assays individually can meet both requirements adequately, however. The difficulty was best overcome by combining EBNA 1 IgA and zta IgG. It was shown that 68.3% of the patients gave a confirmed test results, negative or positive, by both tests. A confirmed negative result was associated with a negative predictive value of 99.1%, providing a clear indication to exclude a diagnosis of NPC; a confirmed positive result was associated with a positive predictive value of 86.8%, providing a clear indication to proceed with diagnostic work-up of NPC. The remaining patients gave equivocal test results, being positive for one or the other test, which were associated with a positive predictive value of 43.3% and 24.2%, respectively.


Subject(s)
Antibodies, Viral/blood , Carcinoma/diagnosis , DNA, Viral/blood , Enzyme-Linked Immunosorbent Assay , Epstein-Barr Virus Infections/diagnosis , Fluorescent Antibody Technique, Indirect , Herpesvirus 4, Human/isolation & purification , Nasopharyngeal Neoplasms/diagnosis , Tumor Virus Infections/diagnosis , Antibody Specificity , Antigens, Viral/immunology , Capsid Proteins/immunology , Carcinoma/blood , Carcinoma/immunology , Carcinoma/virology , Carcinoma, Squamous Cell/blood , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/virology , DNA-Binding Proteins/immunology , Epstein-Barr Virus Infections/blood , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Nuclear Antigens/immunology , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/immunology , Hong Kong , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Lung Neoplasms/blood , Lung Neoplasms/diagnosis , Lung Neoplasms/immunology , Lung Neoplasms/virology , Nasopharyngeal Neoplasms/blood , Nasopharyngeal Neoplasms/immunology , Nasopharyngeal Neoplasms/virology , Neoplasms/blood , Neoplasms/diagnosis , Neoplasms/immunology , Neoplasms/virology , Predictive Value of Tests , Trans-Activators/immunology , Tumor Virus Infections/blood , Tumor Virus Infections/immunology , Viral Proteins/immunology
20.
N Engl J Med ; 347(26): 2097-103, 2002 Dec 26.
Article in English | MEDLINE | ID: mdl-12501221

ABSTRACT

BACKGROUND: It has been difficult to define the burden of influenza in children because of confounding by the cocirculation of respiratory syncytial virus (RSV). In Hong Kong, China, the influenza and RSV infection seasons sometimes do not overlap, thus providing an opportunity to estimate the rate of influenza-related hospitalization in a defined population, free from the effects of RSV. METHODS: In a retrospective, population-based study, we estimated the influenza-associated excess rate of hospitalization among children 15 years old or younger in the Hong Kong Special Administrative Region from 1997 to 1999. Data from a single hospital with intensive use of virologic analyses for diagnosis were obtained to define and adjust for underestimation of the model. RESULTS: Peaks of influenza and RSV infection activity were well separated in 1998 and 1999 but overlapped in 1997. The adjusted rates of excess hospitalization for acute respiratory disease that were attributable to influenza were 278.5 and 288.2 per 10,000 children less than 1 year of age in 1998 and 1999, respectively; 218.4 and 209.3 per 10,000 children 1 to less than 2 years of age; 125.6 and 77.3 per 10,000 children 2 to less than 5 years of age; 57.3 and 20.9 per 10,000 children 5 to less than 10 years of age; and 16.4 and 8.1 per 10,000 children 10 to 15 years of age. CONCLUSIONS: In the subtropics, influenza is an important cause of hospitalization among children, with rates exceeding those reported for temperate regions.


Subject(s)
Hospitalization/statistics & numerical data , Influenza, Human/epidemiology , Acute Disease , Adolescent , Child , Child, Preschool , Female , Hong Kong/epidemiology , Humans , Infant , Male , Respiratory Syncytial Virus Infections/epidemiology , Respiratory Tract Infections/epidemiology , Retrospective Studies
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