Serotype prevalence of Candida albicans from blood culture isolates.

Blood culture isolates of Candida albicans were collected from 102 patients in Seattle, Wash., hospitals (n = 77) and Hong Kong (n = 25). The patients were classified by immune status into two groups. Group I patients were severely immunosppression, and group II patients had underlying risk factors for candidemia but no underlying immunosuppression. Serotyping by Hasenclever tube agglutination was done. In the Seattle area, the odds of fungemia with type B C. albicans were 3.62 times greater than the odds of type B fungemia in group II patients. Although the odds ratio could not be computed for Hong Kong patients, the direction of the relationship in this population was consistent with the data on Seattle patients. Despite the magnitude of the odds ratios, the relative prevalence of type B over type A in group I compared with group II was not significant when analyzed separately by region, probably because of relatively low numbers of isolates in group II. Accepting that the effect of immune status on serotype is equivalent across regions but presupposing that a regional effect on type B prevalence exists, the pooled odds for fungemia with serotype B in group I patients are increased 5.4-fold over those of group II patients. Logistic regression analysis controlling for region gave similar results.     

Cloning and characterisation of malE in Burkholderia pseudomallei

No recombinant protein is available for serodiagnosis or skin test in the diagnosis of melioidosis. This report describes the cloning of the malE gene, which encodes an immunogenic protein of Burkholderia pseudomallei. Bi-directional DNA sequencing of malE revealed that the gene contained a single open reading frame encoding 416 amino acid residues with a predicted molecular mass of 44.4 kDa. BLAST analysis showed that the putative protein encoded by malE is homologous to the maltose-binding protein (MBP) of other bacteria. It has 48% and 63% amino acid identity and similarity with the MBP of Brucella abortus, and malE complementation assay showed that it partially complemented the function of the MBP of Escherichia coli. Several highly conserved regions among the MBP of B. pseudomallei, Br. abortus, Salmonella enterica serotype Typhimurium, E. coli and Enterobacter aerogenes were observed. These regions represent signatures A, B, C, D and F identified in the MBP of E. coli. Further sequence analysis revealed that the first 24 amino acid residues of the MBP of B. pseudomallei probably represent the N-terminal signal peptide of the protein. Similar to the signal peptide of the MBP of E. coli, Ent. aerogenes and S. Typhimurium, the MBP of B. pseudomallei contains two basic residues in the first eight amino acids, followed by a hydrophobic core, with the last three amino acids in the signal peptide being Ala-Gln-Ala, conforming to the consensus sequence Ala-X-Ala at positions -3 to -1 relative to the site of proteolytic cleavage for recognition by signal peptidase I. Further studies on serodiagnosis of melioidosis with recombinant MBP should be performed.     

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.     

Typhoid fever associated with acute appendicitis caused by an H1-j strain of Salmonella enterica serotype Typhi

While most strains of Salmonella enterica serotype Typhi, the etiologic agent of typhoid fever, have only a phase 1 flagellar antigen, H1-d, variations of the flagellar antigen have been observed. Although H1-j strains (one of the flagellar antigen variants) account for 10 to 50% of S. enterica serotype Typhi strains found in Indonesia, there have been no published data to suggest its existence in other parts of the world. We describe a case of typhoid fever associated with acute appendicitis caused by an S. enterica serotype Typhi H1-j strain in a Chinese woman in Hong Kong. A gram-negative, motile rod was recovered from her blood and stool cultures. Conventional biochemical tests and the Vitek system (GNI+) showed that the bacterium was S. enterica serotype Typhi. The isolate agglutinated with poly(O), 9O, Vi and H1-j Salmonella antisera but not with poly(H) antisera. The patient developed antibodies against only S. enterica serotype Typhi O antigens but not against H1-d antigen by the Widal test. Flagellin C gene (fliC) sequencing showed a 261-bp deletion in the fliC gene of the isolate, confirming that the isolate possessed the H1-j antigen. The patient had no past history of travel to Indonesia or personal contact with any Indonesian. She recovered with appendectomy and antibiotic treatment. Further studies should be performed to determine the prevalence of this unusual S. enterica serotype Typhi strain in our locality.     

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.     

Evaluation of NucliSens EasyQ HIV-1 assay for quantification of HIV-1 subtypes prevalent in South-east Asia.

BACKGROUND: Monitoring anti-retroviral therapy requires that viral load assays for human immunodeficiency virus type 1 (HIV-1) be applicable to diverse HIV-1 subtypes. OBJECTIVES: To evaluate NucliSens EasyQ HIV-1 assay for quantitation of common HIV-1 subtypes prevalent in South-east Asia. STUDY DESIGN: One hundred and nineteen plasma samples collected in Hong Kong and Cambodia were used to compare the performance of NucliSens EasyQ HIV-1 and COBAS Amplicor HIV-1 Monitor version 1.5 assays. Viral RNA extracted from the NucliSens MiniMAG was also used for HIV-1 subtyping. RESULTS: Performance of NucliSens EasyQ correlated well with COBAS Amplicor (r=0.777, p<0.001) and the small mean difference (0.0462log(10)IU/mL) obtained in the Bland and Altman model indicated good agreement between two assays. The NucliSens EasyQ assay demonstrated a 95% sensitivity at 500IU/mL and 100% specificity. Reproducibility of this assay was within log(10)2-4IU/mL and had a coefficient of variation between 2.3% and 10.4%. Among the 109 specimens included in the analysis, HIV-1 subtyping identified 64 CRF01_AE, 38 subtype B, 3 subtype C, 3 CRF07_BC and 1 subtype G viruses. CONCLUSIONS: Performance of NucliSens EasyQ was comparable to COBAS Amplicor for HIV-1 viral load monitoring. RNA extracts from NucliSens MiniMAG could be used for HIV-1 viral load monitoring, subtyping and drug resistance mutations detection. Our findings highlight the versatility of both NucliSens EasyQ and COBAS Amplicor in monitoring prevalent subtypes and rare circulating recombinant forms (CRFs) in the South-east Asia region.     

Microchip module for blood sample preparation and nucleic acid amplification reactions

A computer numerical control-machined plexiglas-based microchip module was designed and constructed for the integration of blood sample preparation and nucleic acid amplification reactions. The microchip module is comprised of a custom-made heater-cooler for thermal cycling, a series of 254 microm x 254 microm microchannels for transporting human whole blood and reagents in and out of an 8--9 microL dual-purpose (cell isolation and PCR) glass-silicon microchip. White blood cells were first isolated from a small volume of human whole blood (<3 microL) in an integrated cell isolation--PCR microchip containing a series of 3.5-microm feature-sized "weir-type" filters, formed by an etched silicon dam spanning the flow chamber. A genomic target, a region in the human coagulation Factor V gene (226-bp), was subsequently directly amplified by microchip-based PCR on DNA released from white blood cells isolated on the filter section of the microchip mounted onto the microchip module. The microchip module provides a convenient means to simplify nucleic acid analyses by integrating two key steps in genetic testing procedures, cell isolation and PCR and promises to be adaptable for additional types of integrated assays.     

Photodynamic therapy induced Fas-mediated apoptosis in human carcinoma cells

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. Human poorly (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells undergo rapid apoptosis when treated with PDT sensitized with Hypocrellin A (HA) and Hypocrellin B (HB). It has been shown that these compounds have a strong photodynamic effect on tumors and viruses. The initiating events of PDT sensitized HA and HB-induced apoptosis are poorly defined. In the current study, we sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events are an early event in HA and HB-treated PDT induced apoptosis. Loss of mitochondrial transmembrane potential, release of cytochrome c, involvement of caspases-8 and -3 and the status caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells. Photoactivation of HA and HB enhanced both CD95/CD95L expression and induced CD95-signaling dependent cell death in all tumor cell lines studied. CD95/ CD95L expression appeared within 2 h following light activation and appeared to be a primary event in PDT induced apoptosis. Furthermore, these results indicate that release of mitochondrial cytochrome c into the cytoplasm is a secondary event following the activation of initiator caspase-8 preceding caspase-3 activation, cleavage of PARP and DNA fragmentation. Cytochrome c appeared in the cytosol within 2-3 h post PDT. Cleavage of PARP was observed at 3-4 h following PDT and caspase-3 specific inhibitor DEVD-CHO and broad-spectrum caspases inhibitor z-VAD-fmk blocked caspase-3 activation and PARP cleavage suggesting that caspase-3 plays an important role in HA and HB-induced apoptosis.