Measures of site resourcing predict virologic suppression, immunologic response and HIV disease progression following highly active antiretroviral therapy (HAART) in the TREAT Asia HIV Observational Database (TAHOD).

OBJECTIVES: Surrogate markers of HIV disease progression are HIV RNA in plasma viral load (VL) and CD4 cell count (immune function). Despite improved international access to antiretrovirals, surrogate marker diagnostics are not routinely available in resource-limited settings. Therefore, the objective was to assess effects of economic and diagnostic resourcing on patient treatment outcomes. METHODS: Analyses were based on 2333 patients initiating highly active antiretroviral therapy (HAART) from 2000 onwards. Sites were categorized by World Bank country income criteria (high/low) and annual frequency of VL (> or = 3, 1-2 or <1) or CD4 (> or = 3 or <3) testing. Endpoints were time to AIDS/death and change in CD4 cell count and VL suppression (<400 HIV-1 RNA copies/mL) at 12 months. Demographics, Centers for Disease Control and Prevention (CDC) classification, baseline VL/CD4 cell counts, hepatitis B/C coinfections and HAART regimen were covariates. Time to AIDS/death was analysed by proportional hazards models. CD4 and VL endpoints were analysed using linear and logistic regression, respectively. RESULTS: Increased disease progression was associated with site-reported VL testing less than once per year [hazard ratio (HR)=1.4; P=0.032], severely symptomatic HIV infection (HR=1.4; P=0.003) and hepatitis C virus coinfection (HR=1.8; P=0.011). A total of 1120 patients (48.2%) had change in CD4 cell count data. Smaller increases were associated with older age (P<0.001) and 'Other' HIV source exposures, including injecting drug use and blood products (P=0.043). A total of 785 patients (33.7%) contributed to the VL suppression analyses. Patients from sites with VL testing less than once per year [odds ratio (OR)=0.30; P<0.001] and reporting 'Other' HIV exposures experienced reduced suppression (OR=0.28; P<0.001). CONCLUSION: Low measures of site resourcing were associated with less favourable patient outcomes, including a 35% increase in disease progression in patients from sites with VL testing less than once per year.

Deferred modification of antiretroviral regimen following documented treatment failure in Asia: results from the TREAT Asia HIV Observational Database (TAHOD).

OBJECTIVE: The aim of the study was to examine the rates and predictors of treatment modification following combination antiretroviral therapy (cART) failure in Asian patients with HIV enrolled in the TREAT Asia HIV Observational Database (TAHOD). METHODS: Treatment failure (immunological, virological and clinical) was defined by World Health Organization criteria. Countries were categorized as high or low income by World Bank criteria. RESULTS: Among 2446 patients who initiated cART, 447 were documented to have developed treatment failure over 5697 person-years (7.8 per 100 person-years). A total of 253 patients changed at least one drug after failure (51.6 per 100 person-years). There was no difference between patients from high- and low-income countries [adjusted hazard ratio (HR) 1.02; P=0.891]. Advanced disease stage [Centers for Disease Control and Prevention (CDC) category C vs. A; adjusted HR 1.38, P=0.040], a lower CD4 count (>or=51 cells/microL vs. or=400 HIV-1 RNA copies/mL vs. <400 copies/mL; adjusted HR 2.69, P<0.001) were associated with a higher rate of treatment modification after failure. Compared with patients from low-income countries, patients from high-income countries were more likely to change two or more drugs (67%vs. 49%; P=0.009) and to change to a protease-inhibitor-containing regimen (48%vs. 16%; P<0.001). CONCLUSIONS: In a cohort of Asian patients with HIV infection, nearly half remained on the failing regimen in the first year following documented treatment failure. This deferred modification is likely to have negative implications for accumulation of drug resistance and response to second-line treatment. There is a need to scale up the availability of second-line regimens and virological monitoring in this region.

Development of a marine fish model for studying in vivo molecular responses in ecotoxicology.

A protocol for fixation and processing of whole adult marine medaka (Oryzias melastigma) was developed in parallel with in situ hybridization (ISH) and immunohistochemistry (IHC) for molecular analysis of in vivo gene and protein responses in fish. Over 200 serial sagittal sections (5microm) can be produced from a single adult medaka to facilitate simultaneous localization and quantification of gene-specific mRNAs and proteins in different tissues and subcellular compartments of a single fish. Stereological analysis (as measured by volume density, V(v)) was used to quantify ISH and IHC signals on tissue sections. Using the telomerase reverse transcriptase (omTERT) gene, omTERT and proliferating cell nuclear antigen (PCNA) proteins as examples, we demonstrated that it is possible to localize, quantify and correlate their tissue expression profiles in a whole fish system. Using chronic hypoxia (1.8+/-0.2 mgO(2)L(-1) for 3 months) as an environmental stressor, we were able to identify significant alterations in levels of omTERT mRNA, omTERT protein, PCNA (cell proliferation marker) and TUNEL (apoptosis) in livers of hypoxic O. melastigma (p<0.05). Overall, the results suggest that O. melastigma can serve as a model marine fish for assessing multiple in vivo molecular responses to stresses in the marine environment.

Outpatient parenteral antibiotic therapy in Singapore.

Outpatient parenteral antibiotic therapy (OPAT) remains in its infancy in Singapore, with the first patients enrolled 4 years ago. Singapore's three largest hospitals, with over 3000 inpatient beds, now have designated and approved OPAT services. This study reviews the demographic, clinical and cost data of all patients enrolled in 2005 to facilitate benchmarking between services in Singapore and abroad and also to identify common needs for further development. In 2005, 225 OPAT enrollments in 208 different patients resulted in 4050 days of OPAT care. Orthopaedic diagnoses constituted 40% of admissions. Vancomycin was the most frequently used antibiotic (34%). The re-admission rate was 8.9%, but complications of OPAT care were only occasionally implicated. An estimated $207,200 was saved by patients despite there being significant financial disincentives to subsidised patients. OPAT is a safe, cost-efficient system that is becoming increasingly accepted in Singapore by patients, clinicians and management. Our three services have evolved independently into very similar practices. There is potential for further innovation, including outreach and carer-delivered dosing. However, major financial disincentives require review.

A frequent activated smoothened mutation in sporadic basal cell carcinomas.

Basal-cell carcinomas (BCCs) are the most common cancer in Caucasians. It has been reported that the patched gene is inactivated in 30-40% sporadic BCCs and 20% sporadic medulloblastomas via loss of heterozygosity and nonsense mutations. Recently, two activating smoothened mutations have been found in the sporadic basal cell carcinomas. One, at base pair 1604 (G-to-T transversion) of exon 9, changes codon 535 from tryptophan to leucine, and the other, at base pair 1685 (G-to-A transition) of exon 10, changes codon 562 from arginine to glutamine (Xie et al., 1998). In our study, 1604G-->T was found in 20 out of 97 (20.6%) sporadic BCCs. The high prevalence indicates that 1604G is the mutation hot spot in our tumor samples. This mutation was detected in all three histological subtypes of BCCs, suggesting that smoothened mutation is an early event during the development of the tumor. Our finding of a high smoothened mutation rate, together with high frequent patched gene mutations reported recently, indicates that activation of the hedgehog signal transduction pathway is the most common and early event in the development of sporadic BCCs. Additionally, to determine whether smoothened, like patched, is also involved in the carcinogenesis of medulloblastomas, we screened medulloblastoma samples for these two mutations by restriction analysis. We have found the 1604G-->T mutation in 1 out of 21 medulloblastomas. This result confirmed smoothened gene involvement in the carcinogenesis of medulloblastoma.

Hippocampal involvement in dengue fever.

Flaviviruses are among the most important emerging viruses known to man. Dengue is the most common flavivirus infection in Singapore, and is transmitted between humans by the Aedes mosquito. We report a 25-year-old man with dengue fever complicated by selective hippocampal involvement manifesting as amnesia. This has not been described in the literature previously. Dengue polymerase chain reaction and serology were positive. Magnetic resonance imaging of the brain showed bilateral hippocampal involvement.

Wound infections in tsunami survivors: a commentary.

Survivors of the December 2004 Indian Ocean tsunami sustained a variety of wound infections, ranging from common pathogens to rarely seen organisms. This article discusses the likely microbiology potentially seen in wound infections with exposure to freshwater, seawater, soil, faecal or other contamination, and attempts to provide an organising framework for choosing empiric antibiotics for such infections. Therapy for less frequently encountered clinical entities is also discussed, including tetanus, cutaneous and septicaemic melioidosis, post-traumatic mucormycosis, Vibrio vulnificus and Aeromonas hydrophila.

Isolation of specific IgM monoclonal antibodies by affinity chromatography using alkaline buffers.

Comparative studies on the efficacies of various buffers in eluting absorbent-bound antibodies revealed the denaturative effect of 3 M KSCN on all three IgM antibodies but not on an IgG protein, and the generally weak eluting power of glycine-HCl buffer, pH 2.5, on these monoclonal antibodies. A recently described medium consisting of 50% (v/v) ethylene glycol in an alkaline buffer, pH 10.5, was found to be relatively efficient for elution in all cases. However, subsequent studies on one of the IgM antibodies showed that alkali alone could effect elution, with recovery of active protein improving on increasing the pH, till the maximum (38%) at pH 11.0, after which denaturation occurred. Addition of ethylene glycol to the medium facilitated the elution; however, at pH greater than 10.0, the solvent potentiated the denaturative effect of the medium. Since pH 11.0 was found to be the highest pH in which all three IgM antibodies examined were stable, 0.1 M glycine-NaOH buffer, pH 11.0, may be a useful eluent for IgM (and other) antibodies in general.

Monoclonal IgM/A hybrid antibodies: artifacts due to anti-idiotype (T15) antibodies in commercial anti-alpha sera.

A mouse hybridoma antibody, with specificity for Trichinella spiralis, was found to react against both anti-mu and anti-alpha sera obtained from two to three commercial sources. It also had a unique electrophoretic mobility, bound to staphylococcal protein A, and was comprised of a single type of heavy chain (mu-like) and light chain (kappa). However, Northern blot analysis of RNA extracted from the corresponding hybridoma cells revealed only mu but no alpha message. Subsequent studies on the protein, as well as on a similar "hybrid" antibody previously described, using antisera raised to the antibody but which had been absorbed against normal mu and alpha determinants, suggested an idiotype common to both antibodies: this was intuitively guessed, and later confirmed, to be the phosphorylcholine-specific T15 idiotype. It was further shown that anti-T15 antibodies prevailed in the commercial anti-alpha sera and these (rather than alpha-chain specific antibodies) were responsible for the cross-reactivities of the sera with the hybrid antibodies.

The antigen-specific immunoglobulin G receptor is more sensitive to stimulation than the IgM receptor in transfected B cells.

A murine lymphoma cell line (M12.4) was transfected with immunoglobulin (Ig) genes encoding a T15+ (idiotype) IgM antibody or an idiotypically identical IgG antibody. Three transfectant clones of each class which showed similar (albeit distinguishable) levels of membrane expression of the transfected genes were used in the study. The response of each cell population to stimulation with anti-T15 antibodies was followed by measurement of the change in the intracellular Ca++ concentration. The IgG transfectants were found to be significantly more responsive to such stimulation than the IgM cells. In contrast, there was no difference in their response to a nonspecific reagent, the calcium ionophore A23187.

Production of anti-phosphorylcholine antibodies of the T15 idiotype in CBA/N xid mice: investigation of the defect using a T15 immunoglobulin transgene.

A notable defect in CBA/N xid mice is their relative inability to make antibodies to phosphorylcholine (PC), particularly those of the T15 idiotype which predominate in the anti-PC responses of immunologically normal mice. To investigate the basis of this defect, we introduced functionally rearranged genes encoding a T15+ PC-binding immunoglobulin G antibody into the germline of these animals. Expression of these genes in the xid cells was observed, shown by the existence of a distinct population of T15+ cells (3 x 10(6)) in the spleen of the transgenic animals, and the presence of PC-binding T15+ IgG antibodies (1-15 micrograms/ml) in the serum. Mixed antibody molecules were also found, however, which were composed of both transgene-encoded and endogenously-derived chains. Existence of the T15+ cells in these animals seemed normal, since these were not depleted (to any great extent) and were immunocompetent as well. The latter was shown by the increased T15+ antibody production in the transgenic animals when stimulated with a PC-associated thymus-independent type 1 (TI-1) antigen and anti-idiotype antibodies, but not with the pneumococcal TI-2 antigen. This is similar to the PC-specific (T15-) responsiveness of normal CBA/N xid mice. Based on these results, we argue that a reason why T15+ antibodies are not normally made by CBA/N xid animals is because T15+ genes are not utilized or, as with any T15+ precursors present, selected for in these animals, in contrast to normal mice where the Lyb-5 or CD5 cells (which are absent in CBA/N xid animals) are known to be specially endowed to make such antibodies.

Structural analysis of a phosphorylcholine-binding antibody which exhibits a unique carrier specificity for Trichinella spiralis.

A phosphorylcholine (PC)-binding IgG (Mab2) antibody produced by a hybridoma derived from a BALB/c mouse which had been immunized against Trichinella spiralis was found to bind to the immunizing antigen (TSC) but not to other PC-associated antigens such as pneumococcal antigen (PNC) and PC-conjugated ovalbumin (PC-OVA). Sequence analysis of the protein revealed the presence of a heavy chain (VH) which was very similar (differing in only four amino acids) to that of the M511 myeloma protein, and a light chain (VL) which was completely identical to that of the M167 myeloma protein. Several M511/M167+ proteins, including the prototypic M511 protein and PC-binding proteins of other families (TEPC 15 and W3207), were examined in their binding to the various PC-associated antigens. These were found to be largely indiscriminate although subtle differences were observed for some antigens with some of the antibodies. A comparison of the VH sequences of Mab2 and these proteins revealed that of the differences seen, the single most important substitution in Mab2 which could contribute to the unique specificity of the molecule is the glycine residue at 49H. None of the other proteins, including other PCV-binding proteins published to-date, which utilize the same VH segment (99 in total), has this substitution.

A human and a mouse anti-idiotypic antibody specific for human T14(+) anti-DNA antibodies reconstructed by phage display.

Little is known about human anti-idiotypic antibodies. Phage display methodology was used to reconstruct these antibodies from lupus patients, which recognize a subset (T14(+)) of anti-DNA antibodies. Antigen-specific B cells were isolated from the blood using a peptide based on a complementarity determining region (V(H)CDR3) of the prototypic T14(+) antibody. cDNA fragments of the V(H) and V(L) genes prepared from the cells were expressed as phage displayed single chain Fv (scFv) fragments using the pCANTAB-5E phagemid vector. From a reactive clone obtained, the Ig genes used were identified to be V(H)3, D5-D3, J(H)4b, V(kappa)I and J(kappa)2. The heavy chain was highly mutated, especially in CDR3, which bears mutations mostly of the replacement type; this region is also unusual in being extremely long due to a D-D fusion. In contrast, a mouse hybridoma antibody, made to the same T14(+) peptide and transformed as a scFv fragment, uses a short V(H)CDR3 comprising five amino acids, three of which are tyrosines. Tyrosines may be important for antigen binding because two of these also exist in the human V(H)CDR3. The light chains of both antibodies may also contribute to the specificity of the protein, because their V(L) segments, including the CDRs, are highly homologous to each other.

A one-step two-particle latex immunoassay for the detection of Salmonella typhi endotoxin.

A simple and rapid test (LIMM, short for latex immunoassay) is described for detecting Salmonella typhi endotoxin. It involves the simultaneous binding of the antigen by two types of reagent particles contained in a micro-tube: an indicator latex particle coated with a monoclonal antibody specific for the O-9 determinant on the endotoxin, and a magnetic bead coated with another monoclonal antibody specific for a different O-determinant. At the end of-the test, the magnetic beads are sedimented by use of a magnet, and the result is read based on the turbidity of the indicator latex suspension. Compared to a similar assay developed previously which uses only a single particle reagent (i.e., a tube agglutination system), LIMM was found to be slightly more sensitive especially when using short (less than 30 min) incubation times, and was at all times easier to read. The sensitivity of LIMM, in fact, increased with increasing time of incubation. When compared to the sensitivity (25 ng/ml) of a conventional slide latex agglutination test performed using the same indicator latex reagent, this parameter was 0-, 4.9-, 12.5- and 28.7-fold better after 5, 15, 30 and 60 min of incubation in the LIMM.

A spectrophotometric method for evaluating a latex agglutination assay of Salmonella typhi lipopolysaccharide.

A spectrophotometer set at a wavelength of 400 nm was used to read reaction mixtures containing a 0.01% suspension of antibody-sensitized latex particles and different amounts of soluble antigen, after incubation in tubes for 30 min. The antigen used was Salmonella typhi lipopolysaccharide, and the antibody was an O-9-specific monoclonal antibody. Agglutination was indicated by a fall in the turbidimetric measurement compared to control, unagglutinated latex. It was observed that agglutination increased with increasing concentrations of antigen to a maximum at 0.25-1.0 micrograms/ml, after which, the amount of agglutination decreased at a similar rate as far as 1 mg/ml of antigen, when no agglutination occurred at all. Thus, a bi-symmetrical curve was obtained, suggestive of a precipitation reaction. At the 'equivalence point', the turbidity of the reaction mixture compared to control fell from OD400 1.1 to OD400 0.3. A similar inhibition of reaction at 'antigen excess' was observed visually in the reaction mixtures. Conventional slide tests performed in parallel could also be inhibited, but at higher antigen levels. Inhibition could be achieved with a commercially available latex used for the detection of Neisseria meningitidis antigen.

A tube latex test based on colour separation for the detection of IgM antibodies to either one of two different microorganisms.

A simple two stage assay was developed for the detection of IgM antibodies to either one of two microorganisms chosen arbitrarily for this study. Salmonella enteritidis and Trichinella spiralis. In the first stage, magnetic polystyrene beads (Dynabeads) coated with anti-mu (mouse) antibodies were incubated with the test material for 45 min to capture the IgM antibodies. In the second stage, indicator latex particles were incubated with the Dynabeads for 30 min and the results read following settlement of the Dynabeads under the influence of a magnet. Two types of indicator particles were used: blue-coloured (sensitized with Trichinella antigen) and red-coloured (sensitized with Salmonella antigen). These were mixed in suitable proportions to form a purple suspension. Reaction of either type of latex particles due to binding to the adsorbed IgM antibodies resulted in the settlement of that particle and hence a change of colour in the suspension to either red (if Trichinella-specific antibodies alone were present) or blue (if Salmonella-specific antibodies alone were present). When applied to the sera (used at 1/10 dilution) of both normal and immunized mice, the assay was positive for all but one (18) immune sera, and negative for all but one (9) normal sera.

An improved latex agglutination-inhibition test for the detection of human chorionic gonadotropin in urine.

A commercially available slide latex agglutination-inhibition test for pregnancy was used in a tube modification for the detection of human chorionic gonadotropin (hCG) in urine. The tube method used 100 microliters sample and small quantities of reagents which were mixed continuously for 30 min. The results obtained, based on flocculation, were 15-30-fold better than those obtained by slide agglutination using spiked samples or urine samples from pregnant women. The lowest concentration of hCG detected in the spiked samples was 31 IU/l. When 43 clinical specimens were examined, the tube method detected four more samples than the 18 detected by the slide method. A good correlation (r = 0.96) was observed between the two methods in the determination of the hCG content in the positive samples.

A comparison of immunological methods for the detection of Trichinella spiralis antigen.

Eight immunological methods all using the same monoclonal antibody reagent were compared for the detection of Trichinella spiralis antigen. These were based on: (1) the direct adsorption of the antigen to the immunoadsorbent (nitrocellulose membrane, polyvinyl chloride strip or microplate); (2) capture of the antigen by antibodies pre-sensitized on the immunoadsorbent; and (3) latex agglutination. The methods found suitable were: (a) capture radioimmunoassay (capture-RIA) (sensitivity: less than 0.5 microgram/ml antigen); (b) direct enzyme immunoassay (direct-ELISA) (less than 0.5 microgram/ml); (c) tube latex agglutination test (2.2 micrograms/ml); and (d) direct immunodot assay (8.8 micrograms/ml). However, the performance of the direct-ELISA was greatly affected by the presence of each of three extraneous substances (bovine serum albumin (BSA), lipopolysaccharide (LPS), normal swine muscle homogenate (NSM) added to the antigen sample. The direct immunodot assay was also affected by the presence of BSA or LPS, whereas both the capture-RIA and the tube latex agglutination methods were affected by the presence of NSM only. The findings are discussed with a view of detecting T. spiralis larvae directly from pork samples by immunological means.

A two-particle turbidometric latex immunoassay for the detection of specific IgM antibodies.

A simple two stage assay using latex particles as a reaction indicator has been developed for the detection of IgM antibodies to Trichinella spiralis. In the first stage, magnetic polystyrene beads (Dynabeads) coated with T. spiralis antigen were incubated for 30 min with the test serum. After washing, in the second stage, the assay was developed for 1 h using anti-mu-coated latex particles. After sedimentation of the Dynabeads the turbidity of the resultant latex suspension was measured spectrophotometrically at a wavelength of 400 nm. A decrease in turbidity of more than 20% from that of the control, unreacted, suspension was considered positive. Using an IgM phosphorylcholine-binding monoclonal antibody which was reactive with T. spiralis, the sensitivity of the assay was determined to be 110 ng/ml of antibody. This was 20-fold less than the sensitivity achieved in an indirect enzyme-linked immunosorbent assay (ELISA). When the assay was applied to sera obtained from CBA/N or BALB/c mice, which were either normal or immunized against T. spiralis, the expected results were obtained with titers up to 1/640 observed, and confirmed (r = 0.93, P less than 0.001) in the ELISA.

Some interesting isolates from a diagnostic laboratory.

Citrobacter koseri, Plesiomonas shigelloides, Edwardsiella tarda, Yersinia enterocolitica, Alkalescens dispar, Vibrio parahaemolyticus, and Vibrio alginolyticus were seven interesting microorganisms isolated recently in our diagnostic laboratory.