Predictors of clinical progression in HIV-1-infected adults initiating combination antiretroviral therapy with advanced disease in the Asia-Pacific region: results from the TREAT Asia HIV observational database.

The majority of HIV-infected patients in developing countries commences combination antiretroviral therapy (cART) with advanced disease. We examined predictors of disease progression in patients initiating cART with CD4 count ≤200 cells/mm(3) in the TREAT Asia HIV Observational Database. The main outcome measure was progression to either an AIDS-defining illness or death occurring 6 months after initiation of cART. We used survival analysis methods. A total of 1255 patients contributed 2696 person years of follow-up; 73 were diagnosed with AIDS and 9 died. The rate of progression to the combined end point was 3.0 per 100 person years. The factors significantly associated with a higher risk of disease progression were Indian ethnicity, infection through intravenous drug use, lower CD4 count, and hemoglobin ≤130 g/dL at 6 months. In conclusion, measurements of CD4 count and hemoglobin at month 6 may be useful for early identification of disease progression in resource-limited settings.

Effects of once-daily darunavir/ritonavir versus lopinavir/ritonavir on metabolic parameters in treatment-naive HIV-1-infected patients at week 96: ARTEMIS.

In the ARTEMIS trial, 689 treatment-naïve, HIV-1-infected adults received darunavir/ritonavir (DRV/r) 800/100 mg every day or lopinavir/ritonavir (LPV/r) 800/200 mg total daily dose plus fixed-dose tenofovir/emtricitabine. Week 96 metabolic parameters are reported. Adverse events (AEs) classed as metabolism/nutrition disorders were observed in 14% of DRV/r and 22% of LPV/r patients. Lipid-related AEs were reported in fewer DRV/r (8%) than LPV/r (16%) patients. A small increase in glucose and insulin levels was observed at week 96 in both groups. Lipoma was the only lipodystrophy-related AE reported in >1% of patients (DRV/r, n = 1; LPV/r, n = 4) and no grade 3 or 4 lipodystrophy-related AEs were reported. No clinically relevant changes from baseline were seen in anthropometric measurements in either group. Median mid-waist/hip ratio at week 96 was comparable to baseline in both arms. Over 96 weeks, DRV/r had a similar effect on glucose and insulin levels but a more favourable lipid profile than LPV/r in treatment-naïve, HIV-infected patients.

Isolation of the first three cases of Clostridium difficile polymerase chain reaction ribotype 027 in Singapore.

INTRODUCTION: The incidence of Clostridium (C.) difficile infection (CDI) was on the rise from 2001 to 2006 in Singapore. Recent unpublished data suggests that its incidence had remained stable or decreased in most local public hospitals between 2006 and 2010. It is, however, not known if the polymerase chain reaction (PCR) ribotype 027 strains have been circulating, although reports suggest that this strain is emerging in Asia, with the first cases reported from Japan in 2007, as well as in Hong Kong and Australia in 2009. We initiated a culture-based surveillance to detect this epidemic strain in Singapore. METHODS: From September 2008 to December 2009, all non-duplicate toxin-positive stool samples from the three largest public hospitals in Singapore were collected for culture and further analysis. RESULTS: Out of the 366 samples collected, 272 viable isolates were cultured. Of these, 240 tested toxin-positive and ten tested positive for the binary toxin gene; 35 different PCR ribotypes were found. Three isolates that tested positive for binary toxin contained the same PCR ribotyping pattern as the C. difficile 027 control strain. All three had the 18-bp deletion and single nucleotide tcdC deletion at position 117. Susceptibility testing was performed, demonstrating susceptibility to erythromycin and moxifloxacin. CONCLUSION: We report the first three isolates of C. difficile 027 from Singapore. However, their susceptibility patterns are more consistent with the historical 027 strains. Rising CDI incidence may not be associated with the emergence of the epidemic 027 strain at this time.

Rabies post-exposure prophylaxis in travellers returning from Bali, Indonesia, November 2008 to March 2010.

Since 2008, when the outbreak of rabies in Bali began, 45 patients have attended GeoSentinel or EuroTravNet sites for rabies post-exposure prophylaxis (PEP), representing 12.6% of all travellers seen for PEP in all network clinics during the same time period. This suggests that Bali is emerging as a commonly visited destination among travellers presenting for rabies PEP. The data demonstrate that the majority of animal-related injuries in travellers returning from Bali are associated with exposure to monkeys, and not dog bites/scratches. The clinical implications of this are discussed.

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.

BRE is an antiapoptotic protein in vivo and overexpressed in human hepatocellular carcinoma.

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P<2.2e-16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.

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.

Extremely low exposure of a community to severe acute respiratory syndrome coronavirus: false seropositivity due to use of bacterially derived antigens.

Estimates of seropositivity to a new infectious agent in a community are useful to public health. For severe acute respiratory syndrome (SARS), the figures are conflicting. Herein, we screened 12,000 people in a community stricken by SARS 10 months previously and found 53 individuals (0.44%) who had immunoglobulin G antibodies to the SARS coronavirus (SARS-CoV) nucleocapsid (N) produced in bacteria. However, only seven of these (group 1) had sera which also reacted with the native N antigen expressed in SARS-CoV-infected Vero cells, N-transfected 293T cells, and tissues of infected SARS patients. Of these, six individuals had had SARS previously. The remaining person, as well as the 46 other individuals (group 2), were healthy and had no history of SARS. Group 1 antibodies recognized epitopes located slightly differently in N from those of group 2 antibodies, and a mouse hybridoma antibody resembling the former type was generated. Unusually, group 2 antibodies appeared to recognize cross-reactive bacterial epitopes that presumably were posttranslationally modified in eukaryotes and hence were probably not induced by SARS-CoV or related coronaviruses but rather by bacteria. The N antigen is thus highly unique. The extremely low rate (0.008%) of asymptomatic SARS infection found attests to the high virulence of the SARS-CoV virus.

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.

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.

Expression of human BRE in multiple isoforms.

BRE, a putative stress-modulating gene, found able to down-regulate TNF-alpha-induced NF-kappaB activation upon overexpression, is now shown in human cells expressed as multiple mRNA isoforms. A total of six isoforms are produced by alternative splicing predominantly at either end of the gene. Predicted from the cDNA sequences of these isoforms, three of them (alpha(a), alpha(b), and alpha(c)) code for BRE of different C-terminus, and the other three (beta(a), beta(b), and beta(c)) may possibly be the nonfunctional counterparts. All human cells examined coexpress all the predominant splice variants, albeit at different ratios. Comparing with normal cells, immortalized human cell lines uniformly express higher levels of BRE. Interestingly, peripheral blood monocytes responded to LPS by down-regulating the expression of all the BRE isoforms, which was however less obvious in the cell line counterpart, THP-1. Isoform alpha(a), which codes for the canonical BRE with a C-terminal peroxisomal targeting sequence, is the most abundant transcript. We propose that the function of BRE and its isoforms is to regulate peroxisomal activities.

Molecular mimicry: anti-DNA antibodies may arise inadvertently as a response to antibodies generated to microorganisms.

The origin of anti-DNA antibodies remains speculative. We argue that some of these antibodies may arise inadvertently in nature during the course of a normal immune response due to their induction by antibodies which bear structures (mimotopes) that mimic DNA. These antibodies are not necessarily DNA specific but, like the T15 idiotype (id)-positive antibodies which bind to phosphorylcholine, are produced normally to some environmental or microbial antigen. Such a mimotope was found in a T15(+) antibody at the highly specific region encoded principally by the D gene, DFL16.1. This mimotope was also found in human antibodies that are encoded by DXP'1, the human counterpart of DFL16.1 and which is used commonly in anti-DNA antibodies. The mimotope is closely related to the epitope responsible for the T15 id and appears to be cryptic or normally hidden in the native protein. The existence of such a common, conserved sequence raises questions about how easily anti-DNA antibodies can be generated in nature and what purpose these proteins may serve. Molecular mimicry with regard to autoimmunity must thus be viewed as existing not necessarily between the infectious agent and self-antigens, but also between the antibodies induced by the organism and the self-antigens.

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.

Strand bias in Ig somatic hypermutation is determined by signal sequence within the variable region.

Ig genes undergo hypermutation with a nucleotide preference of A over T for mutation on the coding strand. As only with concomitant strand bias can such nucleotide bias be observed, Ig gene hypermutation is generally accepted as a strand-specific process, for which the mechanistic basis remains unknown. It has previously been shown that different non-Ig sequences replacing the LVJ region of an Ig transgene to various extents are targeted for hypermutation with similar mutation frequencies. However, the nucleotide bias characteristic of Ig hypermutation was not found in two of the three such sequences studied. To test whether it is the DNA sequences of the non-Ig substrates that determine the pattern of nucleotide bias in hypermutation or whether the LVJ sequence may contain element(s) that confer strand bias, we have added back all the replaced LVJ sequences to one of the transgenes, L(kappa)-Vgpt*, that expresses no strand bias in hypermutation and studied the outcome. The results show that the gpt sequence in the presence of the complete LVJ sequence hypermutates differently from the same sequence in L(kappa)-Vgpt* where 84% of the LVJ was replaced. The main difference is the resumption of strand bias characteristic of Ig hypermutation. Thus, whether or not a substrate sequence manifests strand bias in hypermutation is not inherently determined by the substrate DNA sequence. This indicates the presence of special element(s) within the LVJ that confer strand bias.

The effect of lifestyle changes on results of 24-h ambulatory oesophageal pH monitoring.

OBJECTIVES: Twenty-four-hour pH monitoring is the 'gold standard' investigation of gastro-oesophageal reflux disease. It has been suggested that results may be influenced by lifestyle alteration during the study. The aim of this study was to determine the influence of lifestyle alteration and anxiety on outcome in pH monitoring. METHODS: One hundred consecutive patients attending for pH monitoring were recruited. Post-intubation anxiety was quantified (STAI form Y-1). On completion a questionnaire was recorded comprising six questions regarding alteration to eating/drinking (FOOD) (score 0-6) and six regarding posture and activity (ACTIVITY) (score 0-6). The higher the score the greater the alteration of lifestyle. A 10-point visual analogue scale (VAS) scored the degree of distress caused by the experience. pH studies were positive if exposure times at pH < 4 were greater than established normal values or if the Symptom Index was > 50%. Comparisons were made between patients with positive and negative studies regarding FOOD, ACTIVITY, VAS and STAI scores. RESULTS: Patients with positive pH studies had mean FOOD (2 vs 2; P> 0.05), ACTIVITY (2 vs 2; P> 0.05), VAS (3 vs 3; P> 0.05) and STAI (31 vs 32; P> 0.05) scores which were not significantly different from those of patients with negative pH studies. CONCLUSIONS: Lifestyle alteration was minimal in patients undergoing pH monitoring, nor was the experience distressing. There was no difference in lifestyle alteration and anxiety between those with positive and negative pH profiles.