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Abstract Background Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample 33P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance. Results The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 μg and less) and has a sound reproducibility. Conclusions Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner.

Abstract Background During HIV infection and/or antiretroviral therapy (ART), monocytes and macrophages exhibit a wide range of dysfunctions which contribute significantly to HIV pathogenesis and therapy-associated complications. Nevertheless, the molecular components which contribute to these dysfunctions remain elusive. We therefore applied a parallel approach of genome-wide microarray analysis and focused gene expression profiling on monocytes from patients in different stages of HIV infection and/or ART to further characterise these dysfunctions. Results Processes involved in apoptosis, cell cycle, lipid metabolism, proteasome function, protein trafficking and transcriptional regulation were identified as areas of monocyte dysfunction during HIV infection. Individual genes potentially contributing to these monocyte dysfunctions included several novel factors. One of these is the adipocytokine NAMPT/visfatin, which we show to be capable of inhibiting HIV at an early step in its life cycle. Roughly half of all genes identified were restored to control levels under ART, while the others represented a persistent dysregulation. Additionally, several candidate biomarkers (in particular CCL1 and CYP2C19) for the development of the abacavir hypersensitivity reaction were suggested. Conclusions Previously described areas of monocyte dysfunction during HIV infection were confirmed, and novel themes were identified. Furthermore, individual genes associated with these dysfunctions and with ART-associated disorders were pinpointed. These genes form a useful basis for further functional studies concerning the contribution of monocytes/macrophages to HIV pathogenesis. One such gene, NAMPT/visfatin, represents a possible novel restriction factor for HIV. Background Both macrophages and T lymphocyte subsets express the CD4 receptor and either the CXCR4 and/or the CCR5 coreceptor which confer susceptibility to infection with the Human Immunodeficiency Virus (HIV). Upon infection, CD4+ T lymphocytes typically succumb to the cytopathic effect of the virus 1, and the gradual depletion of the CD4+ T lymphocyte pool has been considered a hallmark of HIV infection and the development of the Acquired Immune Deficiency Syndrome (AIDS) since the early days of the HIV pandemic. Macrophages, on the other hand, do not tend to suffer from the cytopathic effects mediated by the virus 23, but instead develop a wide array of dysfunctions which contribute significantly to the pathogenesis of HIV infection. Despite the recognition of macrophage contribution to HIV pathogenesis early on in HIV research 45, most studies have focused and continue to focus on T lymphocyte depletion and/or dysfunction, and many of the molecular mechanisms underlying the macrophage dysfunction during HIV infection remain poorly characterised. Nevertheless, as pointed out by other authors 6, in the combination Antiretroviral Therapy (ART) era where viral suppression in T lymphocytes is increasingly more efficient, the understanding of the viral mechanisms in other reservoir cells such as macrophages becomes ever more crucial. Aberrant HIV-induced macrophage behaviour can be classified as relatively straightforward loss of function, such as reduced phagocytosis 78 and antigen presentation 9, or as more complex dysfunction. Such dysfunctions include a direct contribution to the establishment, spread and persistence of the infection: as long-living primary target cells of HIV with a wide-spread dissemination and a persistent failure to enter apoptosis upon infection 1011, they represent an important cellular reservoir for the virus 12. Additionally, macrophages exacerbate disease progression by contributing to T lymphocyte depletion: HIV infected macrophages have been documented to participate in the killing of uninfected CD4+ and CD8+ T lymphocytes, while at the same time protecting infected CD4+ T lymphocytes from apoptosis 13. Furthermore, infected and uninfected macrophages can contribute to sustained chronic immune activation during HIV infection, e.g. through the perturbation of cytokine and chemokine networks 141516. With the acknowledged notion of chronic immune activation as a paradoxical driving force of immune suppression 17, this pro-inflammatory macrophage phenotype during HIV infection may be a crucial parameter in disease progression. Yet other macrophage dysfunctions are associated with more peripheral HIV- or ART-associated disorders such as atherosclerosis 18, lipodystrophy 19, and metabolic syndrome during HIV infection and/or combination ART 2021. Monocytes, for their part, are much less permissive to infection with HIV, both in vitro 22 and in vivo, where estimates of infected circulating monocytes are consistently low 2324. Circulating monocytes represent the most accessible primary model for macrophage dysfunction during HIV infection, however, and are furthermore of sufficient importance to study in their own right. Infectious virus can be recovered from circulating monocytes, both in untreated patients 24 and in patients undergoing long-term successful combination ART 25. Additionally, the circulating monocyte pool as a whole does seem to be affected during HIV infection, despite the low frequency of actually infected monocytes. Transcriptome studies, in particular, show a form of hybrid phenotype exhibiting both increased and decreased pro-inflammatory features 2627. This modulation of the non-infected monocyte population could be due to the virus itself through mechanisms which do not require direct infection 28, or to other factors contributing to (aberrant) immune activation occurring during HIV infection, such as perturbed cytokine networks 29 or other inflammatory stimulants 30. Several key factors in the described dysregulated processes have been identified 1831, but many molecular components remain elusive. Furthermore, other aspects of HIV and combination ART pathogenesis in which monocyte/macrophage dysfunction is involved may only now be emerging or remain yet to be discovered, in particular in view of the limited number of studies focussing on the monocyte response to ART 32. In order to generate novel hypotheses rather than test pre-existing ones in the context of monocyte-HIV interactions, we performed a transcriptome analysis on monocyte samples from patients in different stages of HIV infection and/or combination ART treatment, using a parallel approach of genome-wide microarray analysis and focused gene expression profiling to identify broad areas of monocyte dysfunction and to pinpoint genes which are potentially involved in one or several of these dysfunctions. In particular the factors which are exploited by the monocyte/macrophage to communicate with and/or modulate other immune cells were of interest, as they represent a particularly relevant population 3334 which is a primary target for intervention.

Despite the availability of effective direct‐acting antiviral (DAA) treatments for Hepatitis C virus (HCV) infection, many people remain undiagnosed and untreated. We assessed the cost‐effectiveness of a Médecins Sans Frontières (MSF) HCV screening and treatment programme within a primary health clinic in Karachi, Pakistan. A health state transition Markov model was developed to estimate the cost‐effectiveness of the MSF programme. Programme cost and outcome data were analysed retrospectively. The incremental cost‐effectiveness ratio (ICER) was calculated in terms of incremental cost (2016 US$) per disability‐adjusted life year (DALY) averted from the provider's perspective over a lifetime horizon. The robustness of the model was evaluated using deterministic and probabilistic sensitivity analyses (PSA). The ICER for implementing testing and treatment compared to no programme was US$450/DALY averted, with 100% of PSA runs falling below the per capita Gross Domestic Product threshold for cost‐effective interventions for Pakistan (US$1,422). The ICER increased to US$532/DALY averted assuming national HCV seroprevalence (5.5% versus 33% observed in the intervention). If the cost of liver disease care was included (adapted from resource use data from Cambodia which has similar GDP to Pakistan), the ICER dropped to US$148/DALY, while it became cost‐saving if a recently negotiated reduced drug cost of $75/treatment course was assumed (versus $282 in base‐case) in addition to cost of liver disease care. In conclusion, screening and DAA treatment for HCV infection are expected to be highly cost‐effective in Pakistan, supporting the expansion of similar screening and treatment programmes across Pakistan. [ABSTRACT FROM AUTHOR]

Background: Cholera remains a leading cause of infectious disease outbreaks globally, and a major public health threat in complex emergencies. Hygiene kits distributed to cholera case-households have previously shown an effect in reducing cholera incidence and are recommended by Médecins Sans Frontières (MSF) for distribution to admitted patients and accompanying household members upon admission to health care facilities (HCFs). Methods: This process evaluation documented the implementation, participant response and context of hygiene kit distribution by MSF during a 2018 cholera outbreak in Kasaï-Oriental, Democratic Republic of Congo (DRC). The study population comprised key informant interviews with seven MSF staff, 17 staff from other organisations and a random sample of 27 hygiene kit recipients. Structured observations were conducted of hygiene kit demonstrations and health promotion, and programme reports were analysed to triangulate data. Results and conclusions: Between Week (W) 28–48 of the 2018 cholera outbreak in Kasaï-Oriental, there were 667 suspected cholera cases with a 5% case fatality rate (CFR). Across seven HCFs supported by MSF, 196 patients were admitted with suspected cholera between W43-W47 and hygiene kit were provided to patients upon admission and health promotion at the HCF was conducted to accompanying household contacts 5–6 times per day. Distribution of hygiene kits was limited and only 52% of admitted suspected cholera cases received a hygiene kit. The delay of the overall response, delayed supply and insufficient quantities of hygiene kits available limited the coverage and utility of the hygiene kits, and may have diminished the effectiveness of the intervention. The integration of a WASH intervention for cholera control at the point of patient admission is a growing trend and promising intervention for case-targeted cholera responses. However, the barriers identified in this study warrant consideration in subsequent cholera responses and further research is required to identify ways to improve implementation and delivery of this intervention. [ABSTRACT FROM AUTHOR]

Introduction: Cholera remains a frequent cause of outbreaks globally, particularly in areas with inadequate water, sanitation and hygiene (WASH) services. Cholera is spread through faecal-oral routes, and studies demonstrate that ingestion of Vibrio cholerae occurs from consuming contaminated food and water, contact with cholera cases and transmission from contaminated environmental point sources. WASH guidelines recommending interventions for the prevention and control of cholera are numerous and vary considerably in their recommendations. To date, there has been no review of practice guidelines used in cholera prevention and control programmes. Methods: We systematically searched international agency websites to identify WASH intervention guidelines used in cholera programmes in endemic and epidemic settings. Recommendations listed in the guidelines were extracted, categorised and analysed. Analysis was based on consistency, concordance and recommendations were classified on the basis of whether the interventions targeted within-household or community-level transmission. Results: Eight international guidelines were included in this review: three by non-governmental organisations (NGOs), one from a non-profit organisation (NPO), three from multilateral organisations and one from a research institution. There were 95 distinct recommendations identified, and concordance among guidelines was poor to fair. All categories of WASH interventions were featured in the guidelines. The majority of recommendations targeted community-level transmission (45%), 35% targeted within-household transmission and 20% both. Conclusions: Recent evidence suggests that interventions for effective cholera control and response to epidemics should focus on case-centred approaches and within-household transmission. Guidelines did consistently propose interventions targeting transmission within households. However, the majority of recommendations listed in guidelines targeted community-level transmission and tended to be more focused on preventing contamination of the environment by cases or recurrent outbreaks, and the level of service required to interrupt community-level transmission was often not specified. The guidelines in current use were varied and interpretation may be difficult when conflicting recommendations are provided. Future editions of guidelines should reflect on the inclusion of evidence-based approaches, cholera transmission models and resource-efficient strategies. [ABSTRACT FROM AUTHOR]

Introduction: Improving access to HIV testing is a key priority in scaling up HIV treatment and prevention services. Home-based voluntary counselling and testing (HBT) as an approach to delivering wide-scale HIV testing is explored here. Methods and Findings: We conducted a systematic review and random-effects meta-analysis of studies published between 1 January 2000 and 24 September 2012 that reported on uptake of HBT in sub-Saharan Africa, to assess the proportion of individuals accepting HBT and receiving their test result. Our initial search yielded 1,199 articles; 114 were reviewed as full-text articles, and 19 publications involving 21 studies (n = 524,867 individuals offered HBT) were included for final review and meta-analysis. The studies came from five countries: Uganda, Malawi, Kenya, South Africa, and Zambia. The proportion of people who accepted HBT (n = 474,377) ranged from 58.1% to 99.8%, with a pooled proportion of 83.3% (95% CI: 80.4%-86.1%). Heterogeneity was high ([τ.sup.2] = 0.11). Sixteen studies reported on the number of people who received the result of HBT (n = 432,835). The proportion of individuals receiving their results out of all those offered testing ranged from 24.9% to 99.7%, with a pooled proportion of 76.7% (95% CI: 73.4%-80.0%) ([τ.sup.2] = 0.12). HIV prevalence ranged from 2.9% to 36.5%. New diagnosis of HIV following HBT ranged from 40% to 79% of those testing positive. Forty-eight percent of the individuals offered testing were men, and they were just as likely to accept HBT as women (pooled odds ratio = 0.84;95% CI: 0.56-1.26) ([τ.sup.2] = 0.33). The proportion of individuals previously tested for HIV among those offered a test ranged from 5% to 66%. Studies in which Conclusions: HBT could substantially increase awareness of HIV status in previously undiagnosed individuals in sub-Saharan Africa, with over three-quarters of the studies in this review reporting >70% uptake. It could be a valuable tool for treatment and prevention efforts. Please see later in the article for the Editors' Summary., Introduction Testing for HIV is the first step in the cascade of care for HIV-positive individuals who need treatment. Knowledge of HIV status is also an important part of HIV [...]