TB-related deaths among adults in Guinea-Bissau.

SETTING: In an urban demographic, high TB burden surveillance site in Guinea-Bissau, most deaths occur at home, and information on cause of death (CoD) is lacking.OBJECTIVE: To examine CoD and the proportion of TB deaths in three groups: among patients examined for TB without a verified diagnosis after diagnostic workup, described as "assumed TB-negative" (aTBneg), among patients with a confirmed diagnosis of TB and in a sample of the background population.DESIGN: Verbal autopsies (VAs) were obtained for registered deaths occurring between 1 January 2010 and 15 June 2016. All deaths among aTBneg and patients with TB, and a sample of deaths in the background population were included.RESULTS: VAs were obtained from 104/112 aTBneg patients, 140/155 patients with TB, and 172/219 from the general population. The leading CoD was TB in respectively 20%, 69% and 9% of the cohorts. HIV/AIDS-related deaths were the most frequent CoD among aTBneg patients (45%) and in the background population (27%), and accounted for 9% of patients with TB.CONCLUSION: TB was shown to be a frequent CoD, not only among patients diagnosed with TB, but also among aTBneg patients and the background population. This indicates a low TB case detection rate.

Impact of HIV-1, HIV-2, and HIV-1+2 dual infection on the outcome of tuberculosis.

BACKGROUND: HIV-1 infection has been shown to impact the outcome of patients with tuberculosis (TB), but data regarding the impact of HIV-2 on TB outcomes are limited. The aim of this study was to assess the impact of HIV types on mortality among TB patients in Guinea-Bissau and to examine the predictive ability of the TBscoreII, a clinical score used to assess disease severity. METHODS: In a prospective follow-up study, we examined the prevalence of HIV-1, HIV-2, and HIV-1+2 co-infection in TB patients in Guinea-Bissau, and the impact on outcomes at 12 months of follow-up. We included all adult TB patients in an observational TB cohort at the Bandim Health Project (BHP) in Guinea-Bissau between 2003 and 2013 and assessed survival status at 12 months after the start of treatment. RESULTS: A total 1312 patients were included; 499 (38%) were female (male/female ratio 1.6). Three hundred and seventy-nine patients were HIV-infected: 241 had HIV-1, 93 had HIV-2, and 45 were HIV-1+2 dual infected. The HIV type-associated risk of TB was 6-fold higher for HIV-1, 7-fold higher for HIV-1+2 dual infection, and 2-fold higher for HIV-2 compared with the HIV-uninfected. Of the patients included, 144 (11%) died, 62 (12%) among females and 82 (9%) among males (hazard ratio (HR) 0.91, 95% confidence interval (CI) 0.64-1.30; p=0.596). Compared to male patients, female patients were younger (1 year younger, 95% CI 0.5-2; p=0.04), reported a longer duration of symptoms (14 days longer, 95% CI 4-25; p=0.003), and had a higher TBscoreII (0.5 points more, 95% CI 0.3-0.7; p<0.001). More females than males were HIV-infected (36% vs. 25%; p<0.001) and more females had a body mass index (BMI) <15 kg/m(2) (11% vs. 6%; p<0.001) and a mid upper arm circumference (MUAC) <200 mm (13% vs. 7%; p < 0.001). HIV infection increased the mortality risk, with HIV-1 infection displaying the highest HR (5.0, 95% CI 3.5-7.1), followed by HIV-1+2 (HR 4.2, 95% CI 2.2-7.8) and HIV-2 (HR 2.1, 95% CI 1.2-3.8). A TBscoreII ≥4 was associated with increased mortality (HR 2.2, 95% CI 1.5-3.1). Significantly increased HRs were found for signs of wasting; a BMI <18 kg/m(2) was associated with a HR of 1.8 (95% CI 1.3-2.6) and a MUAC <220 mm with a HR of 3.8 (95% CI 2.7-5.2). CONCLUSION: The HIV type-associated risk of TB was much higher for HIV-1 patients and higher but less so for HIV-2 patients, compared with the HIV-uninfected. Clinical severity at presentation was also higher for HIV-infected patients, although less so for HIV-2-infected patients, and all HIV-infected patients had a poorer outcome than the uninfected; mortality was 4-5-fold higher for HIV-1 and dually infected patients and two-fold higher for HIV-2-infected patients. These differences between HIV types did not disappear after adjusting for CD4 count.

Profiles of a high-aspect-ratio grating determined by spectroscopic scatterometry and atomic-force microscopy.

The new and fast scatterometry method called optical diffraction microscopy is compared with atomic-force microscopy by use of cross-section scanning-electron microscope images as references. The sample is a high-aspect-ratio grating with a period of approximately 1000 nm. To allow the atomic-force microscope to track all parts of the grating profile, the grating is investigated at different tilt angles. The measured quantities of the profile include sidewall angle gamma (approximately 90 degrees), groove height h (approximately 2000 nm), and degree of filling f (approximately 40%). The two methods, which respond to quite different material properties, give consistent results within standard uncertainties of u(gamma)

Hybridisation of short DNA molecules investigated with in situ atomic force microscopy.

By introducing the complementary DNA (cDNA) strand to a molecular layer of short single stranded DNA (ssDNA), immobilised on a gold surface, we have investigated hybridisation between the two DNA strands through the technique of in situ atomic force microscopy (AFM). Before introduction of cDNA, the ssDNA molecular layer was modulated with the spacer molecule mercaptohexanol (MCH), which makes the ssDNA molecules more accessible for hybridisation. With in situ AFM, we have monitored the formation of a smooth, mixed molecular layer containing ssDNA and MCH. Furthermore, the hybridisation between the two DNA strands has been studied. Introduction of the cDNA strand resulted in an increase in smoothness and thickness of the molecular layer. Both the increase in order and thickness of the molecular layer can be expected if hybridisation occurs, since double stranded DNA molecules have a more rigid and elongated structure than ssDNA molecules.

Identification and characterization of Saccharomyces cerevisiae strains isolated from West African sorghum beer.

The occurrence and characterization of yeasts isolated from sorghum beer produced in Ghana and Burkina Faso, West Africa, were investigated. The yeasts involved in the fermentations were found to consist of Saccharomyces spp. almost exclusively. Of the isolates investigated, 45% were identified as Saccharomyces cerevisiae, whereas more than half of the isolates (53%) had physiological properties atypical of S. cerevisiae or any other member of the complex sensu strictu, as they were able to assimilate only glucose, maltose and ethanol as carbon sources. Both ITS-PCR RFLP and PFGE strongly indicated that these isolates were related to S. cerevisiae, regardless of their phenotypic characteristics. Sequencing of the D1/D2 domain of the 26S rDNA confirmed the close relatedness to S. cerevisiae with 0.5% nucleotide differences. The MAL1 and MAL3 loci were found for all isolates as the only recognized MAL loci. Besides, for 40% of the isolates the MAL61 probe hybridized to a position of about 950 kbp, which has not formerly been described as a MAL locus. The results showed that the spontaneous fermentation of West African sorghum beer is dominated by a variety of strains of S.cerevisiae not previously described, among which starter cultures should be selected.

Phenotypic and genetic diversity of Saccharomyces contaminants isolated from lager breweries and their phylogenetic relationship with brewing yeasts.

A taxonomic study was carried out for isolates of Saccharomyces spp. identified as contaminants ("wild yeast") in 24 different lager breweries. With reference to the current taxonomy all isolates were found to belong to the Saccharomyces sensu stricto complex and 58% of the isolates were further identified as S. cerevisiae, 26% as S. pastorianus and 3% as S. bayanus. The remaining isolates (13%) could not be identified to the species level based on their phenotypic characteristics. However, some of these isolates were identified as S. cerevisiae by HaeIII restriction digest of PCR-amplified intergenic transcribed spacer (ITS) regions. Chromosome length polymorphism (CLP) was evident among the Saccharomyces brewing contaminants with chromosome profiles typical of Saccharomyces sensu stricto. Based upon cluster analysis of their chromosome profiles the majority of the brewing contaminants could be grouped as either S. cerevisiae or S. pastorianus/S. bayanus. Further, the technique was able to differentiate between almost all brewing contaminants and to separate them from any specific lager brewing yeast. The diversity of the Saccharomyces brewing contaminants clearly demonstrated by their CLP was further reflected by MAL genotyping. For the majority of the isolates more than two MAL loci were found with MAL1, MAL2 MAL3, MAL4 and MAL11, MAL31, MAL41 as the dominant genotypes. For all isolates MAL11 and MAL31 were found whereas MAL61 only was found for one isolate. The high number of MAL loci found in the SaccharomYces brewing contaminants indicate their adaptation to a maltose-enriched environment.

Detection and identification of wild yeasts in lager breweries.

Wild yeasts were detected in 41 out of 101 brewery yeast samples investigated using six different selective principles. Malt extract, yeast extract, glucose, peptone (MYGP) agar supplemented with 195 ppm CuSO4 was found to be the most effective selective principle, detecting wild yeasts in 80% of the contaminated samples. Both Saccharomyces and non-Saccharomyces wild yeasts were detected on this medium. Lysine medium, crystal violet medium and incubation of non-selective media at 37 degrees C detected wild yeasts in 46-56% of the contaminated samples. On using actidione medium, only 20% of the wild yeasts were detected. The combined use of MYGP supplemented with 195 ppm CuSO4 and one of the other selective principles did not improve the recovery of the wild yeasts. The wild yeasts found consisted of Saccharomyces cerevisiae (57%), Pichia spp. (28%) and Candida spp. (15%). Using the API ID 32 C kit, 35 different assimilation profiles were obtained for the 124 wild yeast isolates investigated. All isolates were capable of glucose assimilation, whereas only 79% of the isolates assimilated saccharose, 75% maltose, 70% galactose, 65% raffinose and 65% lactate. Lactose, inositol, rhamnose and glucuronate were not assimilated by any of the isolates. The differences in assimilation pattern did not reflect any differences in recovery by the selective principles investigated. The majority of the wild yeast isolates investigated were capable of growth in wort and beer, indicating their possible role as spoilage organisms. The Sacch. cerevisiae isolates were found to be the most hazardous, with some isolates being capable of extensive growth in bottled beer within seventeen days at ambient temperature.

Report from the HLA class II typing by PCR-SSP Multicentre Study.

Results from 360 HLA-DR and -DQ 'low-resolution' typings with polymerase chain reaction sequence-specific primers (PCR-SSP), performed by nine laboratories, were analysed for their overall utility in routinely defining the HLA-DR1-DR18, DR51-DR53 and DQ1-DQ9 specificities in less than 2.5 h. Thirty EDTA blood samples and 10 DNA samples were distributed and analysed by each laboratory. DNA was extracted using a rapid bromide salt extraction protocol. Complete HLA-DR and -DQ typings were performed, three by three, on pre-aliquoted 96-tube PCR trays. When compared with reference typing, 351/360 (98%) correct DR typings were obtained, whereas 320/360 (89%) of the DQ phenotypes were correctly assigned. The time for three complete HLA-DR and -DQ 'low-resolution' typings, including DNA extraction, ranged from 2.0 h to 2.3 h. Unfortunately, an unusually high level of PCR amplification failures was observed (3%), probably due to diffusion and a significant volume loss from some of the pre-aliquoted primer mixes. Consequently, only 52% of the typings were without any amplification failure, and 0-2 amplification failures where found in 88% of the PCR-SSP typings performed. The number of HLA-DR-DQ retypings needed was 7 and 8%, respectively, reflecting the low number of typings where allelic identification was directly affected by the relatively high level of amplification failures in this study. Thus, a 91-98% success rate of correctly identified HLA-DR and -DQ alleles could be maintained, even under suboptimal typing conditions.

Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc3 and N23 gene promoters.

The qualitative and quantitative contributions of four separate cis-acting DNA elements controlling the root nodule-specific soybean leghemoglobin lbc3 gene were analyzed in transgenic Lotus corniculatus plants. Expression from internal deletions in the 5' region between positions -49 and -1956 was monitored from a CAT reporter gene. The strong positive element (SPE; -1090, -947) responsible for high-level expression was demonstrated to be an organ-specific element by deleting proximal nodule-specific control elements. Deletion of the downstream qualitative organ-specific element (OSE; -139, -102) containing the putative nodulin consensus sequences 5'AAAGAT and 5'CTCTT resulted in a low expression level. Efficient SPE enhancement is therefore dependent on the organ-specific element, which by itself does not enhance expression. This quantitative effect of the immediate upstream region carrying the consensus sequences was also found in hybrid promoter studies using the soybean nodulin N23 gene promoter, suggesting the involvement of these motifs in a regulatory mechanism for nodulin genes. Deletion of the lbc3 negative element (NE, -102, -49) linking the SPE and OSE onto the TATA box did not lead to unregulated expression. These results indicate that interaction between positive, negative and neutral qualitative elements controls lbc3 expression. Binding of the nuclear protein NAT2 at the lbc3 weak positive element (WPE; -230, -170) is probably not directly required for this mechanism.

5' Analysis of the soybean leghaemoglobin lbc(3) gene: regulatory elements required for promoter activity and organ specificity.

The soybean leghaemoglobin lbc(3) gene promoter was analysed in transgenic Lotus corniculatus plants. Hybrid-promoter constructions and 5' deletions were studied using chimeric genes composed of the various promoters, the chloramphenicol acetyltransferase (CAT) coding sequence and the lbc(3) 3' flanking region. A 5' Bal31 deletion series mapped a strong positive regulatory element between -1100 and -950. A weaker element located between -230 and -170 defined the minimum 5' region required for detectable promoter activity. Reactivation of inactive promoters with deletion endpoints between -230 and the transcription initiation site was obtained employing the constitutive cauliflower mosaic virus (CaMV) 35S enhancer. The position of cis regulatory element(s) required for nodule-specific expression was defined to 37 bp between -139 and -102. This region contains sequences conserved in other leghaemoglobin and nodulin genes. No indispensable control elements were found on the lbc(3) 3' flanking region.