4 cm in diameter) to a bulk density of 1.4 g cm−3. The major characteristics of the research site are given in Table 1 according to Gerwin et al. (2009). Five grams of labelled litter material (L. corniculatus or C. epigejos) was mixed into the first centimetre of the soil in each microcosm. Microcosms without litter application served as controls. In total, 75 microcosms were randomly placed and incubated at 10 °C in the dark. The soil water content was estimated by weekly weighing and was maintained DNA-PK inhibitor at 55% of the maximum water-holding capacity throughout the experiment. For each treatment (L. corniculatus, C. epigejos, control), 15 microcosms
were prepared for harvests with five independent replicates after 4, 12 and 40 weeks of litter incubation. For microbial analyses, the detritusphere in the first 2 cm of each column was harvested. To quantify the litter degradation rates, additional 15 microcosms for L. corniculatus
and C. epigejos were incubated under the same conditions. Five grams of the labelled litter material (L. corniculatus or C. epigejos) was filled into nylon bags (10 × 10 cm, mesh size 40 μm) and placed into these microcosms, 1 cm below soil surface. At all sampling times, individual litter bags were removed from five independent microcosms; the total amount of litter was air-dried and weighed in order to calculate the litter degradation rates. The total 13C, C and N contents at individual harvesting times were analysed using an Euro EA (Eurovector, HSP targets Milan, Italy) coupled with an isotope ratio mass spectrometer MAT 253 (Thermo Electron, Bremen, Germany). Microbial biomass C was estimated after chloroform fumigation–extraction (Cmic) according to Joergensen (1995). The total organic C content and the δ13C in the CaCl2 Progesterone extracts were measured
using on-line coupling of liquid chromatography and stable isotope ratio MS (Thermo Electron), according to Krummen et al. (2004). PLFA analyses were based on Zelles et al. (1995) and have been described in detail elsewhere (Esperschütz et al., 2009). Fatty acids are presented by the number of C atoms, followed by the number of double bonds. The positions of double bonds are indicated by ‘ω’ and the number of the first double-bonded C atoms from the ω end of the C chain. Anteiso- and iso-branched fatty acids are indicated by ‘ant’ and ‘iso’, followed by the number of C atoms. Branched fatty acids in which the position of the double bond was unknown were indicated by the prefix ‘br’. Methyl groups on the 10th C atom from the carboxyl end of the molecule were indicated by ‘10ME’. Cyclopropane fatty acids were indicated by the prefix ‘cyc’, while dicyclopropylic PLFA were indicated by ‘dic’. Even-chained, saturated fatty acids were abbreviated with the prefix ‘nor’. A univariate anova was carried out using spss 11.
In our previous study on the ultrastructure of M. oxyfera, neither TEM nor electron tomography showed the presence of ICM in M. oxyfera cells under the current growth conditions (Wu et al., 2012). This observation raised the question regarding the actual Veliparib purchase intracellular location of the pMMO enzyme in M. oxyfera. Here, we show that, consistent with the previous observation, M. oxyfera does not develop ICM under the current growth conditions. Ultrathin section of M. oxyfera cells incubated with α-pMmoB showed gold particles both at and close to
the cytoplasmic membrane (Figs 4 and 5). These results together with the presence of membrane spanning regions in the pMmoB sequence (Fig. 1b) indicate that the pMMO enzyme is most likely located at the cytoplasmic
membrane of M. oxyfera cells. In conclusion, our results suggest that pMMO and NirS enzymes are located in the cytoplasmic membrane and the periplasm of M. oxyfera cells, respectively. Double-labelling experiments showed the co-occurrence of both pMMO and NirS in single M. oxyfera cells. Our results validate the presence of key enzymes in methane- and nitrite-converting pathways in the M. oxyfera metagenome assembly. We would like to thank Katinka van de Pas-Schoonen for support in maintaining the M. oxyfera enrichment culture, Harry R. Harhangi, Huub Op den Camp and Jan T. Keltjens for stimulating discussions, Sarah Neumann for support in the production of the antisera and Geert-Jan Janssen for support at the general instruments facility. L.v.N. Copanlisib chemical structure is supported by the Netherlands Organization for Scientific Research (VENI grant 863.09.009), M.L.W. by a Horizon grant (050-71-058), M.S. by ERC 242635 and M.S.M.J. by ERC 232937. “
“Streptococcus pneumoniae, the leading etiological agent of pneumonia, shares a high degree of DNA Nintedanib (BIBF 1120) sequence homology with the viridans group of streptococci. The clinical and pathological manifestations may
present with different features, and discrimination between S. pneumoniae and its close viridans cocci relatives, such as Streptococcus mitis and Streptococcus oralis, is still quite difficult. The 445-bp sequences of the N-terminal region of rpoA from nine S. pneumoniae, seven S. mitis, ten S. oralis, and two related strains were determined and compared with their respective 16S rRNA gene sequences to establish their usefulness in phylogenetic analysis. Pairwise comparisons of rpoA sequences among the species showed higher rates of evolution with lower similarities (92.3–100%) than those of 16S rRNA genes (96.8–100%). The rpoA-based phylogeny generated deeper branches and presented improved discriminatory resolution than the 16S rRNA gene-based phylogeny.
96, respectively. Of the 129/167 joints that responded to treatment at 3 months, 116/129 (90%) had reached the 36-month time point at the time of analysis. Of these 116 patients, 68/116 (59%; 95%CI 49–67%) had a sustained clinical response at 36 months. A strong trend was demonstrated between the degree of initial clinical response and duration ABT-199 order of response with 90% of
complete responders compared to 55% of moderate responders (P = 0.0005) and 55% of moderate responders compared to 23% of mild responders (P = 0.01) maintaining their response at 36 months. This trend was maintained across all arthopathy groups (Table 4). Potentially serious complications
occurred in 2/167 cases (1%) in the first 3 months post-treatment. One intra-articular hemorrhage occurred in a hemophilia patient despite Akt assay appropriate factor replacement prior to the procedure and one extra-articular injection occurred during an attempted hip joint injection. No cases of skin necrosis occurred and the two reported cases did not appear to have any long-term adverse sequelae. We demonstrated an overall satisfactory clinical response rate to yttrium synovectomy of 57% across all arthropathies treated between January 2000 and December 2010. Large joint monoarthropathies demonstrated a particularly favorable response with 85% achieving a satisfactory clinical response compared to 52% for rheumatoid, psoriatic and hemophilic arthropathies combined (P = 0.006). A strong relationship between the degree of initial response and duration of response was demonstrated with those patients achieving a complete response in the first 3 months having a much higher likelihood (90%) of a sustained response at 36 months. We demonstrated no difference in response rates to yttrium synovectomy pre- and post-availability of newer generation DMARDs at our institution, with 41% of rheumatoid P-type ATPase and psoriatic arthropathies achieving satisfactory clinical response pre-2005
compared to 57% post-2005 (P = 0.25). Similarly, no difference in response rates was demonstrated pre- and post-introduction of routine factor replacement therapy in hemophiliac patients at our institution, with 50% of hemophilic arthropathies achieving satisfactory clinical response pre-2005 compared to 44% post-2005 (P = 0.96). Yttrium synovectomy was well tolerated in all patients in our study with a low overall complication rate of 1%. Importantly, no major adverse clinical outcomes were encountered such as skin necrosis or ulceration. The overall satisfactory clinical response rate of 57% in our study is concordant with the literature, with response rates commonly reported in the range of 50–80%.
pseudotuberculosis YpIII strain, RT-PCR (using an sraG-specific oligo in reverse transcription) was used to examine SraG RNA level at different growth phases. As shown in Fig. 1, compared with the expression patterns in
the sraG deletion mutant and the SraG complementing strains, the transcription levels of SraG are invariable under all tested growth stages. A secondary structure of SraG was predicted (Fig. S1) by RNAstructure software (Reuter & Mathews, 2010). To investigate the targets of SraG, we next performed 2D gel analysis to compare the whole-cell protein patterns of WT with ΔsraG from cultures grown to exponential phase. Expressions of 16 proteins having more than 1.5-fold changes between ΔsraG and WT (Table 1 and HSP assay Fig. S2). Among these proteins, seven were down-regulated and nine were up-regulated. We next performed semi-quantitative RT-PCR to compare the mRNA levels of these candidate targets. Among these potential targets, only pnp and YPK_1205 (encoding an hypothetical protein) showed significantly different mRNA levels (Fig. S3). To confirm the different expression level of YPK_1205 in WT and ΔsraG (Fig. 2a), we constructed a single-copy translational fusion of YPK_1205 with lacZ (named 1205zST). β-Galactosidase activities
were tested when isogenic strains were grown to mid-log phase. Expression of 1205zST in the ΔsraG strain was 2.6-fold higher than that in WT (Fig. 2b). Western blotting also confirmed higher YPK_1205 protein level in ΔsraG (Fig. 2c). Mitomycin C mw To further confirm that YPK_1205 mRNA is negatively regulated by SraG, we next performed RT-PCR and observed higher levels of YPK_1205 transcript (based on cDNA level generated by reverse transcription from total RNA) in ΔsraG than in either WT or the SraG complemented strain (Fig. 2d). These results are consistent with the result observed by 2D gel analysis and indicate that SraG negatively regulates YPK_1205 mRNA. The YPK_1205 gene is located downstream of YPK_1206. Inverse RT-PCR was used to examine whether YPK_1206 and YPK_1205 were cotranscribed (there is a 57-bp intergenic
region between them, Fig. 3a). As shown in Fig. 3(b), a region including both the YPK_1206 and YPK_1205 fragment was amplified from one cDNA template, these indicating that the two genes are indeed in one operon. Similar experiments confirmed that YPK_1206-1205 is not cotranscribed with YPK_1204 or YPK_1207 (data not shown). The next question was whether expression of YPK_1206 is also regulated by SraG. We therefore constructed a translational fusion of YPK_1206 with lacZ (1206zST), which was transconjugated into both WT and ΔsraG. Expression of 1206zST was 1.6-fold higher in ΔsraG than in WT (Fig. 3c, columns 1 and 2), indicating that YPK_1206 expression is also negatively regulated by SraG. RT-PCR was used to determine the YPK_1206 mRNA level in WT, ΔsraG and the complementary strains.
cckA and chpT mutants demonstrated a nearly complete loss in RcGTA activity (Fig. 3a). These findings initially suggested that a loss in either ChpT or CckA resulted in a decrease in RcGTA expression, possibly because of the loss of phosphorelay to CtrA. However, western blot analysis of the cultures demonstrated that both cckA and chpT mutants were expressing the RcGTA capsid protein at wild-type levels, but the protein was not detected in the culture supernatants (Fig. 3b). The extracellular levels of the major capsid protein and RcGTA activity were restored to the mutants upon complementation with the plasmid-borne genes. The gene transfer activity of the sciP mutant was lower than wild type (Fig. 3a) but this difference was not statistically different (Table S2). Introduction of the ctrAD51E
allele restored RcGTA expression and increased activity in the ctrA and ctrA/sciP mutants > twofold relative selleck compound to wild type (Fig. 3a). An increase in activity was also observed in both the wild-type (2.4-fold) and sciP mutant (1.6-fold) strains containing ctrAD51E. TSA HDAC order CtrAD51E increased RcGTA activity and extracellular capsid protein levels slightly in the cckA and chpT mutants (Fig. 3c). The ctrAD51A gene yielded surprising results as all strains expressing this version of CtrA showed a large increase in capsid protein levels inside the cells relative to wild type (Fig. 3d). The wild type and sciP mutant containing CtrAD51E also demonstrated significant increases in RcGTA activity (Fig. 3a). However, unlike the CtrAD51E protein, activities in the ctrA and ctrA/sciP mutants remained very low (Fig. 3a), which agreed with observed low extracellular capsid
levels (Fig. 3d and f). Introduction of the ctrAD51A allele caused an increase in RcGTA activity and extracellular capsid levels in both the cckA and chpT mutants (Fig. 3a and d). Viable cell counts were performed with the different strains on the same cultures used for the gene transfer bioassays and western blots. None of the strains were affected for growth rate and all reached the same approximate cell density at stationary phase as determined by culture turbidity (data not shown). The ctrA/sciP, chpT, and cckA mutations were found to have Sclareol no significant effect on the number of colony-forming units (Fig. 4). Unexpectedly, the ctrA mutant showed a significant increase (1.6-fold; P < 0.01) in colony-forming units relative to wild type (Fig. 4). Conversely, the sciP mutant was found to have a significant decrease (~0.5 of wild type; P < 0.01) in colony-forming units (Fig. 4). All anova results are available in Table S3. The introduction of the ctrAD51E and ctrAD51A genes had no effect (Fig. S1). Our experiments with R. capsulatus mutant strains lacking putative orthologs of proteins involved in a pathway controlling CtrA activity in C.
riparius endosymbionts (target organism) were obtained from homogenized internal genitalia of
female P. riparius. Probe specificities were evaluated with such cell suspensions of the Pseudomonas-like endosymbiont and the closely related P. aeruginosa (nontarget organism). A minimum of 300 DAPI-positive cells of randomly chosen areas on microscopic slides were evaluated. Scanning electron microscopy (SEM) studies of P. riparius eggs were carried out with a Philips FEI XL 30 ESEM. Subsequent to dehydration in ethanol (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 30 min each), specimens were coated with gold (Edwards S150B). DNA was extracted using Qiagen DNA extraction kits (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. A 157-base pair fragment of pks-gene encoding a ketosynthase involved in pederin biosynthesis was amplified with primers KS1F (5′-TGGCATCGT GGGGAAAGGCTG-3′) and KS1R (5′-GGCGCAGGTGCTGACACGC-3′) compound screening assay (pks-PCR; Piel, 2002). Primers were purchased from MWG-Eurofins (Ebersberg, Germany). PCR was performed in a total volume of 50 μL containing 24.8 μL PCR-H2O, 10.0 μL 5 × Q-Solution, 5.0 μL 10 × PCR buffer, 5.0 μL ddNTP Mix (2 mM), 2.5 μL of each primer (10 pmol μL−1), 0.2 μL Taq (5 U) (Qiagen). Thermal cycling was at 96 °C for Pifithrin-�� price 5 min followed by 35 cycles of denaturation at 96 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 1 min. Several potential
for 16S rRNA gene-directed oligonucleotide probes were identified on the 16S rRNA gene sequence of the Pseudomonas-like bacterial endosymbiont of P. riparius (accession number: AJ316018; Kellner, 2002a). On the basis of different probe many parameters (e.g. length of probe, hybridization temperature, GC content, Escherichia. coli position, etc.), the probe with target site 444–461 (E. coli numbering according to Brosius et al., 1981) was selected (Table 1) and checked with the probe match-tool (the arb project: http://www.arb-home.de) for specificity. The probe (PAE444) was complementary to the target sequence of the Paederus endosymbiont and displayed high probe accessibility within its target region according to a 16S rRNA gene secondary structure model of E. coli (Fuchs et al., 1998; Behrens et al., 2003). PAE444 exhibited only two mismatches to the closest related nontarget sequence (P. aeruginosa). Thus, a competitor (cPAE444) complementary to the P. aeruginosa sequence was designed in order to achieve full mismatch discrimination (Table 1; Manz et al., 1992). PAE444 coverage alone was experimentally analysed by whole cell hybridization with cell suspensions of endosymbionts (extracted from P. riparius tissue, see Materials and methods) and pure cultures of P. aeruginosa. Probe dissociation curves were recorded to determine stringent hybridization and washing conditions. The nonhybridizing probe NON338 (Manz et al.
The extent to which lipid effects
and overall tolerability differ between treatments with atazanavir and darunavir and whether atazanavir-induced hyperbilirubinaemia may result in more favourable metabolic effects are issues that remain to be resolved. A 96-week randomized clinical trial was carried out. The primary endpoint was change in total cholesterol at 24 weeks. Secondary endpoints were changes in lipids other than total cholesterol, insulin sensitivity, total bilirubin, estimated glomerular filtration rate, and CD4 and CD8 cell counts, and the proportion of patients with plasma HIV RNA < 50 HIV-1 Ruxolitinib price RNA copies/mL and study drug discontinuation because of adverse effects at 24 weeks. Analyses were intent-to-treat. One hundred and seventy-eight patients received once-daily treatment with either atazanavir/ritonavir (n = 90) or darunavir/ritonavir (n = 88) plus tenofovir/emtricitabine. At 24 weeks, mean total cholesterol had increased by 7.26 and 11.47 mg/dL in the atazanavir/ritonavir and darunavir/ritonavir arms, respectively [estimated difference −4.21 mg/dL; 95% confidence Ipilimumab order interval (CI) −12.11 to +3.69 mg/dL; P = 0.75]. However, the ratio of total to high-density lipoprotein (HDL) cholesterol tended to show a greater decrease with atazanavir/ritonavir
compared with darunavir/ritonavir (estimated difference −1.02; 95% CI −2.35 to +0.13; P = 0.07). Total bilirubin significantly Florfenicol increased with atazanavir/ritonavir (estimated difference
+1.87 mg/dL; 95% CI +1.58 to +2.16 mg/dL; P < 0.01), but bilirubin changes were not associated with lipid changes. Secondary endpoints other than total bilirubin were not significantly different between arms. Atazanavir/ritonavir and darunavir/ritonavir plus tenofovir/emtricitabine did not show significant differences in total cholesterol change or overall tolerability at 24 weeks. However, there was a trend towards a lower total to HDL cholesterol ratio with atazanavir/ritonavir and this effect was unrelated to bilirubin. "
“The aim of the study was to assess the seroprevalence of hepatitis E virus (HEV) infection in an HIV-infected population, as determined by HEV immunoglobulin G (IgG) antibodies (anti-HEV). The design of the study was cross-sectional. Serum anti-HEV IgG was determined by enzyme immunoassay in 238 HIV-infected patients consecutively attending our out-patient clinic between April and May 2011. In HEV-seropositive patients, HEV RNA was analysed by nested reverse transcriptase–polymerase chain reaction (RT-PCR). Associations between anti-HEV and liver cirrhosis, route of HIV infection, hepatitis B virus (HBV) and hepatitis C virus (HCV) serological markers, age, sex and alanine aminotransferase (ALT) levels were examined by univariate and multivariate analysis. One hundred and forty patients (59%) had chronic liver disease (99% were HBV- and/or HCV-coinfected).
, 2004). ROS was measured VX-809 purchase essentially as described by Ackerley et al. (2006) excepting that incubation with H2DCF-DA was carried out for 30 min, and fluorescence of the dye was measured by a Hitachi F-3010 spectrofluorometer (excitation at 485 nm and emission at 530 nm). The specificity of H2DCF for different ROS species is limited (Setsukinai et al., 2003), and our assay could detect hydrogen peroxide (H2O2), hydroxyl radical (˙OH), and superoxide anion (). TSB-6 cells were grown in LB at 37 °C without chromate till OD600 nm of 0.25. Then, one-half of these cells were heat stressed by transferring to 65 °C. Both the control and heat-stressed cells were grown for another 24 h.
The cells were harvested and soluble extracts prepared as described previously. Ammonium sulfate was then added to the soluble extracts to 90% saturation. The mixture was centrifuged at 12 000 rpm for 30 min and the supernatant discarded. The pellet was dissolved in 20 mM sodium phosphate buffer and dialyzed against 20 mM sodium phosphate buffer, pH 7.0. For the first-dimension electrophoresis, IPG strips of 7 cm length
and nonlinear pH range 4–7 (Bio-Rad) were rehydrated with 150 μg protein in 125 μL of rehydration buffer (provided with the kit) for 16 h. Isoelectric focusing was carried out in a PROTEAN IEF Cell (Bio-Rad) at 4 kV for 1 h with linear voltage amplification and finally to 20 kVh with rapid click here amplification. Before SDS-PAGE in the second dimension, of the focused strips were equilibrated
at room temperature first with a buffer containing 20% v/v glycerol, 0.375 M Tris–HCl, pH 8.8, 6 M urea, 2% (w/v) SDS, 130 mM DTT and then with a second buffer containing 20% (v/v) glycerol, 0.375 M Tris–HCl, pH 8.8, 6 M urea, 2% (w/v) SDS, and 135 mM iodoacetamide. Electrophoresis was carried out using 10% SDS polyacrylamide gels in a Mini-PROTEAN 3 system (Bio-Rad) at constant 200 V for 35 min. The gels were stained in 0.1% (w/v) Coomassie Brilliant Blue R-250. 2D gel images were obtained by VersaDoc™ (Model 4000) Imaging System (Bio-Rad). The spots were detected, analyzed, and assessed for reproducibility with PDQuest Advanced 2D Analysis software (version 8.0.1; Bio-Rad). Three independent experiments were performed with control and heat-stressed samples, and spots present in each of the three replicate gels of both samples were considered. Spots obtained from the control were taken as standard to determine the fold changes in the corresponding spots obtained from heat-stressed samples. Protein spots were excised from gels and subjected to in-gel digestion essentially as described by Shevchenko et al. (2006) using 25 ng μL−1 of trypsin and without the active extraction step. Mass spectrometry of the digested sample was carried out following a published protocol (Sinha & Chattopadhyay, 2011). Similarity searches to identify the proteins were performed using mascot search engine (version 3.5; Matrix Science, London, UK; www.matrixscience.com).
Comparing UT205 draft genome against H37Rv, CDC1551, F11 and KZN genomes, we also identify UT205 large indels (more than 30 bases) that affected either intergenic or coding regions (Table 2). To compare the differences within the protein coding, we undertook a complete orthologous genes comparison against the H37Rv predicted coding sequences using a global alignment
protocol of the fasta36 package, GGSEARCH. All predicted 3701 CDS DAPT ic50 of UT205 were translated into proteins and compared with the predicted 3998 proteome of H37Rv. For this analysis, all the PPE,vPE-PGRS and genes with sequence ambiguities or gaps (Ns) were excluded. Global protein identity analysis showed that 3271 (88.38%) of the UT205 display 100% identity with H37Rv. The remaining 430 (11.62%) proteins showed changes in at least one amino acid. From those, 388 proteins (10.48%) have an identity between 99.99% and 90%, 15 between 89.99% and 60% (0.41%) identity and 27 < 60% (0.73%) identity. Changes in protein-coding genes were owing to substitutions that introduced premature GSK2118436 ic50 stop codons, or indels that changed the translation frame and generated either truncated or longer proteins owing to the modification of the original stop triplet. Compared to H37Rv, insertions that
modify CDS sequences ranged from 1 to 531 bases (Table 3). The most affected genes, with < 90% identity are listed in Table 3. A detailed analysis of the regulon DosR in the UT205 strain was carried out. Of the 48 genes that compose this regulon, eight genes present modifications. These modifications involve complete gene deletions (such as in the case of Rv1996), CHIR-99021 mouse indels or SNPs in other seven genes (Table 4). The most interesting case involves the 3649 bp deletion, affecting the Rv1996/Rv1997 operon. This deletion eliminates Rv1996 genes and also the intergenic region upstream up to Rv1992c, where the DosR regulated promoter of this operon should be. This implies that both, Rv1996 and Rv1997, should not be expressed owing to a complete deletion and the
loss of the promoter region, respectively (Fig. 3). Pathogen adaptations to its human population hosts have been described in M. tuberculosis (Gagneux et al., 2006; Gagneux & Small, 2007), indicating that this species is more genetically diverse than originally believed. In-depth genomic analysis of Latin American species of M. tuberculosis has not been published so far, and some specific adaptation to this population should be expected, as observed in other human populations. Whole genome shotgun sequencing analysis of UT205 strain showed several differences with reference strains. IS6110 insertion elements were polymorphic compared to other LAM and no LAM reference strains, with novel insertions sites. Nucleotide large sequence polymorphisms showed insertions and deletions that could be specific for the Colombian strains.
Diagnoses were recorded at three different time points: (1) the working diagnosis at the emergency room, (2) the discharge diagnosis, and (3) the final diagnosis evaluated at least 1 year after discharge (>1 diagnosis/patient possible on each occasion). Complications and significant underlying diseases were recorded separately. The final clinical or etiological diagnosis of all patients was defined by the same infectious diseases specialist (H. S.), who had access to all
the results. Diagnoses were listed in the order of relevance to the symptoms as judged by the specialist. The diagnoses RG7422 were coded according to the classification used by GeoSentinel3: a standardized list of 588 possible individual diagnoses categorized under 21 broad syndromes was used. Septicemia was defined as a symptomatic condition with a positive blood culture. Unknown bacterial infection was defined as a clinical picture, C-reactive protein (CRP) (CRP median 136, range 50–275 mg/L),
and a timely response RG7420 purchase to systemic antibiotic therapy, all compatible with bacterial infection. Potentially life-threatening illness was defined as a disease potentially leading to death if left without specific or supportive treatment. The countries visited were grouped into five regions: Sub-Saharan Africa, Southeast Asia, Central Asia and Indian Subcontinent, South and Central America and the Caribbean, Other (North Africa, West Asia, Northeast Asia), modified from GeoSentinel.3 ifenprodil Chi-square tests, t-tests, and Mann–Whitney tests served to test for differences between the groups. The binary and
multinomial logistic regression models served to identify explanatory variables to the outcome variables. Variables that were found to have p value less than 0.2 were included in the multivariable models. To identify independent risk factors, forward and backward selection with Akaike information criteria (AIC) was used. One variable (duration of the trip) had 72 missing values of the 462, and to take that into account in the model, we used multiple imputation with an assumption that the missingness process was missing at random (MAR). The analysis was carried out with SPSS 18.0.2 (SPSS, Inc., Chicago, IL, USA). The demographic and travel data are presented in Table 1.