Our sample was kept clustered together with R conorii conorii (formerly R conorii Malish strain), the agent of classic MSF, in a distinct clade from R conorii israelensis and R conorii caspia subspecies. MDV3100 datasheet The configuration of similarity tree constructed based on gltA was compatible with that of ompA. The present diagnosis of R conorii conorii causing disease with a severe course in our patient confirms previous observations.[4, 5, 8, 9] Severe or fatal cases can be related to advanced age, underlying chronic diseases, or delay of appropriate

treatment.[4, 8] Febrile hemorrhagic syndrome is a frequent manifestation of a wide variety of viral or bacterial infections, and a proper laboratory study to a precise identification of the agent is crucial. Rickettsial diseases have Alectinib chemical structure been frequently related in international travelers throughout the world in the last decades, most of them coming from sub-Saharan Africa.[1, 9, 10] In Brazil, only one fatal case of spotted fever group rickettsiosis caused by R conorri conorii had been reported, in a South African traveler.[10] This case is the first report of MSF in Brazil

imported from Portugal, where R conorii is endemic. This study reinforces once more the importance of health surveillance in alerting local and tourism authorities to provide essential information to international travelers. This reasearch was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors state they have no conflicts of interest to declare. “
“We report a case of severe perniosis in a long-distance cyclist. This case demonstrates the importance of

identifying those at risk of cold-related injuries who are about to embark on extensive travel in cold environments. Perniosis is a moderately severe form of cold injury occurring in the setting of nonfreezing cold and humid conditions. It is a cutaneous inflammatory condition that presents as erythematous painful papules, typically bilateral and located on the dorsum of fingers, toes, nose, ears, 4-Aminobutyrate aminotransferase thighs, or buttocks. Symptom onset is within hours of the cold exposure and can be associated with digital edema, tenderness, and intense pruritus. Acute perniosis usually resolves within several days, while severe cases can lead to blistering, ulceration, scarring, or superinfections and can take weeks to heal.1–3 A 27-year-old male from Australia was cycling across Mongolia with his partner, both of them doctors, during April and May 2010. The patient described spending up to 8 hours on his bicycle per day, always wearing full-length gloves. Average temperatures for April 2010 over the cycled route were: maximum −3°C, minimum −9°C; and for May, maximum 15°C and minimum 2°C. The patient had no formal past medical history, but described short episodes in the past where his hands became mildly swollen and erythematous following exposure to cold.

Rifaximin prophylaxis reduced risk of developing TD versus placebo (p < 0.0001). A smaller percentage of individuals who received rifaximin

versus placebo developed all-cause TD (20% vs 48%, respectively; p < 0.0001) or TD requiring antibiotic therapy (14% vs 32%, respectively; p = 0.003). More individuals in the rifaximin group (76%) completed treatment without developing TD versus those in the placebo group (51%; p = 0.0004). Rifaximin provided a 58% protection rate against TD and was associated with fewer adverse events than Vemurafenib purchase placebo. Conclusions. Prophylactic treatment with rifaximin 600 mg/d for 14 days safely and effectively reduced the risk of developing TD in US travelers to Mexico. Rifaximin chemoprevention should be considered

for TD in appropriate individuals traveling to high-risk regions. An estimated 40% of the 50 million individuals traveling from industrialized to developing countries each year develop travelers’ diarrhea (TD).1 This acute infectious SB431542 research buy illness is characterized by the passage of 7 to 13 watery stools over 2 days, accompanied by one or more additional enteric symptom.1,2 Based on microbiologic evaluation, enteric bacterial pathogens are thought to cause approximately 80% of TD cases, with strains of enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E coli (EAEC) responsible for the majority of cases.3–5 Invasive bacterial pathogens including Shigella and Campylobacter contribute to approximately 4% to 20% of TD cases.5–7 Although TD is often self-limiting, lasting on average for 4 days, the negative consequences of acquiring this illness can be substantial, including disruption of travel plans and increased risk for development of postinfectious

complications,8 such as postinfectious irritable bowel syndrome (PI-IBS)9–14 and inflammatory bowel disease (IBD).15 Antibiotic chemoprophylaxis provides substantial protection from TD and prevents potentially severe complications.16 However, the guidelines recommended by the National Institutes of Health consensus panel in 1985 discouraged the routine administration of systemic antibiotics as 4��8C chemoprophylaxis for TD because of the potential adverse effects associated with administration and concern that overprescribing could contribute to the growing epidemic of antibiotic resistance.17 The ideal chemoprevention agent would achieve the efficacy of systemic antibiotics without the potential adverse effects and antibiotic resistance associated with these agents. Rifaximin (Xifaxan®; Salix Pharmaceuticals, Inc., Morrisville, NC, USA) is a gut-selective, nonsystemic antibiotic18 that has a low risk for development of clinically relevant antibiotic resistance.19 It is indicated for the treatment of TD caused by noninvasive strains of E coli2 and has demonstrated efficacy in treating TD in clinical studies.

2 On the basis of the patient’s clinical symptoms during the early stage of infestation, and taking into account the results obtained from the different diagnostic tests, a presumptive diagnosis of gnathostomiasis was initially reached, followed by one of sparganosis. Since these diseases are very rare in Spain, serological tests were not immediately available, Entinostat in vivo but empirical treatments were administered. The morphological features of the fragment of a surgically extracted larva suggested an infestation by Hypoderma spp. The identification of the different species of Hypoderma relies on the examination of larval morphological features,16,17 but the small size of the fragment hindered complete identification.

However, the presence of high anti-H lineatum antibody titers in the patient’s serum (detected by ELISA at different times) was indicative of infestation www.selleckchem.com/products/Bleomycin-sulfate.html by Hypoderma larvae, supporting the previous morphological suspicion of myiasis. The assessment of cross-reactivity with antigens of other members of the Hypodermatinae subfamily, ie, Hypoderma bovis, Hypoderma tarandi, Hypoderma diana, and Przhevalskiana silenus (see Monfray and Boulard18; Boulard et al.19) is useful when performing ELISA prepared with H lineatum antigens, even though they may not be endemic in the patient’s country of origin. Repeated treatment with ivermectin seemed to be effective since the patient quickly became asymptomatic and

the eosinophil count normalized. Ivermectin is effective in the treatment of several myiases, and it is a good alternative when surgical removal is unfeasible.20 This is important since Hypoderma larvae can migrate within the body to involve in the central nervous system21 or, more often, to the eyes, where they cause ophthalmomyiasis.22 In our case, two parasite larvae were surgically removed. Considering that the swellings did not have any breath hole and the larval size, a diagnosis of fly first instars (LI), ready to moult to second Phosphoprotein phosphatase instars (LII) was made. Furthermore, after the first and second round of ivermectin treatment, new painful swellings appeared probably due to other undetected parasites,

and it was not until the third ivermectin round that the patient became asymptomatic. Although cases of human myiasis are uncommon in Europe, if symptoms are indicative this disease should be kept in mind by physicians examining immigrants and travelers returning from endemic areas such as Ladakh. While serological analysis is useful in the diagnosis of myiasis-causing Hypoderminae larvae in travelers not previously exposed to larval infestation, molecular identification is important. In this work, the sequencing of a partial mitochondrial cox1 gene sequence confirmed H sinense to be the causal agent. Human cases of infestation by Hypoderma spp. have previously been reported, with H bovis and H lineatum or H tarandi as the agents most frequently identified.

He quickly did these colonization tests in the rat lung and intestine and, while it appeared that a mutant deleted for the arcDABC operon had a lower colonization ability compared with the wild type, the effects measured were not significant and therefore not published. However, Gerd remained convinced that P. aeruginosa was a successful pathogen in the CF lung because of its ability to deal with hypoxic conditions. He eventually managed to assemble compelling evidence for the fact that the mucus layer in learn more the CF lung becomes depleted of measurable oxygen and nevertheless supports persistent growth of P. aeruginosa (Worlitzsch et al. J Clin

Invest 109: 317–325, 2002). This important work has been cited more than 500 times and has led to further important discoveries, but has also been misinterpreted by some researchers who believed that P. aeruginosa would adopt Selleck ABT 737 a purely anaerobic lifestyle (i.e. using nitrate respiration and fermentation) in the CF lung. However, the main energy source of P. aeruginosa in this environment is still aerobic respiration, which occurs via the two cbb3 terminal oxidases whose high affinity for oxygen allows the bacterium to grow at submicromolar oxygen concentrations. Such low oxygen levels are undetectable with a Clark electrode.

In more recent studies, Gerd and his collaborators found that the so-called mucoid conversion of P. aeruginosa is strongly stimulated by oxygen depletion. Mucoidy is due to overproduction of the exopolysaccharide alginate by P. aeruginosa and is a hallmark of persistent infection. It turns out that alginate export is controlled by a novel oxygen sensor acting at a post-translational level. As experiments on this mechanism are still ongoing, Gerd was not able to see their completion and publication, which saddened him a great deal. But he stayed optimistic PIK3C2G and passionate about

this work up to his last days. Gerd was always keen to translate his research into the diagnosis, prevention, and treatment of infections with P. aeruginosa, particularly the chronic airways infections in individuals with CF. Gerd developed hygienic measures and devices to control the spread of P. aeruginosa in the hospital environment, and he and Christiane Wolz, his PhD student at that time, were the first in the late 1980s who demonstrated the nosocomial transmission between unrelated CF patients at rehabilitation centers with a molecular probe. Based on his early discovery made in Niels Høiby’s laboratory that in serial CF sera, antibody titers against secreted virulence effectors are inversely correlated with the clinical outcome, he commercialized an ELISA that still is the standard for Pseudomonas serology at central European CF centers. Gerd was involved in the first clinical trial on aerosolized tobramycin to eradicate P.

Typhimurium, but present in S. Typhi (Parkhill et al., 2001; Pickard et al., 2003;

Bueno et al., 2004). In S. Typhi, it is 134 kb in size, corresponding to approximately 150 genes inserted between duplicated pheU tRNA sequences (Hansen-Wester & Hensel, 2002; Pickard et al., 2003). This island contains the Vi capsule biosynthesis genes (Hashimoto et al., 1993), whose production is associated with virulence (see section below), a type IVB pilus operon Target Selective Inhibitor Library screening (Zhang et al., 2000) and the SopE prophage (ST44) encoding the SPI-1 effector SopE (Mirold et al., 1999). SopE is also encoded in S. Typhimurium’s genome, but within the temperate SopE prophage (Hardt et al., 1998) located at a different location (sopE is absent in most S. Typhimurium strains, including

S. Typhimurium strain LT2, but present and located on a prophage in S. Typhimurium strains SL1344 and 14028) (Hardt et al., 1998; Mirold et al., 1999; Pelludat et al., 2003). At the SPI-7 location in S. Typhimurium LT2, we find a single complete Selleckchem PLX-4720 pheU tRNA sequence and STM4320 (a putative merR family bacterial regulatory protein) (Fig. S1g). SPI-8 is an 8 kb DNA fragment found next to the pheV tRNA gene that is part of SPI-13 and will be discussed in that section (Fig. S1l) (Parkhill et al., 2001; Hensel, 2004). SPI-9 is a 16 kb locus present in both serovars (Fig. S1h). This island contains three genes encoding for a T1SS and one for a large protein, sharing an overall 40% nucleotide identity to siiCDEF genes from SPI-4 (Morgan et al., 2004, 2007). The large protein-coding ORF (STM2689) in S. Typhimurium strain LT2 was first suggested to be a pseudogene (McClelland et al., 2001; Morgan et al., 2004). However, a subsequent study showed an undisrupted gene coding a putative 386 kDa product Immune system renamed BapA (Latasa et al., 2005). SPI-10 is an island found next to the leuX tRNA gene at centisome 93. This locus is completely different in each serovar and has been termed SPI-10 only in S. Typhi. In S. Typhimurium, it is substituted by a 20 kb uncharacterized island without any SPI

annotation (Fig. S1i), comprising functionally unrelated genes that share little homology to sequences from the genomic databases (Edwards et al., 2001; Bishop et al., 2005). However, a possible relationship of these genes with DNA repair has been proposed (Porwollik & McClelland, 2003). Deletion of this island in S. Typhimurium strain 14028 caused attenuation of virulence in mice (Haneda et al., 2009). In S. Typhi’s genome, this island corresponds to a 33 kb fragment (Parkhill et al., 2001) carrying a full P4-related prophage, termed ST46 (Edwards et al., 2001; Thomson et al., 2004; Bishop et al., 2005). ST46 harbours the prpZ cluster as cargo genes encoding eukaryotic-type Ser/Thr protein kinases and phosphatases involved in S. Typhi survival in macrophages (Faucher et al., 2008). There is also a complete, but inactivated sefABCDR (S. Enteritidis fimbriae) fimbrial operon (Fig S1i).

Mary’s, London; M Fisher, Royal Sussex County Hospital, Brighton; C Leen, Western General Hospital, Edinburgh. Virology group: B Clotet, R Paredes (central co-ordinators) plus ad hoc virologists from participating sites in the EuroSIDA study. Steering committee: F Antunes, B Clotet, D Duiculescu, J Gatell, B Gazzard, A Horban, A Karlsson, C Katlama,

B Ledergerber (Chair), A D’Arminio Monforte, A Phillips, Sirolimus molecular weight A Rakhmanova, P Reiss (Vice-Chair), J Rockstroh. Coordinating centre staff: J Lundgren (project leader), O Kirk, A Mocroft, N Friis-Møller, A Cozzi-Lepri, W Bannister, M Ellefson, A Borch, D Podlekareva, J Kjær, L Peters, J Reekie, J Kowalska. “
“For potential CMV and antiretroviral drug–drug interactions please refer to Table 5.1. Since the advent of potent antiretroviral therapy in 1996 the incidence, clinical features and long-term prognosis

of CMV retinitis have changed dramatically. Highly active antiretroviral treatment (HAART) has significantly decreased the number of patients with CD4 counts of <50 cells/μL and therefore the proportion of patients at risk Pirfenidone supplier of developing CMVR, as well as significantly prolonging disease-free intervals in patients with pre-existing CMVR [1–3]. In spite of improvements in the era of potent antiretroviral treatments, CMVR remains a significant clinical problem as well as the leading cause of ocular morbidity for patients with AIDS [4]. Despite improvements in immune function (immune reconstitution) due to HAART, new cases of CMVR continue to occur because of late diagnosis of HIV, poor adherence or poor tolerance of treatment and failure of antiretroviral treatment. CMVR usually presents in persons who are severely immunosuppressed with CD4 counts Sunitinib of <50 cells/μL. It may affect one eye at first, but without systemic treatment or improvement of the immune system the other eye

usually becomes affected [5]. Symptoms depend on the site and severity of retinal involvement of CMV. Common clinical presentations include floaters, blind spots, blurred vision or a sudden decrease in vision. However, approximately 15% of patients with active CMVR are asymptomatic. Routine screening with dilated indirect ophthalmoscopy is recommended at 3-monthly intervals in patients with CD4 counts less than 50 cells/μL [6]. CMVR is a clinical diagnosis. Virological confirmation is not ordinarily required. Visualization of the retina should be performed through a dilated pupil to enable peripheral lesions to be seen. Once the diagnosis of CMVR is suspected urgent assessment is required by an ophthalmologist to confirm the diagnosis and advise on appropriate treatment.

Therefore, self-reported depressive symptoms did not improve the SVM prediction accuracy. Including data on CART CPE also failed to improve the prediction. For the scenario where log10 HIV RNA was included, the accuracy of the prediction was 75% for impairment and 72% for NP nonimpairment. These same accuracies were also achieved for the scenario where detectable vs. undetectable HIV RNA was used. Hence inclusion of CPE did not improve

prediction accuracy. Our study was conducted with the intention of generating an extra-brief tool to assist HIV physicians in referring HIV-positive persons at risk for NP impairment. We believe that our study provides a preliminary but robust solution to this first objective. Indeed, we found that our SVM-derived CHIR-99021 mw models yielded adequate prediction accuracy for NP impairment (sensitivity 78%; n=28/36) and NP nonimpairment (specificity 70%; n=43/61). These figures are certainly adequate for use of the algorithm as an adjunct clinical tool. Moreover, we believe that the predictions were quite good in comparison with predictions of HAND provided by brief paper-and-pencil NP instruments. Davis et al. [28] reported

70% sensitivity and 71% specificity for the HIV-dementia scale. Carey et al. [29] showed 78% sensitivity, 85% specificity and 83% overall prediction accuracy using two selected NP tests. The California Computerised Assessment Package (Calcap), a brief cognitive computerized test, yielded 68% sensitivity and 77% specificity [30]. Lastly, the brief computerized battery CogState demonstrated 81% sensitivity, http://www.selleckchem.com/products/gsk1120212-jtp-74057.html 70% specificity, and an overall prediction accuracy of 77% [31]. These accuracy rates provide preliminary support for application of these models in a clinical setting. In addition, this algorithm can be easily implemented on a web-interface platform (under construction) for which the HIV physician will only have to input

the necessary characteristics [for example when using the model determined from detectable levels of HIV RNA the required characteristics are: age in years; current CD4 T-cell count; presence or absence of past CNS HIV-related diseases (yes or no); and current CART duration in months]. The expected duration of the screening (computation G protein-coupled receptor kinase of the algorithm including data entry with interactive instructions) is about 3 min. Here we have shown that it is the inclusion of easily ascertainable clinical factors that makes the algorithm practical. However, while the inclusion of the factors might be obvious, the relative weighting of each is certainly not. This study also contributes to the body of evidence on the use of SVM as a robust tool for data classification problems [18]. SVM methods have been increasingly used in a wide variety of medical classification problems.

“
“The present study

aimed to identify the genes involved in the pathogenesis of systemic lupus erythematosus (SLE) in Arabs by investigating a panel of 84 genes related to the t helper (Th)17-related regulatory network and to further explore the expression levels of serum interleukin (IL)-17A and IL-17F in a studied cohort. A comparative analysis of gene expression profile in SLE and lupus nephritis (LN) patients against that of healthy controls (HC) was performed. A quantitative real-time polymerase chain reaction (PCR) (Th17 autoimmunity and inflammation) array analysis was performed in peripheral white blood cells of 66 SLE patients under specific medical treatment and 30 age/gender/ethnically matched healthy controls. Statistical analysis was carried out using the RT2 Profiler TM PCR Data Analysis tool. The analysis of Th17 pathway revealed 14 genes (IL-17A, IL-17C, IL-17D, IL-17F, IL-18, IL-12RB2, IL-23R, selleck kinase inhibitor CCL2, CCL20, CXCL5, MMP3, RORC, STAT4 and TRAF6) that are differentially expressed in SLE and HC (fold change [FC] < 2, find more P < 0.0006). No significant difference in expression profiles was observed between SLE and LN. A significant difference in serum concentration

of IL-17A (P = 0.002) and IL-17F (P = 0.002) was observed between SLE (13.91 ± 4.25) and LN (18.26 ± 4.24). Our study is the first to investigate a panel of 84 genes related to Th17 regulatory pathway in Arab SLE subjects and the first to explore the effect of current immunosuppression regimens on Th17 regulatory pathway. It paves the way for understanding the etiology of SLE and autoimmune diseases in general. “
“Aims:  The long-terms complications of immunosuppressive and anti-inflammatory treatment in idiopathic inflammatory myositis (IIM) are unknown. We sought to determine the complications of these treatments in a large cohort of patients with biopsy-proven IIM. Methods:  A South Australian database for patients with biopsy-proven IIM was established. Clinical details of patients

including treatment received were recorded. Results:  Forty-three MycoClean Mycoplasma Removal Kit patients with dermatomyositis (DM), 184 with polymyositis (PM) and 117 with inclusion body myositis (IBM) were registered on the database. The prevalence of hypertension and diabetes in this population was 62% and 29%, respectively, considerably higher than the background prevalence of 9.4% and 4%, making detection of treatment-related adverse effects difficult. Hypertension and ischemic heart disease were more likely to be present prior to the diagnosis of IIM rather than following it. Hypertension and diabetes occurred more frequently following the diagnosis of myositis, in patients with DM compared with PM or IBM. Conclusions:  We report a novel association of IIM with hypertension, diabetes and ischemic heart disease, indicating that a comprehensive assessment of vascular risk factors is essential in IIM.

riparius endosymbiont (Fig. 2a–d). The granular layer was found on all electron-microscopically investigated eggs (n=20), which had been oviposited buy Afatinib by P. riparius. In order to check whether this granular layer is already applied to the eggshell in the ovaries during oogenesis or somewhere else in the internal female genitalia

during egg passage, several eggs (n=9) were prepared out of the common oviduct and analysed by electron microscopy. These eggs always exhibited a strongly folded, smooth surface, indicating that a granular layer was absent (Fig. 3c and d). In order to approve these findings molecularly, two eggs from the common oviduct (cf. Fig. 3e) and five already oviposited eggs from different female beetles (n=10) were analysed by pks PCR. pks gene fragments indicating Paederus endosymbionts were amplified from all oviposited eggs, but not from eggs originating from the common oviduct, indicating that the endosymbionts are applied to the egg shell inside the efferent duct (cf. Fig. 3e). Many endosymbiotic bacteria are still unable to grow in vitro, potentially because of specific nutrients present exclusively within the source/host

habitat and are not available in conventional culture media (Lewis, 2007; Davey, 2008). FISH allows the visualization of prokaryotic cells in their natural environment regardless of their culturability. The FISH method targets rRNA, which is essential to basic cellular metabolism and is thought to degrade soon after cell death (Nocker & Camper, 2009). Thus, this method is a very powerful tool for the detection and localization find more of unknown bacterial communities from a range of different habitats (Amann et al., 1995, 2001; Berchtold et al., 1999; Darby Thalidomide et al., 2005;

Davidson & Stahl, 2006; Ferrari et al., 2006, 2008; Vartoukian et al., 2009), such as endosymbiotic bacteria that reside in invertebrates like insects within specific cells or symbiotic organs. Consequently FISH may facilitate isolation and potential cultivation of newly detected or previously uncultivable bacteria, as could be demonstrated recently (Vartoukian et al., 2010). A FISH approach using novel oligonucleotide probes was developed and demonstrated that essentially a ‘pure culture’ of the Pseudomonas-like pederin-producing endosymbionts of P. riparius covers the whole surface of P. riparius eggs, which extends previous reports suggesting that the endosymbiont is transmitted to the offspring via the egg (Kellner, 2001a, b, 2002a, b, 2003; Piel, 2002, 2004, 2005). Most bacteria appear to form biofilms, including P. aeruginosa, and such a multicellular mode of growth likely predominates in nature as a protective mechanism against hostile environmental conditions (Costerton et al., 1995; Costerton & Stewart, 2000). Consequently, this ability could also be existent for the Paederus endosymbiont because of its close relationship to P. aeruginosa (Kellner, 2002a; Piel, 2002; Piel et al., 2004).

This survey is the first reported evaluation of how HIV clinicians use the RITA information at an individual patient level. This survey found that RITA results have

become part of the standard of care in the majority of participating centres and that therefore no additional consent is being obtained from patients. Some centres are still experiencing delays in reporting of results and difficulties with accessing results at clinic level. Some sites see only a small number of new diagnoses, and batch samples buy Dabrafenib for testing. Other sites aim to remove samples from patients with a previous positive HIV antibody result, which is recommended by the HPA but may lead to a delay. At the HPA, over 95% of samples are tested and reported within 7 working days. More work is underway to assist local sites to improve turnaround

and reporting times to allow clinicians early access to results. All HPA reports include an interpretation of the avidity score and the need to consider clinical markers in the interpretation of the test. This survey find more indicates that not all clinicians may access this information, highlighting the need for better data sharing at local level to allow effective use of RITA results in clinical practice. Nevertheless, this survey shows that many clinicians have now incorporated RITA as an additional clinical tool when assessing newly diagnosed HIV patients, in particular, those where the clinical picture suggests an acute HIV seroconversion illness or recent infection and when discussing treatment start. In order to facilitate discussions with patients further, the HPA is considering changing the reporting of results by converting the avidity index into a probability score, for example, the probability in per cent that a newly diagnosed patient was infected within the last 6 months.

selleck screening library Reassuringly, clinicians describe the response from patients on learning about the estimated timing of their infection as overwhelmingly positive or neutral and no adverse events have so far been reported in response to communicating a result. In particular, there are currently no reports that RITA results have been referred to during criminal proceedings, which is strongly discouraged by a recent guidance document published jointly by the National AIDS Trust and the HPA [10]. A complementary patient survey by the HPA in collaboration with four clinics is currently underway exploring the experiences of patients when receiving a RITA result indicating probable recent infection. The majority of respondents stated that RITA results could assist in contact tracing and some independently commented that they have started incorporating RITA into local clinic guidelines for contact tracing.