Seroprevalences of antibodies against ToRCH infectious pathogens in women of childbearing age residing in Brazil, Mexico, Germany, Poland, Turkey and China.

Determination of antibodies against ToRCH antigens at the beginning of pregnancy allows assessment of both the maternal immune status and the risks to an adverse pregnancy outcome. Age-standardised seroprevalences were determined in sera from 1009 women of childbearing age residing in Mexico, Brazil, Germany, Poland, Turkey or China using a multiparametric immunoblot containing antigen substrates for antibodies against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), herpes simplex viruses (HSV-1, HSV-2), Bordetella pertussis, Chlamydia trachomatis, parvovirus B19, Treponema pallidum and varicella zoster virus (VZV). Seroprevalences for antibodies against HSV-1 were >90% in samples from Brazil and Turkey, whereas the other four countries showed lower mean age-adjusted seroprevalences (range: 62.5-87.9%). Samples from Brazilian women showed elevated seroprevalences of antibodies against HSV-2 (40.1%), C. trachomatis (46.8%) and B. pertussis (56.6%) compared to the other five countries. Seroprevalences of anti-T. gondii antibodies (0.5%) and anti-parvovirus B19 antibodies (7.5%) were low in samples from Chinese women, compared to the other five countries. Samples from German women revealed a low age-standardised seroprevalence of anti-CMV antibodies (28.8%) compared to the other five countries. These global differences in immune status of women in childbearing age advocate country-specific prophylaxis strategies to avoid infection with ToRCH pathogens.

Eosinophilia prevalence and related factors in travel and immigrants of the network +REDIVI. / Prevalencia de la eosinofilia y factores relacionados en los viajeros e inmigrantes de la red +REDIVI.

The population movements during the last decades have resulted in a progressively increasing interest in certain infectious diseases. Eosinophilia is a common finding in immigrants and travellers. One of the most common causes of eosinophilia is helminth infection, and some intestinal protozoa. The aim of this paper is to describe the epidemiological characteristics of cases with eosinophilia and its association with the presence of parasites in the REDIVI data network. This is a multicentre prospective observational study that includes patients diagnosed with eosinophilia registered in the cooperative network for the study of infectious diseases in travellers and immigrants (+REDIVI) from January 2009 to December 2012. A total of 5,255 episodes were recorded in the network during the study period, and eosinophilia was observed in 8.1-31.3% of cases (depending on the immigration group). There were 60.2% men, with a median age of 31years. There were 72.4% immigrants, and 81.2% were asymptomatic. The most commonly identified parasites were S.stercoralis (34.4%), Schistosoma sp. (11.0%), and hookworm (8.6%). The relationship between eosinophilia and parasite infection was significant for all helminths (except for cutaneous larva migrans). The symptoms and duration of the journey did not significantly determine the presence of eosinophilia. In the case of eosinophilia in a person who has lived in helminth endemic areas, it is advisable to carry out targeted studies to diagnose the infection, regardless of immigration type, length of stay, or the presence of symptoms.

High seroprevalence of Histomonas meleagridis in Dutch layer chickens.

Histomona meleagridis is a protozoan parasite that may cause outbreaks of histomonosis with high mortality, especially in turkey flocks. Chickens are less susceptible to the disease than are turkeys, but are considered to act as an important reservoir. To determine the seroprevalence of H. meleagridis in Dutch layer chicken flocks, a large scale seroepidemiologic study (3376 samples) was performed by sampling 12 organic flocks, 24 free-ranging flocks, 40 flocks with floor housing, and 40 flocks with cage housing. At the end of the laying period, approximately 30 blood samples per flock were collected for serology. The seroprevalence found was high. In every flock, at least one of the samples tested positive while in 87% of the flocks, at least one of the samples was strongly positive. There were no significant statistical differences in seropositivity between the housing types. To confirm the enzyme-linked immunosorbent assay (ELISA) results, a small-scale seroepidemiologic study (576 samples) was performed in 29 additional layer chicken flocks kept in different housing systems. Subsequently, a subset of five seropositive flocks was selected. Five birds were obtained from each of these flocks in order to detect the parasite using culture and PCR. In all five flocks, H. meleagridis was either isolated from (culture), detected in (PCR), or both, the birds sampled. Together with the previously performed validation studies, the latter results confirm that the positive ELISA serology found is genuine. We conclude that the seroprevalence of H. meleagridis in layers is, as anticipated, high.

First description of naturally acquired Tritrichomonas foetus infection in a Persian cattery in Spain.

Tritrichomonas foetus has been identified as the causative agent of feline intestinal trichomonosis, characterized by clinical signs of chronic large bowel diarrhoea. This disease has been reported in cats from the USA, Europe and Australia. However, its epidemiology is still unclear. The aim of the present study was to describe T. foetus infection in a Persian cattery in Spain. T. foetus infection was sequentially diagnosed in 20 cats by direct faecal smear examined under the microscope, specific culture (In Pouch TF medium) and PCR. A standard coprological sedimentation method was also performed in order to screen for other intestinal parasites in all the cats included. In addition, sera were tested for IgG antibodies against Leishmania infantum, Toxoplasma gondii, and for the detection of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Five out of 20 cats were positive for T. foetus (25%), two of them by microscopy, culture and PCR and three by culture and PCR. No association was found between T. foetus infection and age or sex. L. infantum and T. gondii seroprevalence rates were 15% and 10%, respectively. The prevalence of FeLV p27 antigen and of FIV antibodies in the study population was zero. Cystoisospora spp. oocysts were detected in one cat. These preliminary results show that the transmission of T. foetus infection in cluster conditions may occur between asymptomatic cats and young or immunocompromised animals.

Genetic resistance of carp (Cyprinus carpio L.) to Trypanoplasma borreli: influence of transferrin polymorphisms.

In serum most of the iron molecules are bound to transferrin (Tf), which is a highly polymorphic protein in fish. Tf is an essential growth factor for mammalian trypanosomes. We performed a series of experiments with Trypanoplasma borreli to detect putative correlations between different Tf genotypes of common carp (Cyprinus carpio L.) and susceptibility to this blood parasite. Five genetically different, commercially exploited carp lines (Israelian 'D', Polish 'R2' and 'K', Ukrainian 'Ur', Hungarian 'R0') and a reference laboratory cross ('R3xR8') were challenged with T. borreli and parasitaemia measured to determine susceptibility to the parasite. Among the commercial carp lines, Israelian 'D' carp were identified as most and Polish 'R2' carp as least susceptible, and used to produce a next generation and reciprocal crosses. These progenies were challenged with T. borreli and parasitaemia measured. We demonstrated significant effects of genetic background of the carp lines on susceptibility to T. borreli. This genetic effect was preserved in a next generation. We also observed a significant male effect on susceptibility to T. borreli in the reciprocal crosses. Serum samples from a representative number of fish from two infection experiments were used for Tf genotyping by polyacrylamide gel electrophoresis (PAGE), identifying DD, DG and DF as most frequent Tf genotypes. We could detect a significant association of the homozygous DD genotype with low parasitaemia in the least susceptible 'R2' (and 'K') carp lines and the lack of a such an association in the most susceptible 'D' carp line. Upon examination of parasite growth in vitro in culture media supplemented with 3% serum taken from fish with different Tf genotypes, we could show a faster decrease in number of parasites in culture media with serum from DD-typed animals.

Identification of blood parasites in old world warbler species from the Danube River Delta.

Warbler species of the families Sylviidae and Acrocephalidae occurring in the Danube river delta are frequently exposed to blood-sucking arthropods that transmit avian blood parasites. We investigated infections by three genera of hemosporidian parasites in blood samples from six warbler species. Altogether in 17 (32.6%) of 52 blood samples, a PCR product was amplified. The great reed warbler (Acrocephalus arundinaceus) had the highest prevalence, with 63.6% (7/11) infected individuals, whereas no infection was detected in marsh warbler (Acrocephalus palustris). The most common parasite genus was Haemoproteus, which was found in 15.4% (8/52) of individuals. Seven known parasite lineages (five Haemoproteus and two Plasmodium) and two new lineages were recorded (one Leucocytozoon and one Plasmodium).

Histopathological diagnosis of infectious diseases using patients' sera.

It is expected that sera of patients suffering from infectious diseases contain high-titered IgG-type antibodies against the causative pathogen, particularly when inflammatory reactions, such as abscess or granuloma, are histopathologically confirmed in immunocompetent individuals. The present review article describes the usefulness of diluted patients' sera for identifying pathogens, by means of the indirect immunoperoxidase technique, in histopathologic specimens routinely embedded in paraffin. Infectious agents, such as bacteria, fungi, viruses, protozoa, and helminthes, were demonstrable with reliable sensitivity and limited specificity. Endogenous human immunoglobulins in paraffin sections were scarcely detected by the peroxidase-labeled secondary antibody. The method is simple, economic, useful, and beautiful for the histopathologic diagnosis of infectious diseases.

Use of a live vaccine to modulate Cryptobia salmositica infections in Salmo salar.

The vaccine strain of Cryptobia salmositica multiplies in Atlantic salmon Salmo salar and it can modulate the severity of the disease in Cryptobia-infected individuals. Fish injected with the vaccine 3 d post-infection with C. salmositica had lower peak parasitaemias and higher antibody titres than infected fish given the vaccine 7 d post-infection or those infected fish that were not given the vaccine.

Highly Multiplex Real-Time PCR-Based Screening for Blood-Borne Pathogens on an OpenArray Platform.

Molecular diagnostics are increasingly used in the blood bank industry. A device that can combine simultaneous detection of multiple targets with the flexibility of inclusion of emerging pathogens is desirable for testing blood products. A highly multiplexed blood-borne pathogen panel (BBPP) using dual-label probe chemistry (TaqMan assays) was developed for simultaneous detection and discrimination of 17 viral pathogens in human plasma samples and 13 bacterial and protozoan pathogens in human blood samples on the OpenArray platform. The custom BBPP OpenArray plate was tested for specificity and analytical sensitivity with purified nucleic acids from each pathogen and with pathogen-spiked human blood and plasma samples. The results of analytical validation of known samples yielded decision trees for identification of coded samples: pathogens spiked in human plasma or whole blood. Results from coded samples demonstrated no false positives among the plasma or whole blood specimens. Samples not detected were at the lower limit of the detectible range or qualified for retesting as indeterminate. Further demonstration of the performance of the BBPP OpenArray was achieved with clinical samples from a blood donor testing organization. Ninety-five percent of virus-positive samples were correctly identified. These results show that a high-throughput OpenArray PCR platform can be expanded and adapted for higher discrimination and newly emerging agents, enabling consideration for development as a next-generation device for testing blood products.

Testosterone and Haemosporidian Parasites Along a Tropical Elevational Gradient in Rufous-Collared Sparrows (Zonotrichia capensis).

Elevation has been proposed as a dominant ecological variable shaping life history traits and subsequently their underlying hormonal mechanisms. In an earlier meta-analysis of tropical birds, elevation was positively related to testosterone levels. Furthermore, parasitism by avian haemosporidians should vary with elevation as environmental conditions affect vector abundance, and while testosterone is needed for breeding, it is hypothesized to be immunosuppressive and thus could exacerbate haemosporidian infection. Our objective in this study was to examine the relationships between elevation, testosterone levels, and parasitism by avian haemosporidians. We surveyed breeding male rufous-collared sparrows (Zonotrichia capensis) across a wide elevational range along the equator. We measured baseline testosterone levels, haemosporidian infection at four elevations spanning the species' natural range in the Ecuadorian Andes (600, 1500, 2100, 3300 m). Testosterone levels from breeding males were not related to elevation, but there was high intrapopulation variability. Testosterone levels were not related to the probability of parasitism, but our results from one population suggested that the likelihood of being infected by haemosporidian parasites was greater when in breeding condition. In conclusion, even though there is variation in life history strategies among the studied populations, wider divergence in seasonality and life history traits would probably be needed to detect an effect of elevation on testosterone if one exists. Additionally, our results show that variation in testosterone is not related to infection risk of haemosporidians, thus other factors that take a toll on energetic resources, such as reproduction, should be looked at more closely.

Calibration-free concentration analysis of protein biomarkers in human serum using surface plasmon resonance.

In complex biological samples such as serum, determination of specific and active concentration of target proteins, independent of a calibration curve, will be valuable in many applications. Calibration-free concentration analysis (CFCA) is a surface plasmon resonance (SPR)-based label-free approach, which calculates active concentration of proteins using their known diffusion coefficient and observed changes in binding rates at different flow rates under diffusion-limited conditions. Here, for the first time we demonstrate the application of CFCA for determining protein biomarker abundance, specifically serum amyloid A (SAA), directly in the serum samples of patients suffering from different infectious and non-infectious diseases. The assay involves preparation of appropriate reaction surfaces by immobilizing antibodies on CM5 chips via amine coupling followed by serum sample preparation and injection over activated and reference surfaces at flow-rates of 5 and 100 µL/min. The system was validated in healthy and diseased (infectious and non-infectious) serum samples by quantifying two different proteins: ß2-microglobulin (ß2M) and SAA. All concentration assays were performed for nearly 100 serum samples, which showed reliable quantification in unattended runs with high accuracy and sensitivity. The method could detect the serum ß2M to as low as 13 ng/mL in 1000-fold serum dilution, indicating the possible utility of this approach to detect low abundance protein biomarkers in body fluids. Applying the CFCA approach, significant difference in serum abundance of SAA was identified in diseased subjects as compared to the healthy controls, which correlated well with our previous proteomic investigations. Estimation of SAA concentration for a subset of healthy and diseased sera was also performed using ELISA, and the trend was observed to be similar in both SPR assay and ELISA. The reproducibility of CFCA in various serum samples made the interpretation of assay simple and reliable. This study illustrates a significant step forward in rapid monitoring of several protein markers in serum samples, with utility in biomarker validation and other therapeutic applications.

The Gametocytes of Leucocytozoon sabrazesi Infect Chicken Thrombocytes, Not Other Blood Cells.

Leucocytozoon parasites infect a large number of avian hosts, including domestic chicken, and cause significant economical loss to the poultry industry. Although the transmission stages of the parasites were observed in avian blood cells more than a century ago, the specific host cell type(s) that the gametocytes infect remain uncertain. Because all the avian blood cells, including red blood cells (RBCs), are nucleated, and the developing parasites dramatically change the morphology of the infected host cells, it has been difficult to identify Leucocytozoon infected host cell(s). Here we use cell-type specific antibodies to investigate the identities of the host cells infected by Leucocytozoon sabrazesi gametocytes. Anti-RBC antibodies stained RBCs membrane strongly, but not the parasite-infected cells, ruling out the possibility of RBCs being the infected host cells. Antibodies recognizing various leukocytes including heterophils, monocytes, lymphocytes, and macrophages did not stain the infected cells either. Antisera raised against a peptide of the parasite cytochrome B (CYTB) stained parasite-infected cells and some leukocytes, particularly cells with a single round nucleus as well as clear/pale cytoplasm suggestive of thrombocytes. Finally, a monoclonal antibody known to specifically bind chicken thrombocytes also stained the infected cells, confirming that L. sabrazesi gametocytes develop within chicken thrombocytes. The identification of L. sabrazesi infected host cell solves a long unresolved puzzle and provides important information for studying parasite invasion of host cells and for developing reagents to interrupt parasite transmission.

Coagulation abnormalities in 5 cats with naturally occurring cytauxzoonosis.

OBJECTIVE: To characterize hemostasis and determine if disseminated intravascular coagulation (DIC) is present in cats with cytauxzoonosis. DESIGN: Cross-sectional study. SETTING: University teaching hospital. ANIMALS: Five client-owned cats with cytologic and PCR-confirmed cytauxzoonosis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Admission samples were collected for hemostasis testing including platelet count, activated partial thromboplastin time, prothrombin time, fibrinogen, antithrombin (AT), d-dimer, protein C, plasminogen, antiplasmin, factors VII, VIII, IX, X, and XI, von Willebrand factor, and thromboelastography. Results were compiled for combined criteria used to define DIC, and all 5 cats satisfied criteria using a previously described modified scoring system for DIC in cats. The abnormalities found in all 5 cats included thrombocytopenia, low protein C activity, and prolonged prothrombin time; however, none of the cats had low AT activity. None of the cats had clinical signs of hemorrhage despite thrombocytopenia, coagulation factor deficiency (5/5 cats), and thromboelastographic evidence of hypocoagulability (2/5 cats). Three of 5 cats survived to hospital discharge. The nonsurvivors had disseminated cytauxzoonosis with schizont-laden macrophages in vessels of various organs. CONCLUSIONS: This is the first report that comprehensively describes the hemostastic status of cats with naturally occurring infection with Cytauxzoon felis. All 5 cats had laboratory evidence of overt DIC. Unlike human and canine models of sepsis-induced DIC, AT deficiency was not found in this series of cats. Further research is warranted to investigate therapeutic strategies targeting thrombotic DIC to improve survival in cats with cytauxzoonosis.

Immunological activation markers in the serum of African and European HIV-seropositive and seronegative individuals.

OBJECTIVE: The concentration of type 1 and type 2 cytokines and fibroblast-associated apoptosis-1 soluble receptor (sAPO-1/Fas) was analysed in the sera of Ugandan and Italian HIV-1-seropositive and seronegative individuals. The data were compared to determine whether the immunological status of these groups was different. METHODS: Sixty-seven Ugandan and 30 Italian HIV-positive patients were analysed and stratified according to CD4 counts (group 1, > 500 x 10(6)/l; group 2, 200-500 x 10(6)/l; group 3, < 200 x 10(6)/l). Sera from 15 Ugandan and 11 Italian HIV-negative blood donors were also analysed. Serum concentration of type 1 cytokines [interleukin (IL)-2, IL-12, and interferon (IFN)-gamma] and type 2 cytokines (IL-4 and IL-10), and sAPO-1/Fas were measured by enzyme-linked immunosorbent assay. RESULTS: Serum levels of IL-2, IFN-gamma and IL-10 but not of IL-4 and IL-12, were elevated in HIV-positive group 1 and 2 Africans compared with HIV-positive Italian individuals. IL-4 was mildly augmented in HIV-positive group 3 African patients. Serum concentration of sAPO-1/Fas was reduced in HIV-positive Africans compared with HIV-positive Italian individuals. Finally, serum levels of IL-2 and IL-10 were increased and sAPO-1/Fas reduced when sera of HIV-negative African healthy controls were compared with their Italian counterparts. The ratio of type 1/type 2 cytokines was roughly 1.0 in HIV-negative African controls, and much greater than 1.0 in HIV-negative Italian controls. CONCLUSIONS: These preliminary findings indicate that immune activation is present in African HIV infection. Furthermore, these data raise the possibility that abnormal immune activation and increased susceptibility to antigen-induced cell death is present even in HIV-negative African controls.

Neutrophil myeloperoxidase deficiency associated with canine hepatozoonosis.

Hepatozoonosis is a very important disease in dogs in Nigeria. Hepatozoonosis was reported in Nigeria in 18 dogs. The clinical signs included fever, anorexia, loss of weight, lameness, oculonasal discharge and conjunctivitis. Hematologic findings included leukocytosis due to neutrophilia and eosinophilia. Parasitemia varied from 1 to 9% of the circulating neutrophils in the peripheral blood smears of the dogs examined. Hepatozoon canis gametocytes were identified in circulating neutrophils of dogs. Peripheral blood smears from dogs confirmed to have natural H. canis infection were cytochemically stained for myeloperoxidase. Parasitized neutrophils were myeloperoxidase deficient while non-parasitized neutrophils were myeloperoxidase positive. This is considered important, because deficiency of the enzyme may be responsible for poor response of H. canis to chemotherapeutic agents.

Babesia canis infection in canine-red blood cell-substituted SCID mice.

We have developed a mouse model for Babesia canis infection using severe combined immunodeficiency (SCID) mice whose circulating red blood cells had been substituted with canine red blood cells. Substitution of red blood cells in SCID mice was achieved by repetitive transfusions of canine red blood cells, together with administration of an antimouse red blood cell monoclonal antibody. Following inoculation of canine-red blood cell-SCID mice with B. canis, parasites proliferated in the canine red blood cells that had been transfused into the SCID mice, resulting in much higher parasitaemia than that observed in dogs. In an attempt to demonstrate the utility of this mouse model, three antiprotozoal drugs, diminazene diaceturate, clindamycin and oxytetracycline, were examined for their efficacy to inhibit the growth of B. canis in canine-red blood cell-SCID mice. The mouse model clearly showed that diminazene diaceturate and oxytetracycline were capable of eliminating B. canis from the canine-red blood cell-SCID mice, whereas clindamycin exhibited only a static effect as parasitaemia relapsed upon cessation of drug administration.

Lack of basophilia in human parasitic infections.

While basophilia is often found in animal models of parasitic infection, it has not yet been established whether it occurs in parasite-infected humans. We investigated the relationship between basophilia and parasitic infections in humans by reviewing charts from 668 patients with confirmed parasitic infection (472 with only helminths, 146 with only protozoa, and 50 with both helminth and protozoan infections) and from 50 patients without parasitic infections. Basophilia (> 290 cells/mm3 ) occurred in only four of the 668 parasite-infected patients (0.6%), and there were no statistically significant differences in the percentages of patients with basophilia or in the absolute basophil counts among either the helminth-infected, protozoa-infected, or uninfected populations. Analysis with regard to relative basophil levels revealed that basophils constituted more than 3% of the peripheral white blood cell population in only four patients. Thus, basophilia occurs only rarely in human parasitic infections and is consequently not a useful clinical marker in the evaluation of suspected parasitic disease.

[Changes in the blood pictureof the red goat of Maradi as a function of its gastrointestinal parasitism]. / De la variation de la formule sanguine de la chèvre rousse de Maradi en fonction de son parasitisme gastro-intestinal

Numerous "chèvres de Maradi" are bred in Republique du Niger (cap. Niamey), 2 - 10(+6) numbered in 1973. This rustic ruminant is often very parasitized by intestinal nematodes and sporozoa. The most frequent genera are Bunostomum (55%), Trichostrongylus (40%), Strongyloides (27%), Oesophagostomum and Haemonchus (20%), sometimes Moniezia or Stilesia, coccidiosis being endemic and very pathogenic, Eimeria (70%). Polyparasitism is a "modus vivendi" between the host and these various parasites. All modification of the number or the kind of parasites (prevalence of one or two genera) involves a variation of the differential leucocyte count (anthelmintic cure for example). When the normal leucocyte count is 18 to 22 - 10(+3) per mm3, whose neutrophils: 40.73%; acidophils: 2%; basophils: 0.28%; monocytes: 11.28%; lymphocytes (small and big forms): 45.71%, a tapeworm parasitism by adults (Moniezia or Stilesia) or by peritoneal larvae (Cysticercus sp.) involves a light eosinophilia (8%), in morbid cases of coccidiosis, neutrophilia prevails (70%), and a polyparasitism with nematodes and Eimeria is characterized by monocytosis and neutrophilia, the polynuclear eosinophils being very rare. These observations show the necessity to elaborate simultaneously two cures: the first with an anthelmintic product, the second against coccidia, to avoid an uncertain Eimeria proliferation after the nematode destruction. In African breeding conditions, where polyparasitism is very frequent, such a therapeutic schedule is recommended.

[Studies of the ecology of some blood protozoa of wild small mammals in North Germany (author's transl)]. / Untersuchungen zur Okologie einiger Blutprotozoen bei wildlebenden Kleinsäugern in Norddeutschland.

In 1977 and 1978, 696 small mammals of 9 different species were surveyed for protozoal blood parasites. The following parasites were found: Hepatozoon sylvatici in Apodemus flavicollis, H. erhardovae and Trypanosoma evotomys in Clethrionomys glareolus, T. microti and Babesia microti in Microtus agrestis and T. croicidurae in Crocidura russula. H. erhardovae showed regularly high infection-rates (more than 70%) during all the years in 1977 and 1978. H. sylvatici was found only in 5.6% of the yellow-necked mice from March to October. T. microti and T. evotomys were predominately found in the time from July to October. The high infection-rates of the bank vole with B. microti from January to March and July to August indicate a bimodal type of seasonal dynamic. Under natural conditions M. agrestis was the only host for B. microti. Nymphs of the tick Ixodes ricinus were found to be able to transmit B. microti to Syrian golden hamsters (Mesocricetus auratus) in the laboratory.

Infectious diseases manifested in the peripheral blood.

Although primary diagnosis of infectious disease is uncommonly made from morphologic examination of a blood smear in the United States, knowledge of the distinctive morphologic features of various organisms, coupled with an understanding of the clinical and epidemiologic features of various disorders, permits recognition and diagnosis of uncommonly encountered infections. Furthermore, nonspecific manifestations of infection may provide an important clue in guiding a further diagnostic work-up.