Outcome of parasitological examinations in dogs in Germany: a retrospective survey.

Dog faecal samples examined from January 2019 to December 2019 were retrospectively analysed for frequency of endoparasites. The examinations were performed with several different methods: 29,219 samples were examined by flotation method and sodium acetate-acetic acid-formalin concentration (SAFC) technique, 1,330 samples by Baermann-Wetzel migration technique, 12,221 samples using a Giardia coproantigen enzyme-linked-immunosorbent assay (ELISA), 1,180 samples using a Cryptosporidium coproantigen ELISA, 1,671 samples by polymerase chain reaction (PCR) testing for Giardia duodenalis and 447 samples by PCR testing for Cryptosporidium spp.. A total of 7.1% of the samples were positive for parasites in the microscopical examination using the flotation method and SAFC technique. The parasites found included Cystoisospora spp. (2.8%), Giardia duodenalis (2.3%), Ancylostomatidae (1.8%), Toxocara canis (1.6%), Trichuris vulpis (0.7%), Toxascaris leonina (0.5%), Capillaria spp. (0.2%), Angiostrongylus vasorum (0.2%), Crenosoma vulpis (0.1%), Taeniidae (0.1%), Sarcocystis spp. (0.03%), Dipylidium caninum (0.01%), Diphyllobothrium latum (< 0.01%), Spirurida (< 0.01%) and Opisthorchiidae (< 0.01%). Using the Baermann-Wetzel migration technique, Angiostrongylus vasorum was found in 0.75% and Crenosoma vulpis in 0.3% of the samples. ELISAs for Giardia duodenalis and Cryptosporidium spp. revealed 13.9% and 1.0% positive faecal samples, and Giardia duodenalis and Cryptosporidium spp. PCRs 19.4% and 2.0%, respectively. Dogs in the first year of life were more frequently infected with parasites than older animals. In the microscopic examination using flotation method and SAFC technique, the significantly highest detection rates were found in dogs up to six months of age (p < 0.001).

Molecular survey on vector-borne pathogens in clinically healthy stray cats in Zaragoza (Spain).

BACKGROUND: In Europe, feline vector-borne infections are gaining importance because of the changing climate, expanding habitats of potential vectors and expanding pathogen reservoirs. The main objective of this study was to assess the prevalence of vector-borne pathogens (VBPs) in stray cats in Zaragoza, Spain, and to investigate potential risk factors for infection, including feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV). METHODS: Blood samples from stray cats presented to the veterinary faculty in Zaragoza between February 2020 and 2022 were tested by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Anaplasma platys, Bartonella henselae, Ehrlichia canis, Rickettsia spp., haemotropic Mycoplasma spp., Hepatozoon spp., Leishmania infantum, piroplasms and microfilariae at the LABOKLIN laboratory. The cats were also tested for FeLV and FIV by PCR. RESULTS: Nearly half of the cats (158/332, 47.6%) were positive for at least one VBP. Hepatozoon spp. were detected in 25.6%, haemotropic Mycoplasma spp. in 22.9%, B. henselae in 9.3% and L. infantum in 2.1% of the cats. Male sex had a statistically significant association with test results for haemotropic Mycoplasma spp. (odds ratio 1.38 [1.21;1.57]); regionality with Hepatozoon spp., B. henseale and FIV; and seasonality with Hepatozoon spp., haemotropic Mycoplasma spp., L. infantum and FeLV (P ≤ 0.05 each). A strong positive correlation was reported for the amount of rainfall and the number of cats that tested positive for Hepatozoon spp. (ρ = 753, P = 0.05). None of the cats tested positive for A. phagocytophilum, A. platys, E. canis, Rickettsia spp., piroplasms, or microfilariae. Co-infections with multiple VBPs were detected in 56 out of 332 cats (16.9%). Thirty-one of the 332 cats included in the study (9.3%) tested positive for FeLV (6.9%) and for FIV (3.6%). In 20/31 cats (64.5%) that tested positive for FeLV/FIV, coinfections with VBP were detected (P = 0.048, OR 2.15 [0.99; 4.64]). CONCLUSIONS: VBPs were frequently detected in stray cats in Zaragoza. In particular, regionality and seasonality had a statistically significant association with PCR results for most VBPs included in the study.

Feline leishmaniosis with focus on ocular manifestation: a case report.

BACKGROUND: In Europe, feline leishmaniosis is commonly caused by Leishmania infantum. There is little knowledge regarding pathogenesis, ocular manifestations and long-term follow-ups in cats with leishmaniosis. FINDINGS: A 6-year-old female, spayed European Shorthair cat was imported from Spain to Germany 2 years prior to its first clinical presentation. The cat showed lethargy, weight loss, ulcerative lesions on the front limbs and high-grade chronic uveitis. The diagnosis of L. infantum infection was based on the cytological finding of amastigotes in skin lesions, positive qPCR of EDTA-blood and positive PCR of a cyto-brush sample from the conjunctiva. Supportive findings included positive serology by IFAT, serum protein capillary electrophoresis with peaks in alpha2- and gamma-globulin sections and marked elevation of SAA. Enucleation had to be performed on day 288 on both eyes because of blindness, glaucoma and high-grade uveitis. Histologically, high numbers of Leishmania spp. amastigotes were found in histiocytes. IFAT and PCR were positive in the aqueous humor in both eyes, respectively. Feline leukemia virus antigen and feline immunodeficiency virus antibody testings were positive. Hematological and biochemical results revealed mild leukocytosis with lymphocytosis, monocytosis and eosinopenia as well as marked elevation of SAA and hyperglobulinemia. The cat was treated with allopurinol, responded well and was still alive at follow-up on day 288 after first presentation. However, enucleation was necessary because of refractory glaucoma and uveitis.  CONCLUSION: For the first time, ocular evidence of Leishmania IgG antibodies was demonstrated in the aqueous humor of both eyes in cats. There is limited knowledge about the pathogenesis, treatment options and outcomes in cats infected with L. infantum. This case report supports the hypothesis that immunosuppression increases the risk of clinical signs of leishmaniasis in cats. Alpha2- and gamma-globulin peaks in serum protein capillary electrophoresis are supportive criteria for the diagnosis of L. infantum infection. SAA is valuable for monitoring. Regarding ophthalmology, uveitis and glaucoma may have a poor prognosis.

[Feline uroliths: Analysis of frequency and epidemiology in Germany (2016-2020)]. / Harnsteinbefunde bei Katzen aus Deutschland in den Jahren 2016­2020.

OBJECTIVES: This study was aimed to assess the distribution and frequency of uroliths in cats as well as to evaluate gender-, age-, and breed-specific differences. MATERIAL AND METHODS: 3629 uroliths from cats in Germany were analyzed by infrared-spectroscopy during the period 2016-2020. The majority (3300) of the uroliths originated from purebred cats of 22 breeds. RESULTS: Uroliths were prevalent in the order of male neutered (45.3 %), female spayed (35.3 %), male intact (7.4 %), and female intact cats (6.3 %). Median age of the cats with uroliths was 7 years, although it varied slightly depending on types of the urolith. The most frequent uroliths were calcium oxalate (59.5 %), followed by struvite (33.7 %), ammonium urate (2 %), calcium phosphate (1.7 %), cystine (0.7 %) and xanthine (0.4 %). Males (59.4 %) and females (59.2 %) showed nearly identical prevalence of calcium oxalates, whereas it significantly diverged in castrated (60.2 %) versus intact animals (54.4 %; p = 0.01). With regard to struvites, male cats (32.6 %) were less prone than females (35.4 %; p = 0.08) while intact cats (38.2 %) significantly outnumbered the castrated cohorts (33.1 %; p = 0.02). The prevalence for calcium oxalate uroliths increased by 6.6 % during the study period, contrasting a decrease of 5.5 % for struvites. Some breeds (> 10 individuals) showed significantly higher propensities than others for urolith formation. Calcium oxalates were significantly more frequent in British shorthair cats (85.2 %), Ragdoll (75 %), Scottish fold (74.1 %) and Persians (72.4 %). Struvites were prevailing in Norwegian forest cats (48.5 %), British longhair (41.7 %), European shorthair (41.7 %) and Siberian forest cats (36.8 %). Siamese cats had the significantly highest percentage of cystine uroliths (16 %). CONCLUSIONS AND CLINICAL RELEVANCE: The occurrence of urine calculi in cats from Germany was found to be most frequent for calcium oxalate and struvite types. We also found breed-, age-, gender- specific differences in addition to variations depending on the neutering status of the animals.

Anaplasma phagocytophilum in domestic cats from Germany, Austria and Switzerland and clinical/laboratory findings in 18 PCR-positive cats (2008-2020).

OBJECTIVES: Anaplasma phagocytophilum is considered the most significant rickettsial pathogen affecting cats. The organism is transmitted by ticks of the species Ixodes ricinus in Central Europe and can cause granulocytic anaplasmosis in pets, wildlife and humans. The aims of this study were to assess the frequency of positive test results for A phagocytophilum in cats in Germany, Austria and Switzerland, as well as to evaluate clinical and laboratory findings in cats with positive PCR results. METHODS: This study included the results of direct (PCR) and indirect detection methods (immunofluorescence antibody tests [IFAT]) requested by veterinarians in Germany, Austria and Switzerland between 2008 and 2020 from the LABOKLIN laboratory (Bad Kissingen, Germany). The veterinarians treating the PCR-positive cats were contacted by telephone to enquire about their clinical signs, laboratory findings, management and outcomes. RESULTS: In total, 244/1636 cats (15%) tested positive by direct (PCR: n = 27/725 [4%]) and/or indirect detection methods (IFAT: n = 221/956 [23%]). In 18/26 cats with PCR results positive for A phagocytophilum, additional information about clinical signs, laboratory findings, treatment and outcome was obtained. Of these 18 cats, five had comorbidities independent of their infection with A phagocytophilum. The most common clinical signs in PCR-positive cats (total/without comorbidities) were lethargy (83%/92%), fever (83%/85%) and thrombocytopenia (61%/62%). Overall, more than half (57%) of the cats with and without comorbidities recovered clinically. CONCLUSIONS AND RELEVANCE: Infections with A phagocytophilum should be considered as differential diagnoses in cats with tick infestation, lethargy, fever and thrombocytopenia. The clinical signs and laboratory findings are consistent with published case reports in cats. Ectoparasite prophylaxis in cats is recommended throughout the entire year.

Molecular detection of Hepatozoon species infections in domestic cats living in Germany.

OBJECTIVES: Three species of protozoal Hepatozoon species (H felis, H canis and H silvestris) are known to infect cats in Europe. The objective of this study was to determine the prevalence of Hepatozoon species in samples from cats living in Germany that were submitted to a veterinary laboratory. METHODS: The study included cats tested for Hepatozoon species by PCR between 2007 and 2020 by the Laboklin laboratory. Travel history and haematological results were documented for cats with positive test results. From 2018 onwards, a partial 18S rRNA Hepatozoon gene fragment was sequenced from cats with positive PCR results. RESULTS: Sixty-four of 931 cats (7%) tested positive for Hepatozoon species. Sex and age did not have a statistically significant impact. Sequencing was carried out for 16 samples and revealed H felis in all cases. All cats with positive test results and a relevant travel history had been imported from the Mediterranean or south-eastern Europe. There were no autochthonous infections with Hepatozoon species. Leukocytosis, haemoconcentration and anaemia were the most common haematological abnormalities. CONCLUSIONS AND RELEVANCE: Although infections with Hepatozoon species in cats are usually subclinical, it may be useful to screen cats imported from the Mediterranean and south-eastern Europe for these pathogens to prevent local transmission cycles. There was no evidence of autochthonous infections in Germany; however, further investigations regarding a possible transmission of Hepatozoon species from infected cats to blood-feeding arthropods in Germany may be of interest. To avoid potential spread of the pathogens, ectoparasite prophylaxis is advisable.

Signalment and Clinical Data of Cats with Exocrine Pancreatic Insufficiency Diagnosed Using Feline Trypsin-like Immunoreactivity in Routine Diagnostics.

Serum feline trypsin-like immunoreactivity (fTLI) is commonly used to diagnose feline exocrine pancreatic insufficiency (EPI). This study aimed to describe signalment and clinical data of cats with EPI. Determination of TLI was performed using an in-house ELISA; the reference interval was defined using a Reference Limit Estimator. Groups were formed from 4813 cats (2019-2020), based on their fTLI concentration: 1 (<8 µg/L; decreased; n = 275), 2 (8-88 µg/L; reference interval; n = 4256), and 3 (>88 µg/L; increased; n = 282). Males and Domestic Shorthairs were most common in all groups. Group 3 had the highest (13 years), and group 1 had the lowest (9 years), median age. Clinical information was available for 200 cats (decreased fTLI: n = 87, lower reference interval (8-12 µg/L): n = 113). Treatment response was observed in 83% (decreased fTLI) and 66% (lower reference interval). EPI cats displayed weight loss (69%), diarrhoea (68%), vomiting (41%), anorexia (39%), poor hair coat (35%), lethargy (33%), and/or polyphagia (21%). The lower the serum fTLI concentration, the more often good treatment response was reported (p = 0.022) but there were no statistically significant clinical signs. In conclusion, fTLI is a helpful parameter to diagnose EPI but predicting treatment response based on signalment or clinical signs is not possible.

DETECTION OF TESTADENOVIRUSES AND ATADENOVIRUSES IN TORTOISES AND TURTLES IN EUROPE.

Adenoviruses have been regularly detected in squamate reptiles; evidence of infection in chelonians is described much less frequently. The adenoviruses found in turtles and tortoises have been genetically diverse, and have included members of the genus Siadenovirus, a proposed testadenovirus genus, and, in a single case, an Atadenovirus. In this study, samples from 949 chelonians submitted to a diagnostic laboratory were screened for the presence of adenoviruses by polymerase chain reaction (PCR) targeting a portion of the DNA polymerase gene. Adenoviruses were detected in 22 (2.3%) chelonians of different species. Adenovirus-positive species included Hermann's tortoises (Testudo hermanni), spur-thighed tortoises (T. graeca), Horsfield's tortoises (T. horsfieldii), sliders (Trachemys spp.), box turtles (Terrapene spp.) and a black pond turtle (Geochlemys hamiltonii). Sequencing and phylogenetic analyses of the obtained PCR products revealed that the majority of the detected adenoviruses (72.7%) cluster with members of the proposed testadenovirus genus, while the rest (27.3%) cluster with the atadenoviruses. This study significantly expands the known host range of both the proposed testadenoviruses and the atadenoviruses in different chelonian species and families.

[Occurrence of uroliths - age-, breed-, and gender-specific differences in dogs from Germany]. / Auftreten von Harnsteinen ­ alters-, rasse- und geschlechtsspezifische Unterschiede bei Hunden aus Deutschland.

OBJECTIVES: The aim of this study was to assess the distribution and frequency of uroliths in dogs in Germany and to evaluate age-, gender-, and breed-specific differences. MATERIAL AND METHODS: A total of 2772 uroliths from dogs in Germany that had been submitted from veterinary practices during the years 2017-2019 were analyzed by infrared spectroscopy. RESULTS: Uroliths were analyzed as follows: Struvite (44.7 %), calcium oxalate monohydrate (whewellite 24.5 %), cystine (15.2 %), ammonium urate (5.3 %), calcium oxalate dihydrate (weddellite 4.4 %), calcium phosphate (1.6 %), xanthine (1.2 %), whewellite/weddellite (11 %), brushite (0.4 %) and other substances (1.7 %). The dogs' median age (in years) with regards to the urolith type amounted to: struvite 8, calcium oxalate (whewellite and weddellite) 10, cystine 5, brushite 11, xanthine 6.5, and ammonium urate 6 years. Struvite uroliths were most common in intact females (83.8 %) and neutered females (80.8 %), while cystine uroliths (36.7 %) and calcium oxalates (35.7 % whewellite and weddellite) were most common in intact males. Calcium oxalate (50 %), struvite (17.3 %), and cystine uroliths (10.5 %) were found in male neutered dogs. The most frequent breeds with struvite uroliths were Pug (75.9 %), Labrador (73.1 %), Shih Tzu (62.2 %), Dachshund (50.4 %) and Maltese (36 %). Calcium oxalates were most frequently encountered in the breeds Miniature Schnauzer (54.8 %), Jack Russell Terrier (49.5 %), and Yorkshire Terrier (48.2 %). Cystine uroliths were most frequent in the breeds French Bulldog (47.2 %), Bulldogs (44.8 %), Chihuahua (44.1 %) and Dachshund (32.8 %). CONCLUSION AND CLINICAL RELEVANCE: Dogs from Germany displayed age-, gender- and breed-specific differences in the frequency of urine calculi occurrence. Knowledge of the frequent occurrence of the distinct uroliths in Germany as well as potential breed dispositions allow the veterinarian to implement an appropriate preventive treatment plan. This would involve a targeted monitoring program for the prevention and follow-up care of the patient in conjunction with the pet owner.

Increase in cognitive function is seen in many single-operated pediatric patients after epilepsy surgery.

PURPOSE: The recurrent seizures of pediatric drug-resistant epilepsy (DRE) are known to impair brain development and can lead to a loss in cognitive functioning. Surgery is increasingly being used to treat children with DRE. This study investigates the pre- and postoperative cognitive function in a pediatric epilepsy surgery cohort as well as predictive determinants of change in intelligence quotient (IQ) following surgery. METHODS: A consecutive series of 91 Danish children who underwent focal resective epilepsy surgery between January 1996 and December 2016 were included. All underwent preoperative cognitive evaluation and were reevaluated at 1-year and/or 2-year follow-up. Single-operated and multi-operated patients were examined separately. RESULTS: 79 of 91 patients were single-operated. Single-operated patients received less anti-epileptic drugs (AED) and experienced a decrease in seizure frequency postoperatively, p < 0.001. IQ increased postoperatively (IQ change ± standard deviation: 3.3 ± 14.0), p < 0.05. High preoperative seizure frequency was a significant predictor for decreased IQ, p < 0.01. Multi-operated patients did not experience a reduction in AED treatment. Surgery and continued AED treatment did, however, result in significantly better seizure control, p < 0.01. IQ remained unchanged in multi-operated patients. CONCLUSION: Epilepsy surgery allowed for IQ gains in single-operated patients. Preoperative seizure frequency was a significant predictor of IQ change following surgery. Interactions between other, not included, possible predictors remain to be examined. Single-operated patients had the best cognitive outcome. The inclusion of a non-surgical control group is needed to assess the extent of the beneficial effects of surgery on cognitive ability.

A methodological perspective on the cognitive outcome of epilepsy surgery in children and adolescents.

PURPOSE: The purpose of the study was to explore the impact of timing and test specificity of cognitive outcome measures after pediatric epilepsy surgery. METHODS: A consecutive national cohort of 114 children with medically resistant epilepsy having had resective epilepsy surgery were screened for children tested with a complete age-appropriate Wechsler Intelligence test at two or three time-points. This provided 43 children for analyses. Composite subscale scores were assessed in comparison to index and intelligence quotient (IQ) scores. RESULTS: We found a main effect of time in seizure-free children for full-scale IQ (FSIQ); F(2, 42) = 6.49 with higher T2 measures compared with T1 (MDiff = 5.46, p = .006). There was a difference in FSIQ scores between seizure-free and nonseizure-free children at T2; M = 7.31, 95% confidence interval (CI) [0.05 to 14.57], t(38) = 2.04, p = .049, favoring seizure-free children. A statistical difference between composite scale scores and index scores was found with medium to large effect. The correlation of medical treatment (anti-epileptic drug (AED)) change and score differences in FSIQ outcome was significant (p = .041), with less AED correlated with a higher FSIQ. All children with left-temporal surgery had a stable or improved verbal comprehension composite subscale score outcome at T2 regardless of seizure status. CONCLUSION: Our results correspond to some longitudinal studies with outcome measures >2 years, in contrast to short-term studies ≤2 years with a stable outcome. Our study supports the fact that the specificity of the used tests and the timing of assessments after pediatric epilepsy surgery are essential factors for the clinical validity of outcome measures. However, there are further needs of extensive longitudinal studies to provide a better understanding of life-long cognitive development and impact after childhood epilepsy surgery.

CASPR2 autoimmunity in children expanding to mild encephalopathy with hypertension.

OBJECTIVE: To delineate autoimmune disease in association with contactin-associated protein 2 (CASPR2) antibodies in childhood, we reviewed the clinical phenotype of children with CASPR2 antibodies. METHODS: Retrospective assessment of patients recruited through laboratories specialized in autoimmune CNS disease. RESULTS: Ten children with serum CASPR2 antibodies were identified (age at manifestation 18 months to 17 years). Eight children with CASPR2 antibody titers from ≥1:160 to 1:5,120 had complex autoimmune diseases with an age-dependent clinical phenotype. Two children with structural epilepsy due to CNS malformations harbored nonspecific low-titer CASPR2 antibodies (serum titers 1:80). The clinical symptoms of the 8 children with high-titer CASPR2 antibodies were general weakness (8/8), sleep dysregulation (8/8), dysautonomia (8/8) encephalopathy (7/8), neuropathic pain (7/8), neuromyotonia (3/8), and flaccid paresis (3/8). Adolescents (3/8) showed pain, neuromyotonia, and encephalopathy, whereas younger children (5/8) displayed severe hypertension, encephalopathy, and hormonal dysfunction mimicking a systemic disease. No tumors were identified. Motor symptoms remitted with immunotherapy. Mild behavioral changes persisted in 1 child, and autism spectrum disorder was diagnosed during follow-up in a young boy. CONCLUSION: High-titer CASPR2 antibodies are associated with Morvan syndrome in children as young as 2 years. However, CASPR2 autoimmunity mimics systemic disease and hypertensive encephalopathy in children younger than 7 years. The outcome following immunotherapy was mostly favorable; long-term behavioral impairment may occur in younger children.

The immune microenvironment in typical carcinoid lung tumour, a brief report of four cases.

Pulmonary typical carcinoid (TC) is a low-grade, rare lung cancer of neuroendocrine origin. Currently, there is very little information available about the immune cell composition in TC tumours. Here, we analysed by flow cytometry resected tumours from four never-smoker female patients with TC. Twelve distinct immune cell types were identified in TC tumours. The most abundant immune cells were CD8+ T cells, CD4+ T cells, B cells and macrophages, which represented 19.8%, 17.7%, 11.5% and 11% of all tumour-infiltrating CD45+ leucocytes, respectively. Natural killer (NK) cells (8.8%) and neutrophils (3.9%) were also common. Three types of dendritic cells (DCs) were identified (plasmacytoid DCs, CD1c  DCs, and CD141  DCs) which together constituted 1.4% of all immune cells in TC tumours. Small populations of basophils (1.2%), mast cells (0.8%) and eosinophils (0.6%) were also present. Notably, the percentage of leucocytes (of all living cells) was much lower in TC tumours compared to high-grade non-small cell lung cancer (NSCLC) tumours and also compared to non-cancerous lung tissue. We conclude that TC tumours are relatively non-inflammatory, although the immune landscape was found to be very complex.

Histopathological findings and canine pancreatic lipase immunoreactivity in normal dogs and dogs with inflammatory and neoplastic diseases of the pancreas.

BACKGROUND: Diagnosis of pancreatic diseases in dogs is still challenging because of variable clinical signs, which do not always correspond with clinical pathology and histopathological findings. OBJECTIVES: To characterize inflammatory and neoplastic pancreatic diseases of dogs and to correlate these findings with clinical findings and canine pancreatic lipase immunoreactivity (cPLI) results. ANIMALS: Tissue specimens and corresponding blood samples from 72 dogs submitted for routine diagnostic testing. METHODS: Four groups were defined histologically: (1) normal pancreas (n = 40), (2) mild pancreatitis (n = 8), (3) moderate or severe pancreatitis (acute, n = 11; chronic, n = 1), and (4) pancreatic neoplasms (n = 12). An in-house cPLI ELISA (<180 µg/L, normal; >310 µg/L, pancreatitis) was performed. RESULTS: In dogs with normal pancreas, 92.5% of serum cPLI results were within the reference range and significantly lower than in dogs with mild acute pancreatitis, moderate or severe acute pancreatitis and pancreatic tumors. In dogs with moderate or severe acute pancreatitis, cPLI sensitivity was 90.9% (95% confidence interval [CI], 58.7%-99.8%). Most dogs (9/12) with pancreatic tumors (group 4) had additional pancreatic inflammation and cPLI results were increased in 10 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: High cPLI indicates serious acute pancreatitis but underlying pancreatic neoplasms should also be taken into consideration. This study confirms the relevance of histopathology in the diagnostic evaluation of pancreatic diseases.

Bronchoalveolar lavage cytokines are of minor value to diagnose complications following lung transplantation.

Early diagnosis and treatment of acute cellular rejection (ACR) may improve long-term outcome for lung transplant recipients (LTRs). Cytokines have become valuable diagnostic tools in many medical fields. The role of bronchoalveolar lavage (BAL) cytokines is of unknown value to diagnose ACR and distinguish rejection from infection. We hypothesized that distinct cytokine patterns obtained by surveillance bronchoscopies during the first year after transplantation are associated with ACR and microbiologic findings. We retrospectively analyzed data from 319 patients undergoing lung transplantation at University Hospital Zurich from 1998 to 2016. We compared levels of IL-6, IL-8, IFN-γ and TNF-α in 747 BAL samples with transbronchial biopsies (TBB) and microbiologic results from surveillance bronchoscopies. We aimed to define reference values that would allow distinction between four specific groups "ACR", "infection", "combined ACR and infection" and "no pathologic process". No definitive pattern was identified. Given the overlap between groups, these four cytokines are not suitable diagnostic markers for ACR or infection after lung transplantation.

Diagnostic methods for the measurement of human TNF-alpha in clinical laboratory.

Measurement of Tumour Necrosis Factor alpha (TNF-α) in peripheral blood is a useful tool to assess inflammatory responses in a large range of diseases. One of the major challenges for cytokine analysis is the availability of a proper analytical tool with high specificity, accuracy, linearity, precision, stability, and analytical sensitivity. Although available immunoassays are usually robust and reproducible, it is also true that they are not interchangeable. Two ELISA, four flow cytometric bead array (CBA) and four Luminex immunoassays were compared. Correlation between different techniques was almost absent, while some immunoassays based on the same technique showed significant correlation. Among the ten different assays evaluated, just few of them complied with the pre-established acceptance validation criteria. Interestingly, sera and plasma collected from the same healthy donor had significant different reference values. Samples stability was maintained in serum up to one week at four degrees, while plasma was stable only when it was frozen. Since several anti-inflammatory treatments are based on biologics targeting TNF-α (anti-TNF-α antibodies), potential interference with the immunoassays was tested and resulted relevant. This study shows that although each immunoassay presents benefits and drawbacks, just few assays are suitable for the measurement of TNF-α in clinical laboratories, demonstrating that, so far, the measurement of TNF-α in human blood is still not yet harmonised. In addition, we found that false negative results caused by anti-TNF-α treatments should be carefully considered for results interpretation.

Impact of CYP2D6 Polymorphisms on Tamoxifen Treatment in Patients With Retroperitoneal Fibrosis: A First Step Towards Tailored Therapy?

OBJECTIVE: To investigate the influence of CYP2D6 polymorphisms on outcomes and health-related quality of life of patients with retroperitoneal fibrosis (RPF) receiving tamoxifen (TMX). TMX is an effective alternative to corticosteroids for patients with RPF. Conversion of TMX to more potent endoxifen is dependent on enzyme activity of CYP2D6. MATERIALS AND METHODS: CYP2D6 genotyping and phenotype prediction of all patients treated with TMX between 02/2007 and 01/2018 was assessed using multiplex polymerase chain reaction (PCR). Groups were classified by phenotype: extensive (EM) vs poor and intermediate (PM + IM) vs ultrarapid metabolizer (UM). Retrospective evaluation of outcome (including magnetic resonance imaging and positron emission tomography-computed tomography) and health-related quality of life using the SF-36 was performed. RESULTS: A total of 63/194 patients received TMX, 40/63 with complete follow-up were sequenced: Twenty-nine patients with EM phenotype, 8 PM + IM and 3 UM. The median therapy duration was 364.5 days with a mean follow-up of 62.9 months. Seven therapy terminations occurred due to lack of response (17.5%), including all UM patients (P <.001). Magnetic resonance imagings showed a regression of fibrosis for EM and PM + IM in 69% and 62.5% of cases and a progression for UM in 100% (P = .004). In positron emission tomography-computed tomography, glucose utilization of RPF decreased significantly for EM and PM + IM. The physical sum-score of SF-36 improved for EM and PM + IM and decreased for UM (P <.05). The removal of DJ-stents was successful for EM, PM + IM, and UM in 48.3%, 75%, and 0% of cases (P = .0581). CONCLUSION: Contrary to expectations, UM showed the lowest success rate, which concludes that genotyping of RPF-patients may be useful in the sense of a tailored-therapy.

Clonality testing in the lymph nodes from dogs with lymphadenomegaly due to Leishmania infantum infection.

INTRODUCTION: In southern European countries, multicentric lymphoma and leishmaniosis are the main differential diagnoses in dogs presented with generalized lymphadenomegaly. The cytological examination is in some cases inconclusive and polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR) has become a common method to confirm or rule out a lymphoproliferative neoplasia. According to the literature, leishmaniosis may lead to clonal arrangements and therefore to a false diagnosis of lymphoma, but this assumption is made from a single leishmania infected dog. Therefore, the objective of this study was to prospectively evaluate results from PARR in dogs with lymphadenomegaly due to clinical leishmaniosis at the moment of diagnosis. MATERIALS AND METHODS: 31 dogs with a diagnosis of leishmaniosis based on the LeishVet guidelines were included in the study. Samples from enlarged lymph nodes were taken for cytological examination, clonality testing and Leishmania infantum PCR. RESULTS: All 31 dogs had medium to high positive antibody titers against Leishmania spp. and 30/31 had a positive Leishmania PCR from the lymph node. A polyclonal arrangement for B cells (immunoglobulin heavy chain gene) and T cells (T-cell receptor gamma chain gene) antigen receptors was found in 28/31 dogs. Two out of 31 dogs showed a monoclonal arrangement for Ig with high (1:2) and low (1:7) polyclonal background respectively; and one of the 31 dogs showed a monoclonal arrangement for T cell receptor with low (1:3) polyclonal background. CONCLUSION: Infections with Leishmania infantum resulted in clonal rearrangement, and therefore in a possible false diagnosis of lymphoma, in 3 out of 31 dogs (9.7%). Although, PARR is a useful method to differentiate lymphoma from reactive lymphoid hyperplasia in dogs with leishmaniosis, mono-/biclonal results should be interpreted carefully, especially in the presence of any degree of polyclonal background, and together with other clinicopathological findings.

Giardia duodenalis in small animals and their owners in Germany: A pilot study.

Giardia duodenalis is a relevant gastrointestinal protozoan pathogen of humans and animals. This species complex consists of eight genetically different assemblages. Assemblages A and B are pathogenic to humans and pets, thus confer zoonotic potential. The risk of zoonotic transmission has been controversially discussed. The aim of this monocentric cross-sectional pilot study was to investigate G. duodenalis assemblages in humans and pets living in common households in Berlin/Brandenburg (Germany). Samples from dogs, cats and humans sharing the same households were screened for Giardia infection by antigen-detecting assays. All human samples were additionally analysed by a Giardia-specific qPCR. Cyst quantification and sequences of different gene loci (triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), ß-giardin (bg) and for dogs SSUrDNA) were analysed. A total of 38 households (31 households with dogs and seven with cats) with 69 human individuals participated in the study. Initial antigen-detecting assays revealed Giardia-positive results for 13 (39%) canine, one (14%) feline and one human sample. Reanalysis of the human samples by qPCR revealed two more positive specimens (4%). Two of these three samples were identified as assemblage B at all tested loci. Success rate of assemblage typing for pet samples was generally low and comprised mainly the SSUrDNA locus only. Overall, six of 13 Giardia-positive canine samples were typable (2× A, 1× co-infection: A and B, 1× C; 2× D). One pair of samples (dog and human) from the same household had a similar but not identical assemblage B sequence at tpi locus. Assemblage A was also detected in the dog specimen, which hampered sequence analysis. In conclusion, although exhibiting limitations due to the sample size, our study highlights the need for better and standardized typing tools to distinguish G. duodenalis strains with higher resolution in order to perform proper case-control studies for a realistic estimation of zoonotic risk.

Treating osteosarcoma with CAR T cells.

Novel therapies to treat patients with solid cancers that have developed resistance to chemotherapy represent unmet needs of considerable dimensions. In the present review, we will address the attempts to develop chimeric antigen receptor (CAR) targeted immunotherapy against osteosarcoma (OS). This aggressive cancer displays its peak incidence in children and young adults. The main cause of patient death is lung metastases with a 5-year survival as low as 5%-10% in the primary metastatic setting and 30% in the relapse situation, respectively. Effective adjuvant combination chemotherapy introduced more than 40 years ago improved the survival rates from below 20% to around 60% in patients; however, since then, no major breakthroughs have been made. The use of immune checkpoint inhibitors has been disappointing in OS, while other types of immunotherapies such as CAR T cells remain largely unexplored. Indeed, for CAR T-cell therapy to be efficacious, two main criteria need to be fulfilled: (a) CAR T cells should target an epitope selectively expressed on the cell surface of OS in order to prevent toxicities in normal tissues and (b) the target should also be widely expressed on OS metastases. These challenges have already been undertaken in OS and illustrate the difficulties in developing tomorrow's CAR-T treatment in a solid tumour. We will discuss the experiences with CAR-T therapy development and efficacy to combat the clinical challenges in OS.