Establishment of a specimen panel for the decentralised technical evaluation of the sensitivity of 31 rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021.

IntroductionThe detection of SARS-CoV-2 with rapid diagnostic tests (RDT) has become an important tool to identify infected people and break infection chains. These RDT are usually based on antigen detection in a lateral flow approach.AimWe aimed to establish a comprehensive specimen panel for the decentralised technical evaluation of SARS-CoV-2 antigen rapid diagnostic tests.MethodsWhile for PCR diagnostics the validation of a PCR assay is well established, there is no common validation strategy for antigen tests, including RDT. In this proof-of-principle study we present the establishment of a panel of 50 pooled clinical specimens that cover a SARS-CoV-2 concentration range from 1.1 × 109 to 420 genome copies per mL of specimen. The panel was used to evaluate 31 RDT in up to six laboratories.ResultsOur results show that there is considerable variation in the detection limits and the clinical sensitivity of different RDT. We show that the best RDT can be applied to reliably identify infectious individuals who present with SARS-CoV-2 loads down to 106 genome copies per mL of specimen. For the identification of infected individuals with SARS-CoV-2 loads corresponding to less than 106 genome copies per mL, only three RDT showed a clinical sensitivity of more than 60%.ConclusionsSensitive RDT can be applied to identify infectious individuals with high viral loads but not to identify all infected individuals.

Detection of Enterobius vermicularis in greater Berlin, 2007-2017: seasonality and increased frequency of detection.

The pinworm (Enterobius vermicularis) causes mostly mild infections characterised by nocturnal anal pruritus, mainly in children. Still, the infection is stigmatising and sleep disturbances may lead to lack of concentration. For Germany, no epidemiological data are available. Laboratory data of all patients for whom detection of E. vermicularis by cellulose tape test had been requested between 2007 and November 2017 were analysed retrospectively. E. vermicularis was detected in 971/5578 (17.4%) samples collected from 3991 patients. The detection rate increased significantly within the period of investigation. It was higher in male than in female patients (20.0 vs. 15.4%). Children 4 to 10 years old and, if also examined, their relatives were most frequently affected. Control investigations at an interval of at least 1 month, which could indicate insufficient therapy or re-infection, were performed in 90/714 patients (12.6%). While parasite detection in children < 6 years was evenly distributed throughout the year, in older patients, it peaked between October and December. In conclusion, in the area of investigation, the frequency of E. vermicularis is higher in males than in females and is subject to a hitherto undescribed seasonality. The causes of the increased frequency of parasite detection warrant further investigations.

No evidence of carbapenemase-producing Enterobacteriaceae in stool samples of 1,544 asylum seekers arriving in Rhineland-Palatinate, Germany, April 2016 to March, 2017.

INTRODUCTION: Since 2015, increased migration from Asia and Africa to Europe has raised public health concerns about potential importation of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE), specifically those producing carbapenemases (C-PE), into European hospitals. AIMS: To inform infection control practices about ESBL-PE prevalence in asylum seekers and to investigate whether C-PE prevalence exceeds that in the German population. METHODS: Cross-sectional study from April 2016-March 2017. Routinely collected stool samples from asylum seekers were tested for antibiotic resistant Enterobacteriaceae. Country/region of origin and demographic characteristics were explored as risk factors for faecal colonisation. RESULTS: Of 1,544 individuals, 294 tested positive for ESBL-PE colonisation (19.0%; 95% confidence intervals (CI): 17.0-21.0). Asylum seekers originating from Afghanistan/Pakistan/Iran had a prevalence of 29.3% (95% CI: 25.6-33.2), from Syria 20.4% (95% CI: 16.1-25.2) and from Eritrea/Somalia 11.9% (95% CI: 8.7-15.7). CTX-M-15 (79%) and CTX-M-27 (10%) were the most common ESBL determinants. Highest ESBL-PE prevalences were observed in boys under 10 years and women aged 20-39 years (interaction: p = 0.03). No individuals tested positive for C-PE. Faecal C-PE colonisation prevalence in asylum seekers was not statistically significantly different from prevalence reported in German communities. CONCLUSION: In absence of other risk factors, being a newly arrived asylum seeker from a region with increased faecal ESBL-PE colonisation prevalence is not an indicator for C-PE colonisation and thus not a reason for pre-emptive screening and isolation upon hospital admission.

Mucosal Inducible NO Synthase-Producing IgA+ Plasma Cells in Helicobacter pylori-Infected Patients.

The mucosal immune system is relevant for homeostasis, immunity, and also pathological conditions in the gastrointestinal tract. Inducible NO synthase (iNOS)-dependent production of NO is one of the factors linked to both antimicrobial immunity and pathological conditions. Upregulation of iNOS has been observed in human Helicobacter pylori infection, but the cellular sources of iNOS are ill defined. Key differences in regulation of iNOS expression impair the translation from mouse models to human medicine. To characterize mucosal iNOS-producing leukocytes, biopsy specimens from H. pylori-infected patients, controls, and participants of a vaccination trial were analyzed by immunohistochemistry, along with flow cytometric analyses of lymphocytes for iNOS expression and activity. We newly identified mucosal IgA-producing plasma cells (PCs) as one major iNOS(+) cell population in H. pylori-infected patients and confirmed intracellular NO production. Because we did not detect iNOS(+) PCs in three distinct infectious diseases, this is not a general feature of mucosal PCs under conditions of infection. Furthermore, numbers of mucosal iNOS(+) PCs were elevated in individuals who had cleared experimental H. pylori infection compared with those who had not. Thus, IgA(+) PCs expressing iNOS are described for the first time, to our knowledge, in humans. iNOS(+) PCs are induced in the course of human H. pylori infection, and their abundance seems to correlate with the clinical course of the infection.

Highly sensitive and specific detection of Giardia duodenalis, Entamoeba histolytica, and Cryptosporidium spp. in human stool samples by the BD MAX™ Enteric Parasite Panel.

Detection of intestinal protozoan parasites by light microscopy is cumbersome, needs experienced personnel, and may lack sensitivity and/or specificity as compared with molecular-based stool assays. Here, we evaluated the BD MAX™ Enteric Parasite Panel, i.e., a multiplex real-time PCR assay for simultaneous detection of Giardia duodenalis, Entamoeba histolytica, and cryptosporidia (Cryptosporidium parvum and C. hominis), by examining 200 positive human stool samples (138 × G. duodenalis, 27 × E. histolytica, 35 × Cryptosporidium spp.) and 119 controls including 18 samples with E. dispar. The majority of the samples, i.e., 153/200 (76.5%) positive samples and 66/119 (55.5%) controls, were confirmed by multiplex in-house PCR detecting the same parasites as the BD MAX™ Enteric Parasite Panel. The BD MAX™ assay did not yield false-positive results. Sensitivity and specificity were 97.8% (95% CI, 93.3-99.4%) and 100% (95% CI, 97.4-100%) for G. duodenalis, 100% (95% CI, 84.5-100%) and 100% (95% CI, 98.4-100%) for E. histolytica, and 100% (95% CI, 87.7-100%) and 100% (95% CI, 98.3-100%) for cryptosporidia, and similar data were obtained when only the 219 PCR-confirmed samples were analyzed. Thus, the BD MAX™ Enteric Parasite Panel provides a highly sensitive and specific tool for the laboratory diagnosis of three predominant protozoan parasites causing enteritis.

Increasing metronidazole and rifampicin resistance of Helicobacter pylori isolates obtained from children and adolescents between 2002 and 2015 in southwest Germany.

BACKGROUND: Increasing antibiotic resistance has been reported for Helicobacter pylori, but data on the prevalence of antibiotic resistance of H. pylori in pediatric patients and the development of resistance over time are sparse. METHODS: Data for 610 H. pylori isolates obtained between 2002 and 2015 from gastric biopsies of 582 (mainly treatment-naïve) pediatric patients from southwest Germany were analyzed retrospectively regarding the antibiotic susceptibility determined by Etest and patients' characteristics. RESULTS: Overall resistance to metronidazole, clarithromycin, and rifampicin was 28.7%, 23.2%, and 13.3%, respectively, while resistance to amoxicillin was rare (0.8%). Simultaneous resistance to metronidazole and clarithromycin was observed for 7.7% of the isolates, and 2.3% were resistant to metronidazole, clarithromycin, and rifampicin. Differences between primary vs secondary resistance existed for metronidazole (24.7% vs 38.8%, P=.01) and clarithromycin (17.2% vs 54.1%, P=.0001). From 2002-2008 to 2009-2015, resistance to metronidazole increased from 20.8% to 34.4% (P=.003) and to rifampicin from 3.9% to 18.8% (P=.0001); this was not associated with increased numbers of patients previously treated for H. pylori infection in the second study period. In contrast, resistance to clarithromycin did not change significantly over time. Resistance was not associated with age, sex, or family origin in Europe. CONCLUSIONS: The considerable antibiotic resistance of H. pylori isolates argues for standard antibiotic susceptibility testing of H. pylori in pediatric patients prior to the initiation of antibiotic therapy.

Development of visceral leishmaniasis in an HIV(+) patient upon immune reconstitution following the initiation of antiretroviral therapy.

CASE PRESENTATION: Here, we report on a case of VL in an HIV-infected patient from the Republic of Georgia who had moved to Germany 14 years before and who had travelled several times to southern Europe in between. After presenting with typical Pneumocystis jiroveci pneumonia, which was treated appropriately, the patient was started on antiretroviral therapy. Shortly thereafter, however, he developed fever of unknown origin. All laboratory assays for the diagnosis of various infectious agents including serological assays and polymerase chain reaction testing of bone marrow aspirate to diagnose VL did not yield positive results at first. Only upon repetition of these tests, diagnosis of VL could be made and the patient treated accordingly. CASE DISCUSSION: Visceral leishmaniasis (VL) is a common opportunistic infection in HIV-positive patients from endemic countries but occurs rarely following antiretroviral treatment. This case demonstrates that patients who develop VL upon immune reconstitution may not be diagnosed initially by standard laboratory assays for the diagnosis of VL and underlines the necessity to repeat serologic and molecular biologic testing for VL in cases of fever of unknown origin in patients from or with travel history to endemic countries.

Highly specific detection of Cryptosporidium spp. oocysts in human stool samples by undemanding and inexpensive phase contrast microscopy.

To compare phase contrast microscopy (PCM) of unstained slides for the detection of Cryptosporidium spp. oocysts with a commercially available enzyme immunoassay (EIA) for the detection of cryptosporidial antigen in human stool samples, we prospectively analysed by both methods 463 fresh human stool samples obtained from diarrhoeic patients between July and October 2014. Compared with the EIA, the sensitivity, specificity, positive and negative predictive value of PCM were 88.9 % (95 % confidence interval (CI), 66.0-98.1 %), 100 % (95 % CI, 99.0-100 %), 100 % (95 % CI, 77.3-100 %) and 99.6 % (95 % CI, 98.3-100 %), respectively. Additionally, we retrospectively examined with PCM 65 fixed stool samples that had been collected in 2010 from mostly asymptomatic Rwandan children <5 years of age; 14 of these samples had previously yielded positive results with a highly sensitive real-time (RT)-PCR. PCM detected cryptosporidia in 5/14 RT-PCR-positive samples, and notably, also in one of 51 RT-PCR-negative samples, which was subsequently confirmed by acid-fast staining. Positive and negative percent agreement of PCM with RT-PCR were 35.7 % (95 % CI, 16.2-61.4 %) and 98.0 % (95 % CI, 88.7-100 %), respectively. Positive PCM results were associated with higher RT-PCR cycle threshold values (p = 0.044). In conclusion, PCM offers a highly specific, undemanding and inexpensive method for the laboratory diagnosis of acute human cryptosporidiosis independent of the causative Cryptosporidium species.

Antimicrobial resistance of Neisseria gonorrhoeae isolates in south-west Germany, 2004 to 2015: increasing minimal inhibitory concentrations of tetracycline but no resistance to third-generation cephalosporins.

Increasing antimicrobial resistance of Neisseria gonorrhoeae, particularly to third-generation cephalosporins, has been reported in many countries. We examined the susceptibility (determined by Etest and evaluated using the breakpoints of the European Committee on Antimicrobial Susceptibility Testing) of 434 N. gonorrhoeae isolates collected from 107 female and 327 male patients in Stuttgart, south-west Germany, between 2004 and 2015. During the study period, high proportions of isolates were resistant to ciprofloxacin (70.3%), tetracycline (48.4%; increasing from 27.5% in 2004/2005 to 57.7% in 2014/2015; p = 0.0002) and penicillin (25.6%). The proportion of isolates resistant to azithromycin was low (5.5%) but tended to increase (p = 0.08). No resistance and stable minimum inhibitory concentrations were found for cefixime, ceftriaxone, and spectinomycin. High-level resistance was found for ciprofloxacin (39.6%) and tetracycline (20.0%) but not for azithromycin; 16.3% of the isolates produced betalactamase. Thus, cephalosporins can still be used for the treatment of gonorrhoea in the study area. To avoid further increasing resistance to azithromycin, its usage should be limited to patients allergic to cephalosporins, or (in combination with cephalosporins) to patients for whom no susceptibility testing could be performed or those co-infected with chlamydiae.

Role of dendritic cells in the pathogenesis of Whipple's disease.

Accumulation of Tropheryma whipplei-stuffed macrophages in the duodenum, impaired T. whipplei-specific Th1 responses, and weak secretion of interleukin-12 (IL-12) are hallmarks of classical Whipple's disease (CWD). This study addresses dendritic cell (DC) functionality during CWD. We documented composition, distribution, and functionality of DC ex vivo or after in vitro maturation by fluorescence-activated cell sorting (FACS) and by immunohistochemistry in situ. A decrease in peripheral DC of untreated CWD patients compared to healthy donors was due to reduced CD11c(high) myeloid DC (M-DC). Decreased maturation markers CD83, CD86, and CCR7, as well as low IL-12 production in response to stimulation, disclosed an immature M-DC phenotype. In vitro-generated monocyte-derived DC from CWD patients showed normal maturation and T cell-stimulatory capacity under proinflammatory conditions but produced less IL-12 and failed to activate T. whipplei-specific Th1 cells. In duodenal and lymphoid tissues, T. whipplei was found within immature DC-SIGN(+) DC. DC and proliferating lymphocytes were reduced in lymph nodes of CWD patients compared to levels in controls. Our results indicate that dysfunctional IL-12 production by DC provides suboptimal conditions for priming of T. whipplei-specific T cells during CWD and that immature DC carrying T. whipplei contribute to the dissemination of the bacterium.

Simultaneous detection of Entamoeba histolytica/dispar, Giardia duodenalis and cryptosporidia by immunochromatographic assay in stool samples from patients living in the Greater Cairo Region, Egypt.

Gastrointestinal infection due to intestinal parasites is an enormous health problem in developing countries and its reliable diagnosis is demanding. Therefore, this study aimed at evaluating a commercially available immunochromatographic assay (ICA) for the detection of cryptosporidia, Giardia duodenalis, and Entamoeba histolytica/dispar for its usefulness in the Greater Cairo Region, Egypt. Stool samples of 104 patients who presented between October 2012 and March 2013 with gastrointestinal symptoms or for the exclusion of parasites at Kasr-Al-Ainy University Medical School were examined by light microscopy of wet mounts and the triple ICA. Microscopy revealed in 20% of the patients [95% confidence interval (CI), 13.5-29.0%] parasites with Hymenolepis nana, E. histolytica/dispar and Blastocystis hominis being the most frequent ones, but was not able to detect G. duodenalis and cryptosporidia, whereas ICA was positive in 21% (95% CI, 14.3-30.0%) and detected E. histolytica/dispar in 12.5% (95% CI, 7.3-20.4%), cryptosporidia in 6.7% (95% CI, 3.1-13.5%) and G. duodenalis in 15.4% (95% CI, 9.6-23.6%) of the patients. Detection of one or more pathogens was associated with access to water retrieved from a well or pump (p = 0.01). Patients between 20 and 29 years of age (p = 0.08) and patients with symptoms of 5 days or longer (p = 0.07) tended to have a higher risk to be infected than patients of other age groups or with shorter-lasting symptoms. In conclusion, the ICA was easy to perform and timesaving. Importantly, it enabled the detection of cryptosporidia, which cannot be found microscopically in unstained smears, demonstrated a higher sensitivity for the detection of G. duodenalis than microscopy, and was more specific for distinguishing E. histolytica/dispar from apathogenic amoeba.

Reduced prevalence of Giardia duodenalis in iron-deficient Rwandan children.

OBJECTIVE: Acute symptomatic infection with Giardia duodenalis impairs iron absorption, but iron deficiency may protect against infections caused by various micro-organisms including parasites. We therefore examined the association of G. duodenalis infection and iron deficiency in 575 Rwandan children under 5 years of age. METHODS: Giardia duodenalis infection was diagnosed by triplicate microscopy and PCR assays, and iron deficiency was defined as a ferritin concentration <12 ng/ml. RESULTS: Largely asymptomatic G. duodenalis infection was seen in 65.3% of the children and iron deficiency in 17.4%. G. duodenalis infection was less common in iron-deficient children (51%) than in non-deficient children (68%, P = 0.002). In multivariate analysis, the odds of G. duodenalis infection were almost halved in iron-deficient children (adjusted odds ratio, 0.54; 95% confidence interval, 0.33-0.86). CONCLUSION: In this highly endemic setting, there was no evidence that Giardia infection impairs iron status. Rather, iron deficiency appeared to protect against infection with this parasite.

Soil-transmitted helminths in southern highland Rwanda: associated factors and effectiveness of school-based preventive chemotherapy.

OBJECTIVES: Preventive chemotherapy of schoolchildren against soil-transmitted helminths (STHs) is widely implemented in Rwanda. However, data on its actual efficacy are lacking. We assessed prevalence, associated factors and manifestation of STH infection among schoolchildren in southern highland Rwanda as well as cure and reinfection rates. METHODS: Six hundred and twenty-two children (rural, 301; urban, 321) were included preceding the administration of a single dose of 500 mg mebendazole. Before treatment, and after 2 and 15 weeks, STH infection was determined by Kato-Katz smears and by PCR assays for Ascaris lumbricoides. Clinical and anthropometric data, socio-economic status and factors potentially associated with STH infection were assessed. RESULTS: Soil-transmitted helminth (STH) infection was present in 38% of rural and in 13% of urban schoolchildren. Ascaris lumbricoides accounted for 96% of infections. Of these, one-third was detected by PCR exclusively. Factors associated with STH infection differed greatly between rural and urban children. Likewise, STH infection was associated with stunting and anaemia only among urban children. The cure rate after 2 weeks was 92%. Among eight non-cleared A. lumbricoides infections, seven were submicroscopic. Reinfection within 3 months occurred in 7%, but the rate was higher among rural children, and with initially present infection, particularly at comparatively high intensity. CONCLUSIONS: The rural-urban difference in factors associated with STH infection and in reinfection rates highlights the need for targeted interventions to reduce transmission. PCR assays may help in detecting low-level infections persisting after treatment. In southern Rwanda, mebendazole is highly effective against the STH infections predominated by A. lumbricoides.

Giardia duodenalis arginine deiminase modulates the phenotype and cytokine secretion of human dendritic cells by depletion of arginine and formation of ammonia.

Depletion of arginine is a recognized strategy that pathogens use to evade immune effector mechanisms. Depletion depends on microbial enzymes such as arginases, which are considered virulence factors. The effect is mostly interpreted as being a consequence of successful competition with host enzymes for the substrate. However, both arginases and arginine deiminases (ADI) have been associated with pathogen virulence. Both deplete arginine, but their reaction products differ. An ADI has been implicated in the virulence of Giardia duodenalis, an intestinal parasite that infects humans and animals, causing significant morbidity. Dendritic cells (DC) play a critical role in host defense and also in a murine G. duodenalis infection model. The functional properties of these innate immune cells depend on the milieu in which they are activated. Here, the dependence of the response of these cells on arginine was studied by using Giardia ADI and lipopolysaccharide-stimulated human monocyte-derived DC. Arginine depletion by ADI significantly increased tumor necrosis factor alpha and decreased interleukin-10 (IL-10) and IL-12p40 secretion. It also reduced the upregulation of surface CD83 and CD86 molecules, which are involved in cell-cell interactions. Arginine depletion also reduced the phosphorylation of S6 kinase in DC, suggesting the involvement of the mammalian target of rapamycin signaling pathway. The changes were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Comparison of NH(4)(+) and urea revealed distinct immunomodulatory activities of these products of deiminases and arginases, respectively. The data suggest that a better understanding of the role of arginine-depleting pathogen enzymes for immune evasion will have to take enzyme class and reaction products into consideration.

Hookworm-related cutaneous larva migrans in northern Brazil: resolution of clinical pathology after a single dose of ivermectin.

To assess the effect of ivermectin on the morbidity caused by hookworm-related cutaneous larva migrans in patients in hyperendemic areas, we treated 92 patients (with 441 tracks in total) from Manaus, Brazil, with single-dose ivermectin (200 µg/kg). Four weeks later, patients had 60 tracks, and the associated morbidity improved significantly.

Dendritic cells from human mesenteric lymph nodes in inflammatory and non-inflammatory bowel diseases: subsets and function of plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDC) in mesenteric lymph nodes (MLN) may be important regulators of both inflammatory and non-inflammatory mucosal immune responses but human studies are rare. Here we compare pDC from human MLN and peripheral blood (PB) by phenotype and function. MLN from patients with or without inflammatory bowel disease (IBD) undergoing colon surgery and PB from patients with IBD and from controls were used to isolate mononuclear cells. The pDC were analysed by flow cytometry for the expression of CD40, CD80, CD83, CD86, CCR6, CCR7, CX3CR1, CD103 and HLA-DR. Purified pDC from MLN and PB were stimulated with staphylococcus enterotoxin B (SEB), CpG-A, interleukin-3 (IL-3), SEB + IL-3, CpG-A + IL-3 or left unstimulated, and cultured alone or with purified allogeneic CD4(+) CD45RA(+) HLA-DR- T cells. Subsequently, concentrations of IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, interferon-α (IFN-α), IFN-γ and tumour necrosis factor-α (TNF-α) in culture supernatants were determined by multiplex bead array. The PB pDC from IBD patients exhibited an activated and matured phenotype whereas MLN pDC and control PB pDC were less activated. CpG-A and CpG-A + IL-3-stimulated MLN pDC secreted less IL-6 and TNF-α compared with PB pDC from controls. Compared with co-cultures of naive CD4 T cells with PB pDC, co-cultures with MLN pDC contained more IL-2, IL-10 and IFN-γ when stimulated with SEB and SEB + IL-3, and less IFN-α when stimulated with CpG-A. MLN pDC differ phenotypically from PB pDC and their pattern of cytokine secretion and may contribute to specific outcomes of mucosal immune reactions.

Risk of immunodeficiency virus infection may increase with vaccine-induced immune response.

To explore the efficacy of novel complementary prime-boost immunization regimens in a nonhuman primate model for HIV infection, rhesus monkeys primed by different DNA vaccines were boosted with virus-like particles (VLP) and then challenged by repeated low-dose rectal exposure to simian immunodeficiency virus (SIV). Characteristic of the cellular immune response after the VLP booster immunization were high numbers of SIV-specific, gamma interferon-secreting cells after stimulation with inactivated SIV particles, but not SIV peptides, and the absence of detectable levels of CD8(+) T cell responses. Antibodies specific to SIV Gag and SIV Env could be induced in all animals, but, consistent with a poor neutralizing activity at the time of challenge, vaccinated monkeys were not protected from acquisition of infection and did not control viremia. Surprisingly, vaccinees with high numbers of SIV-specific, gamma interferon-secreting cells were infected fastest during the repeated low-dose exposures and the numbers of these immune cells in vaccinated macaques correlated with susceptibility to infection. Thus, in the absence of protective antibodies or cytotoxic T cell responses, vaccine-induced immune responses may increase the susceptibility to acquisition of immunodeficiency virus infection. The results are consistent with the hypothesis that virus-specific T helper cells mediate this detrimental effect and contribute to the inefficacy of past HIV vaccination attempts (e.g., STEP study).

Highly contiguous genomes of human clinical isolates of Giardia duodenalis reveal assemblage- and sub-assemblage-specific presence-absence variation in protein-coding genes.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.

Nocardia farcinica activates human dendritic cells and induces secretion of interleukin-23 (IL-23) rather than IL-12p70.

Studying the interaction of dendritic cells (DCs) with bacteria controlled by T-cell-mediated immune responses may reveal novel adjuvants for the induction of cellular immunity. Murine studies and the observation that nocardias infect predominantly immunosuppressed patients have suggested that these bacteria may possess an adjuvant potential. Moreover, adjuvants on the basis of the nocardial cell wall have been applied in clinical studies. Since the handling of adjuvants by DCs may determine the type of immune responses induced by a vaccine, the present study aimed at investigating the interaction of immature human monocyte-derived DCs with live or inactivated Nocardia farcinica in vitro and determining the cellular phenotypic changes as well as alterations in characteristic functions, such as phagocytosis, induction of T-cell proliferation, and cytokine secretion. Human DCs ingested N. farcinica and eradicated the bacterium intracellularly. DCs exposed to inactivated N. farcinica were activated, i.e., they developed a mature phenotype, downregulated their phagocytic capacity, and stimulated allogeneic T cells in mixed leukocyte reactions. Soluble factors were not involved in this process. To elucidate the potential adjuvant effect of N. farcinica on the induction of T-cell-mediated immune responses, we characterized the cytokines produced by nocardia-exposed DCs and detected substantial amounts of tumor necrosis factor alpha (TNF-α) and interleukin-12 p40 (IL-12p40). However, nocardia-treated DCs secreted only small amounts of IL-12p70, which were significantly smaller than the amounts of IL-23. Thus, N. farcinica activates DCs, but adjuvants based on this bacterium may have only a limited capacity to induce Th1 immune responses.