Homeostasis of cellular amino acids in Acanthamoeba castellanii exposed to different media under amoeba-bacteria coculture conditions.

BACKGROUND: Acanthamoeba castellanii is a free-living protist that feeds on diverse bacteria. A. castellanii has frequently been utilized in studies on microbial interactions. Grazing bacteria also exhibit diverse effects on the physiological characteristics of amoebae, such as their growth, encystation, and cytotoxicity. Since the composition of amoebae amino acids is closely related to cellular activities, it can indicate the overall responses of A. castellanii to various stimuli. METHOD: A. castellanii was exposed to different culture conditions in low-nutrient medium with heat-killed DH5α to clarify their effects. A targeted metabolomic technique was utilized to evaluate the concentration of cellular amino acids. The amino acid composition and pathways were analyzed by two web-based tools: MetaboAnalyst and Pathview. Then, long-term exposure to A. castellanii was investigated through in silico and in vitro methods to elucidate the homeostasis of amino acids and the growth of A. castellanii. RESULTS: Under short-term exposure, all kinds of amino acids were enriched in all exposed groups. In contrast to the presence of heat-killed bacteria, the medium exhibited obvious effects on the amino acid composition of A. castellanii. After long-term exposure, the amino acid composition was more similar to that of the control group. A. castellanii may achieve amino acid homeostasis through pathways related to alanine, aspartate, citrulline, and serine. DISCUSSION: Under short-term exposure, compared to the presence of bacteria, the type of medium exerted a more powerful effect on the amino acid composition of the amoeba. Previous studies focused on the interaction of the amoeba and bacteria with effective secretion systems and effectors. This may have caused the effects of low-nutrient environments to be overlooked. CONCLUSION: When A. castellanii was stimulated in the coculture system through various methods, such as the presence of bacteria and a low-nutrient environment, it accumulated intracellular amino acids within a short period. However, different stimulations correspond to different amino acid compositions. After long-term exposure, A. castellanii achieved an amino acid equilibrium by downregulating the biosynthesis of several amino acids.

Classical Borrelia Serology Does Not Aid in the Diagnosis of Persistent Symptoms Attributed to Lyme Borreliosis: A Retrospective Cohort Study.

OBJECTIVE: The diagnosis of Lyme borreliosis is based on two-tier testing using an ELISA and Western blot. About 5-10% of patients report persistent symptoms of unknown etiology after treatment, resulting in substantial difficulties in further diagnostic workup. This paper presents a study aimed at determining whether serology can differentiate between patients with persistent symptoms attributed to Lyme and other patients with Lyme borreliosis. METHODS: A retrospective cohort study included 162 samples from four subgroups: patients with persistent symptoms of Lyme (PSL), early Lyme borreliosis with erythema migrans (EM), patients tested in a general practitioner setting (GP), and healthy controls (HC). ELISA, Western blots, and multiplex assays from different manufacturers were used to determine inter-test variations in PSL and to compare reactivity against Borrelia-specific antigens among the groups. RESULTS: In comparing the IgG and IgM reactivity by Western blot, IgG was more often positive in the PSL group than in the GP group. The individual antigen reactivity was similar between the PSL and EM or GP groups. Inter-test agreement among the manufacturers was variable, and agreement was higher for IgG testing compared to IgM. CONCLUSIONS: Serological testing is unable to define the subgroup of patients with persistent symptoms attributed to Lyme borreliosis. Additionally, the current two-tier testing protocol shows a large variance among different manufacturers in these patients.

Lyme Borreliosis Serology: A Prospective Cohort Study of Forestry Service Workers in the Netherlands over 8 Years (2008 to 2016) of Follow-Up.

There is little known about the dynamics within responses to Borrelia spp. upon repeated exposure to tick bites and the development of serological markers over time. Most studies have investigated antibody development in risk populations over a short period of time. Therefore, we aimed to study the dynamics of anti-Borrelia antibodies in forestry service workers over 8 years in association with tick bite exposure. METHODS: Blood samples from 106 forestry service workers originally included in the 200 Functional Genomics Project (Radboudumc, Nijmegen, the Netherlands) were followed for 8 years and tested annually for anti-Borrelia antibodies (ELISA and Western blot). IgG seroconversion was related to the number of tick bites in the previous year, which was obtained through annual questionnaires. The hazard ratio for Borrelia IgG seroconversion was calculated using Cox regression survival analysis and a logistic regression model, both adjusting for age, gender and smoking. RESULTS: Borrelia IgG seropositivity in the study population did not vary significantly between years and the average prevalence was 13.4%. Of the 27 subjects that underwent seroconversion during the study period, 22 reconverted from positive to negative. Eleven subjects seroconverted a second time. The total seroconversion rate per year (negative to positive) was 4.5%. Active smoking was associated with IgG seroconversion in the >5 tick bites group (p < 0.05). According to the two models used, the risks of IgG seroconversion in the >5 tick bites group were HR = 2.93 (p = 0.10) and OR = 3.36 (p < 0.0005). CONCLUSIONS: Borrelia IgG seroconversion in forestry service workers was significantly related to increasing tick bite exposure in a survival and logistic regression model adjusting for age, gender and smoking.

An International External Quality Assessment Scheme to Assess the Diagnostic Performance of Polymerase Chain Reaction Detection of Acanthamoeba Keratitis.

PURPOSE: The purpose of this study was to assess the variation in methods and to determine whether an External Quality Assessment Scheme (EQAS) for polymerase chain reaction (PCR) detection of Acanthamoeba keratitis is valuable for the diagnostic process. METHODS: A multicenter EQAS was introduced, covering 16 diagnostic laboratories. Using Acanthamoeba castellanii ATCC strain 30010, 3 sets of samples were prepared, containing different amounts of DNA, cysts, or trophozoites. Samples were masked and sent to the participants with instructions for use and a questionnaire concerning the applied methodologies. Special attention in this questionnaire was given to the used pretreatment methods to assess existing variations in these procedures. RESULTS: A large variation in the methodologies and substantial differences in the diagnostic performance were found between participants. In contrast to the DNA samples where all participants had a perfect score, several false negative results were reported for the samples containing cysts or trophozoites. Only 9 participants had an optimal score, whereas one participant reported all samples as negative, one participant reported failures due to inhibition, and the other 5 reported in total 7 false negative results. A clear correlation was noticed between the PCR detection rate and the number of cysts or trophozoites in the sample. CONCLUSIONS: The results indicate that a pretreatment procedure can be a risky step in PCR-based detections of Acanthamoeba , but it improves the sensitivity and reliability, especially of samples containing cysts. Therefore, participation in an EQAS is informative for routine diagnostic laboratories and can assist in improving the laboratory procedures used for the diagnosis of Acanthamoeba keratitis.

In Vitro Effectiveness of Soft Contact Lens Solutions Available on the Dutch Market against Acanthamoeba Species.

Acanthamoeba keratitis is almost universally associated with contact lens (CL) use. Until today, however, CL solution manufacturing protocols lack testing of anti-amoebic activity. This study investigates the effectiveness of CL solutions available on the Dutch market against trophozoites and cysts of Acanthamoeba castellanii and Acanthamoeba polyphaga. Sixteen CL solutions were tested: 13 multiple purpose solutions (MPS), 2 hydrogen peroxidase solutions (HPS) and 1 povidone-iodine-based solution (PIS). The Spearman-Karber (SK) log reduction method and an XTT colorimetric assay were used to evaluate the effectiveness at the manufacturer's minimum recommended disinfection time (MMRDT) and after eight hours. At the MMRDT, one MPS showed an SK mean log reduction (MLR) of >3.0 against A. castellanii trophozoites. Two additional MPS and both HPS reached this threshold after eight hours. The SK MLR values for A. polyphaga trophozoites were between 1 and 3 at all time points. Using the XTT colorimetric assay, only HPS 1 showed >99.9% reduction (equivalent to 3 log reduction) in metabolic activity of A. castellanii trophozoites after eight hours. For A. polyphaga, both HPS and PIS showed a metabolic reduction of >99.9% after eight hours. Cysts were resistant against all solutions. We conclude that following the manufacturer's guidelines, few solutions provide sufficient effectiveness against Acanthamoeba trophozoites and none against cysts. The results underline the importance of adequate hygiene when handling CLs.

The Performance of Nine Commercial Serological Screening Assays for the Diagnosis of Lyme Borreliosis: a Multicenter Modified Two-Gate Design Study.

In this retrospective study, the performance of nine serological screening assays for Lyme borreliosis (LB) diagnostics was evaluated using a study population of LB cases and controls. Sera derived from 74 well-defined LB cases and 122 controls were included. The LB cases were diagnosed with erythema migrans (EM; n = 11), Lyme neuroborreliosis (LNB; n = 35), Lyme arthritis (LA; n = 20), or acrodermatitis chronica atrophicans (ACA; n = 8). Controls comprised 74 age- and gender-matched healthy individuals and 48 patients with other diseases with anticipated high rates of cross-reactivity. The assays under evaluation were selected based on a literature review and expected continued availability with CE marking under the new in vitro diagnostic regulation (European Union) 2017/746. The overall sensitivity (IgG and IgM results combined) among LB cases ranged between 54.5% (6 of 11) and 90.9% (10 of 11) for EM patients and between 97.1% (34 of 35) and 100% for patients with LNB, LA, and ACA. The positivity rate ranged between 8.1% (6 of 74) and 29.7% (22 of 74) among the healthy controls and between 22.9% (11 of 48) and 64.6% (31 of 48) among the cross-reactivity controls. The IgM results were more heterogeneous than the IgG and IgM/IgG results and did not contribute to the overall sensitivity but substantially increased the positivity rates among the controls. In conclusion, all evaluated Borrelia serological screening assays performed comparably with respect to early- and late-disseminated LB. The addition of an IgM assay to the screening of Borrelia-specific IgG antibodies had no added value for the diagnosis of Lyme borreliosis. IMPORTANCE Serology plays an important role in the diagnosis of Lyme borreliosis. Guidelines prescribe a two-tier testing algorithm in which a highly sensitive screening assay is used for screening and reactive sera are retested with an immunoblot to reduce false positivity rates. Recently, two commonly used screening assays were discontinued, including the very well-performing C6 Lyme enzyme-linked immunosorbent assay (ELISA) (Immunetics). This study provides an evaluation of the performance of nine different Borrelia serology screening assays, eight with expected future availably and the C6 Lyme ELISA, using a well-defined study panel of Lyme borreliosis patients, healthy population controls, and cross-reactivity controls. Evaluation data on multiple assays aid diagnostic laboratories in their choice for a reliable Borrelia serology screening assay to improve their diagnostic algorithm for Lyme borreliosis.

Malaria diagnosis in a malaria non-endemic high-resource country: high variation of diagnostic strategy in clinical laboratories in the Netherlands.

BACKGROUND: Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination of other methods should be used and when. METHODS: Three questionnaires were sent between March 2018 and September 2019 to laboratories subscribing to the external quality assessment scheme for the diagnosis of blood and intestinal parasites of the Dutch Foundation for Quality Assessment in Medical Laboratories in order to investigate how much variation in the laboratory diagnosis of malaria between different clinical laboratories is present in the Netherlands. RESULTS: The questionnaires were partially or fully completed by 67 of 77 (87%) laboratories. Only 9 laboratories reported 10 or more malaria positive patients per year. Most laboratories use a different diagnostic strategy, within office versus outside office hours depending on the screening assay result. Within office hours, 62.5% (35/56) of the responding laboratories perform an immunochromatographic test (ICT) in combination with microscopic examination of thick and thin blood films without additional examinations, such as Quantitative Buffy Coat and/or rtPCR analysis. Outside office hours 85.7% (48/56) of laboratories use an ICT as single screening assay and positive results are immediately confirmed by thick and thin blood films without additional examinations (89.6%, 43/48). In case of a negative ICT result outside office hours, 70.8% (34/48) of the laboratories perform microscopic examination of the thick film the next morning and 22.9% (11/48) confirm the negative ICT result immediately. Furthermore, substantial differences were found in the microscopic examinations of thick and thin blood films; the staining, theoretical sensitivity of the thick film and determination of parasitaemia. CONCLUSIONS: This study demonstrated a remarkably high variation between laboratories in both their diagnostic strategy as well as their methods for microscopic examination for the diagnosis of malaria in a clinical setting, despite existing national and international guidelines. While the impact of these variations on the accuracy of the diagnosis of malaria is yet unknown, these findings should stimulate clinical laboratories to critically review their own diagnostic strategy.

Humoral and cellular immune responses after influenza vaccination in patients with postcancer fatigue.

The aim of this study was to compare humoral and cellular immune responses to influenza vaccination in cancer survivors with and without severe symptoms of fatigue. Severely fatigued (n = 15) and non-fatigued (n = 12) disease-free cancer survivors were vaccinated against seasonal influenza. Humoral immunity was evaluated at baseline and post-vaccination by a hemagglutination inhibition assay. Cellular immunity was evaluated at baseline and post-vaccination by lymphocyte proliferation and activation assays. Regulatory T cells were measured at baseline by flow cytometry and heat-shock protein 90 alpha levels by ELISA. Comparable humoral immune responses were observed in fatigued and non-fatigued patients, both pre- and post-vaccination. At baseline, fatigued patients showed a significantly diminished cellular proliferation upon virus stimulation with strain H3N2 (1414 ± 1201 counts), and a trend in a similar direction with strain H1N1 (3025 ± 2339 counts), compared to non-fatigued patients (3099 ± 2401 and 5877 ± 4604 counts, respectively). The percentage of regulatory T lymphocytes was significantly increased (4.4 ± 2.1% versus 2.4 ± 0.8%) and significantly lower amounts of interleukin 2 were detected prior to vaccination in fatigued compared to non-fatigued patients (36.3 ± 44.3 pg/ml vs. 94.0 ± 45.4 pg/ml with strain H3N2 and 28.4 ± 44.0 pg/ml versus 74.5 ± 56.1 pg/ml with strain H1N1). Pre-vaccination heat-shock protein 90 alpha concentrations, post-vaccination cellular proliferation, and post-vaccination cytokine concentrations did not differ between both groups. In conclusion, influenza vaccination is favorable for severely fatigued cancer survivors and should be recommended when indicated. However, compared to non-fatigued cancer survivors, fatigued cancer survivors showed several significant differences in immunological reactivity at baseline, which warrants further investigation.

Has CXCL13 an added value in diagnosis of neurosyphilis?

In patients with syphilis, central nervous system (CNS) involvement is often difficult to determine. In patients who also are infected with human immunodeficiency virus (HIV), this is even more challenging, as cerebrospinal fluid (CSF) pleocytosis can be attributed to HIV, syphilis, or both. Hence, this study investigated (i) CSF chemokine (C-X-C motif) ligand 13 (CXCL13) as a potential marker to diagnose neurosyphilis in HIV-infected individuals and (ii) the added value of CSF CXCL13 to conventional CSF biomarkers, such as the rapid plasma reagin test (RPR), in diagnosing neurosyphilis. We included 103 syphilis patients from two centers in The Netherlands: 47 non-HIV-infected patients and 56 HIV-infected patients. A positive CSF-RPR was regarded as the gold standard for neurosyphilis. CSF CXCL13 levels were significantly higher in neurosyphilis patients when neurosyphilis was diagnosed by CSF-RPR (P = 0.0002) than in the syphilis control group. The sensitivity and specificity of CSF CXCL13 (cutoff of 76.3 pg/ml) to diagnose neurosyphilis by using positive CSF-RPR as the gold standard were 50% and 90%, respectively. CSF CXCL13 had an added value to CSF-RPR positivity in 70% of HIV-positive patients and in 33% of HIV-negative patients. Our data show that CSF CXCL13 might be a potential additional marker in neurosyphilis when other markers are not conclusive. The added value of CSF CXCL13 measurement to the current neurosyphilis gold standard appears to benefit HIV-positive patients more than HIV-negative patients.

KIR and Human Leukocyte Antigen Genotype Associated Risk of Cytomegalovirus Disease in Renal Transplant Patients.

BACKGROUND: Cytomegalovirus(CMV) infections have a significant effect on morbidity and mortality in kidney transplants. We conducted a study to ascertain the association of natural killer cell killer immunoglobulin-like receptors and human leukocyte antigen (HLA) genotype with risk of CMV disease. METHODS: The 90 CMV-negative patients receiving a first renal transplantation from a CMV-positive donor in this study received triple immunosuppressive therapy and prophylactic CMV treatment for up to 3 months after transplantation. RESULTS: We observed a 43.3% incidence rate of CMV disease within the first year after transplantation. Twenty-seven recipients experienced a rejection episode, 14 of which had CMV disease, mostly after rejection, suggesting that in this group, CMV disease is not a risk factor for rejection. KIR gene or genotype distribution were similar between the CMV diseased and CMV disease-free group. Twenty-seven recipients (30%) carried KIR-AA genotype, of which nine (33%) had CMV disease. Of the remaining 63 (70%) recipients with KIR-BX genotype, 30 (48%) had CMV disease. There was no significant difference between the two genotype groups with regard to occurrence of CMV disease, although there was a trend toward a lower incidence of CMV disease in recipients carrying the KIR-AA genotype. For CMV disease, we found no significant risk associated with the number of activating or inhibitory KIRs. Neither was missing KIR ligands for the inhibitory KIRs (HLA-C1/C2/Bw4) in recipients associated with lower rates of CMV disease. CONCLUSION: In CMV-negative recipients, genotypic analysis of KIR repertoire and HLA ligands does not provide risk factors for primary CMV disease after renal transplantation.

Absolute lymphocyte count predicts the response to new influenza virus H1N1 vaccination in pediatric cancer patients.

We measured the vaccination response to the new H1N1 virus in relation to the lymphocyte count prior to vaccination in pediatric cancer patients. The absolute lymphocyte count above the lower normal limits (LNL) for age prior to vaccination predicts the response to influenza vaccination in pediatric cancer patients treated with chemotherapy.

[Borrelial lymphocytoma]. / Borrelia-lymfocytoom.

BACKGROUND: Borrelial lymphocytoma is a relatively rare but typical presentation of Lyme disease. Predilection sites are the ears in children and chest/nipples in adults. It is treated like an erythema migrans and has a good prognosis. CASE DESCRIPTION: A 16-year-old boy presented with a swollen, red and painful right nipple since several months. An ultrasound showed normal breast tissue. The patient was referred to the pediatric surgeon who performed an incision biopsy. Histopathological examination revealed follicular hyperplasia without signs of malignancy. An infectious cause, most likely Lyme disease, was suspected. Serological analysis and PCR of the tissue confirmed the diagnosis of a borrelial lymphocytoma, and the patient was treated with doxycycline with good result. CONCLUSION: Early recognition of the characteristic clinical presentation of borrelial lymphocytoma, supported by positive results from serologic testing for Lyme disease, avoids the need for additional and invasive diagnostic tests.

Humoral and cellular immune responses after influenza vaccination in patients with chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is a clinical condition characterized by severe and disabling fatigue that is medically unexplained and lasts longer than 6 months. Although it is possible to effectively treat CFS, the nature of the underlying physiology remains unclear. Various studies have sought evidence for an underlying disturbance in immunity. The aim of this study was to compare the humoral and cellular immune responses upon influenza vaccination in CFS patients and healthy controls. RESULTS: Identical antibody titers were observed in CFS patients and healthy controls. Patients and controls demonstrated similar seroprotection rates against all three virus-strains of the influenza vaccine, both pre- and post-vaccination. Functional T cell reactivity was observed in both CFS patients and healthy controls. CFS patients showed a non-significant, numerically lower cellular proliferation at baseline compared to controls. Vaccination induced a significant increase in cellular proliferation in CFS patients, but not in healthy controls. Cytokine production and the number of regulatory T cells were comparable in patients and controls. CONCLUSIONS: The humoral and cellular immune responses upon influenza vaccination were comparable in CFS patients and healthy controls. Putative aberrations in immune responses in CFS patients were not evident for immunity towards influenza. Standard seasonal influenza vaccination is thus justified and, when indicated, should be recommended for patients suffering from CFS.

Impact of bacterial colonization on exhaled inflammatory markers in wheezing preschool children.

Wheeze is a common symptom in preschool children. The role of bacteria, regulatory T (T(reg)) cells and their association with airway inflammation in preschool wheeze is largely unknown. We evaluated inflammatory markers in exhaled breath condensate (EBC), bacterial colonization and circulating T(reg) cells in preschool children with and without recurrent wheeze. We recruited 252 children (aged two to four years) with (N = 202) and without (N = 50) recurrent wheeze. EBC was collected using an efficient closed glass condenser. Inflammatory markers in EBC (Interleukin(IL)-2, IL-4, IL-8, IL-10, IL-13) were assessed using multiplex immunoassay. Nasal and throat swabs were analysed for presence of Streptococcus pneumoniae, Haemophilus (para)influenzae and Staphylococcus aureus. Proportions of T(reg) cells (CD4(+)CD25(high)CD127(-)) were quantified by flow cytometry. Recurrent wheezing children had elevated EBC levels of IL-2, IL-4, IL-10 and IL-13 compared to non-wheezers (odds ratio (95% confidence interval): 1.67 (1.23-2.27): 1.58 (1.15-2.18): 1.47 (1.14-1.90): 1.55 (1.16-2.06), p <0.05, respectively). Bacteria were frequently present in children with and without wheeze, with no difference in prevalence (16-52% versus 16-50%, respectively). Moreover, the proportion of T(reg) cells did not differ between both groups. Wheezing children with bacterial colonization did not significantly differ in exhaled levels of inflammatory markers or proportion of T(reg) cells compared to wheezing children without colonization. The analysis of EBC might serve as a helpful non-invasive tool to early assess airway inflammation in wheezing children. The various elevated exhaled inflammatory markers indicate increased airway inflammation in wheezing preschool children. In the presence of wheeze, we found no evidence for bacterial induced airway inflammation.

The first locally acquired human infection of Echinococcus multilocularis in The Netherlands.

In the northern part of Western Europe, Echinococcus multilocularis is primarily detected in and spreading among foxes. The present case marks E. multilocularis as an emerging pathogen for humans, as it describes the first human case of probably locally acquired E. multilocularis in The Netherlands, with various interesting clinical aspects.

The contribution of water contact behavior to the high Schistosoma mansoni Infection rates observed in the Senegal River Basin.

BACKGROUND: Schistosomiasis is one of the major parasitic diseases in the world in terms of people infected and those at risk. Infection occurs through contact with water contaminated with larval forms of the parasite, which are released by freshwater snails and then penetrate the skin of people. Schistosomiasis infection and human water contact are thus essentially linked, and more knowledge about their relationship will help us to develop appropriate control measures. So far, only few studies have related water contact patterns to infection levels. METHODS: We have conducted detailed direct water contact observations in a village in Northern Senegal during the first years of a massive Schistosoma mansoni outbreak to determine the role of human water contact in the extent of the epidemic.We quantified water contact activities in terms of frequency and duration, and described how these vary with age and sex. Moreover, we assessed the relationship between water contact- and infection intensity patterns to further elucidate the contribution of exposure to the transmission of schistosomiasis. RESULTS: This resulted in over 120,000 recorded water contacts for 1651 subjects over 175 observation days. Bathing was the main activity, followed by household activities. Frequency and duration of water contact depended on age and sex rather than season. Water contacts peaked in adolescents, women spent almost twice as much time in the water as men, and water contacts were more intense in the afternoon than in the morning, with sex-specific intensity peaks. The average number of water contacts per person per day in this population was 0.42; the average time spent in the water per person per day was 4.3 minutes. CONCLUSIONS: The observed patterns of water contact behavior are not unusual and have been described before in various other settings in sub-Saharan Africa. Moreover, water contact levels were not exceptionally high and thus cannot explain the extremely high S. mansoni infection intensities as observed in Northern Senegal. Comparison with fecal egg counts in the respective age and sex groups further revealed that water contact levels did not unambiguously correspond with infection levels, indicating that factors other than exposure also play a role in determining intensity of infection.