We validated the use of stored samples for Chlamydia trachomatis research. C. trachomatis DNA was detected by real-time PCR in clinical (urine and self-taken vaginal swabs) and spiked samples using six different media, five different time points (up to 2 years), and four different temperature conditions. C. trachomatis was detected in all 423 samples, and no clinically relevant degradation impact was detected.
Studies about associations of infections with herpes viruses and other pathogens, such as Chlamydia pneumoniae (CP) and Helicobacter pylori (HP) with cardiovascular disease (CVD), diabetes mellitus (DM), frailty and/or mortality are conflicting. Since high levels of antibodies against these pathogens occur in the elderly, the role of these pathogens in morbidity and mortality of vulnerable elderly was explored.
Blood samples of 295 community dwelling psycho-geriatric patients were tested for IgG antibodies to herpes simplex virus type 1 and 2, varicella zoster virus, Epstein Barr virus (EBV), cytomegalovirus (CMV), human herpes virus type 6 (HHV6), CP and HP. Frailty was defined with an easy-to-use previously described frailty risk score. Relative risks (RR) with 95% confidence intervals were calculated to evaluate associations between CVD, DM, frailty and pathogens. Pathogens as a predictor for subsequent mortality were tested using Kaplan Meier analyses and Cox proportional hazard models. The mean age was 78 (SD: 6.7) years, 20% died, 44% were defined as frail, 20% had DM and 49% had CVD. Presence of CMV antibody titers was associated with frailty, as shown by using both qualitative and quantitative tests, RR ratio 1.4 (95% CI: 1.003-2.16) and RR ratio 1.5 (95% CI: 1.06-2.30), respectively. High IgG antibody titers of HHV6 and EBV were associated with DM, RR ratio 3.3 (95% CI: 1.57-6.49). None of the single or combined pathogens were significantly associated with mortality and/or CVD.
Prior CMV infection is associated with frailty, which could be in line with the concept that CMV might have an important role in immunosenescence, while high IgG titers of HHV6 and EBV are associated with DM. No association between a high pathogen burden and morbidity and/or mortality could be demonstrated.
Herpes viruses; Cytomegalovirus; Frailty; Diabetes mellitus; Morbidity; Mortality
This study presents a novel approach to aid in diagnosis of urinary tract infections (UTIs). A real-time PCR assay was used to screen for culture-positive urinary specimens and to identify the causative uropathogen. Semi-quantitative breakpoints were used to screen for significant bacteriuria (presence of ≥105 CFU/ml of uropathogens) or low-level bacteriuria (containing between 103 and 104 CFU/ml of uropathogens). The 16S rDNA-based assay could identify the most prevalent uropathogens using probes for Escherichia coli, Pseudomonas species, Pseudomonas aeruginosa, Staphylococcus species, Staphylococcus aureus, Enterococcus species and Streptococcus species. 330 urinary specimens were analysed and results were compared with conventional urine culture. Using a PCR Ct value of 25 as semi-quantitative breakpoint for significant bacteriuria resulted in a sensitivity and specificity of 97% and 80%, respectively. In 78% of the samples with monomicrobial infections the assay contained probes to detect the bacteria present in the urine specimens and 99% of these uropathogens was correctly identified. Concluding, this proof-of-concept approach demonstrates that the assay can distinguish bacteriuria from no bacteriuria as well as detect the involved uropathogen within 4 hours after sampling, allowing adequate therapy decisions within the same day as well as drastically reduce consequent urine culturing.
Few studies have been performed on urinary tract infections (UTIs) in men. In the present study, general practitioners (n = 42) from the Dutch Sentinel General Practice Network collected urinary samples from 560 male patients (≥18 years) suspected of UTI and recorded prescribed antibiotic treatment. In this way, the antibiotic susceptibility of Gram-negative uropathogens, including extended-spectrum beta-lactamase (ESBL-) producing Escherichia coli could be determined. In addition, E. coli susceptibility and antibiotic prescriptions were compared with data from a similar UTI study among women and with data collected 7 years earlier. Of 367 uropathogens (66%) identified (≥103 cfu/mL), most were Gram-negative (83%) and E. coli being isolated most frequently (51%). Antibiotic susceptibility to ciprofloxacin, norfloxacin and nitrofurantoin was 94%, 92% and 88%, respectively, whereas co-amoxiclav (76%) and co-trimoxazole (80%) showed lower susceptibilities. One ESBL (0.5%) was found. A significantly higher proportion of female UTIs was caused by E. coli compared with men (72% versus 51%, P<0.05). E. coli susceptibility tended to be lower in men compared with women, although not reaching statistical significance. No changes in E. coli susceptibility were observed over time (all P>0.05). Co-amoxiclav and nitrofurantoin prescriptions increased over time (11% versus 28% and 16% versus 23% respectively, both P<0.05), whereas co-trimoxazole prescriptions decreased (24% versus 14%, P<0.05). In conclusion, given the observed gender differences in uropathogen distribution and (tendency in) E. coli antibiotic susceptibility, empirical male UTI treatment options should be based on surveillance studies including men only. When awaiting the culture result is clinically not possible, fluoroquinolones are advised as first-choice antibiotics for male UTIs in Dutch general practices based on current antibiotic susceptibility data. The prevalence of ESBL-producers was low and no differences were observed in antibiotic susceptibility over a 7-year period. In addition, antibiotic prescriptions changed in accordance with national guidelines during this time period.
Hospital HIV care and public sexual health care (a Sexual Health Care Centre) services were integrated to provide sexual health counselling and sexually transmitted infections (STIs) testing and treatment (sexual health care) to larger numbers of HIV patients. Services, need and usage were assessed using a patient perspective, which is a key factor for the success of service integration.
The study design was a one-group pre-test and post-test comparison of 447 HIV-infected heterosexual individuals and men who have sex with men (MSM) attending a hospital-based HIV centre serving the southern region of the Netherlands. The intervention offered comprehensive sexual health care using an integrated care approach. The main outcomes were intervention uptake, patients’ pre-test care needs (n=254), and quality rating.
Pre intervention, 43% of the patients wanted to discuss sexual health (51% MSM; 30% heterosexuals). Of these patients, 12% to 35% reported regular coverage, and up to 25% never discussed sexual health topics at their HIV care visits. Of the patients, 24% used our intervention. Usage was higher among patients who previously expressed a need to discuss sexual health. Most patients who used the integrated services were new users of public health services. STIs were detected in 13% of MSM and in none of the heterosexuals. The quality of care was rated good.
The HIV patients in our study generally considered sexual health important, but the regular counselling and testing at the HIV care visit was insufficient. The integration of public health and hospital services benefited both care sectors and their patients by addressing sexual health questions, detecting STIs, and conducting partner notification. Successful sexual health care uptake requires increased awareness among patients about their care options as well as a cultural shift among care providers.
HIV; Quality of care; Services integration; Public health care; Hospital care; STI
Antibiotic consumption is associated with adverse drug events (ADE) and increasing antibiotic resistance. Detailed information of antibiotic prescribing in different age categories is scarce, but necessary to develop strategies for prudent antibiotic use. The aim of this study was to determine the antibiotic prescriptions of different antibiotic classes in general practice in relation to age.
Retrospective study of 22 rural and urban general practices from the Dutch Registration Network Family Practices (RNH). Antibiotic prescribing data were extracted from the RNH database from 2000–2009. Trends over time in antibiotic prescriptions were assessed with multivariate logistic regression including interaction terms with age. Registered ADEs as a result of antibiotic prescriptions were also analyzed.
In total 658,940 patients years were analyzed. In 11.5% (n = 75,796) of the patient years at least one antibiotic was prescribed. Antibiotic prescriptions increased for all age categories during 2000–2009, but the increase in elderly patients (>80 years) was most prominent. In 2000 9% of the patients >80 years was prescribed at least one antibiotic to 22% in 2009 (P<0.001). Elderly patients had more ADEs with antibiotics and co-medication was identified as the only independent determinant for ADEs.
The rate of antibiotic prescribing for patients who made a visit to the GP is increasing in the Netherlands with the most evident increase in the elderly patients. This may lead to more ADEs, which might lead to higher consumption of health care and more antibiotic resistance.
Molecular DNA-based diagnostics are increasingly being used for diagnosis of viral infections. For enteric viruses, PCR assays have also been developed. The aims of this study were to compile and evaluate a comprehensive panel of PCR assays for diagnosis of viruses causing diarrheal disease and to evaluate its use in a largely pediatric population in a 750-bed university medical center. The PCR panel was designed to include assays for detection of adenovirus, astrovirus, enterovirus, norovirus, parechovirus, rotavirus, and sapovirus. The results of the PCR panel were evaluated in relation to conventional viral diagnostics consisting of viral culture and/or rotavirus and adenovirus rapid antigen tests on samples that were taken for routine diagnostics. Comparing conventional with PCR-based testing, the number of viruses detected increased dramatically from 25 to 106 when PCR assays were used. This increase was due mainly to detection of previously undetected viruses, i.e., astrovirus, norovirus, and sapovirus. In 24% of the samples, norovirus was detected. Also, the lower detection limit of PCR-based adenovirus, enterovirus, parechovirus, and rotavirus diagnostics further increased the detection rate. By focusing on samples from patients with complaints of gastroenteritis, detection of a causative agent was increased from 49% by conventional tests to 97% by molecular diagnostics. However, many samples containing low viral loads were found in patients with complaints other than intestinal complaints. In conclusion, the proposed comprehensive PCR panel with appropriate cutoff values can be used for sensitive, rapid, and clinically relevant diagnosis of gastrointestinal viruses.
Over 90% of all antibiotics in Europe are prescribed in primary care. It is important that antibiotics are prescribed that are likely to be effective; however, information about antibiotic resistance in the community is incomplete. The aim of our study is to investigate the appropriateness of antibiotic prescribing in primary care in Europe by collecting and combining patterns of antibiotic resistance patterns and antibiotic prescription patterns in primary care. We will also evaluate the appropriateness of national antibiotic prescription guidelines in relation to resistance patterns.
Antibiotic resistance will be studied in an opportunistic sample from the community in nine European countries. Resistance data will be collected by taking a nose swab of persons (N = 4,000 per country) visiting a primary care practice for a non-infectious disease. Staphylococcus aureus and Streptococcus pneumoniae will be isolated and tested for resistance to a range of antibiotics in one central laboratory. Data on antibiotic prescriptions over the past 5 years will be extracted from the electronic medical records of General Practitioners (GPs). The results of the study will include the prevalence and resistance data of the two species and 5 years of antibiotic prescription data in nine European countries.
The odds of receiving an effective antibiotic in each country will be calculated as a measure for the appropriateness of prescribing. Multilevel analysis will be used to assess the appropriateness of prescribing. Relevant treatment guidelines of the nine participating countries will be evaluated using a standardized instrument and related to the resistance patterns in that country.
This study will provide valuable and unique data concerning resistance patterns and prescription behaviour in primary care in nine European countries. It will provide evidence-based recommendations for antibiotic treatment guidelines that take resistance patterns into account which will be useful for both clinicians and policy makers. By improving antibiotic use we can move towards controlling the resistance problem globally.
Prozone means false-negative or false-low results in antigen-antibody reactions, due to an excess of either antigen or antibody. The present study prospectively assessed its frequency for malaria rapid diagnostic tests (RDTs) and Plasmodium falciparum samples in an endemic field setting.
From January to April 2010, blood samples with P. falciparum high parasitaemia (≥ 4% red blood cells infected) were obtained from patients presenting at the Provincial Hospital of Tete (Mozambique). Samples were tested undiluted and 10-fold diluted in saline with a panel of RDTs and results were scored for line intensity (no line visible, faint, weak, medium and strong). Prozone was defined as a sample which showed no visible test line or a faint or weak test line when tested undiluted, and a visible test line of higher intensity when tested 10-fold diluted, as observed by two blinded observers and upon duplicate testing.
A total of 873/7,543 (11.6%) samples showed P. falciparum, 92 (10.5%) had high parasitaemia and 76 were available for prozone testing. None of the two Pf-pLDH RDTs, but all six HRP-2 RDTs showed prozone, at frequencies between 6.7% and 38.2%. Negative and faint HRP-2 lines accounted for four (3.8%) and 15 (14.4%) of the 104 prozone results in two RDT brands. For the most affected brand, the proportions of prozone with no visible or faint HRP-2 lines were 10.9% (CI: 5.34-19.08), 1.2% (CI: 0.55-2.10) and 0.1% (CI: 0.06-0.24) among samples with high parasitaemia, all positive samples and all submitted samples respectively. Prozone occurred mainly, but not exclusively, among young children.
Prozone occurs at different frequency and intensity in HRP-2 RDTs and may decrease diagnostic accuracy in the most affected RDTs.
Broad-range real-time PCR and sequencing of the 16S rRNA gene region is a widely known method for the detection and identification of bacteria in clinical samples. However, because of the need for sequencing, such identification of bacteria is time-consuming. The aim of our study was to develop a more rapid 16S real-time PCR-based identification assay using species- or genus-specific probes. The Gram-negative bacteria were divided into Pseudomonas species, Pseudomonas aeruginosa, Escherichia coli, and other Gram-negative species. Within the Gram-positive species, probes were designed for Staphylococcus species, Staphylococcus aureus, Enterococcus species, Streptococcus species, and Streptococcus pneumoniae. The assay also included a universal probe within the 16S rRNA gene region for the detection of all bacterial DNA. The assay was evaluated with a collection of 248 blood cultures. In this study, the universal probe and the probes targeting Pseudomonas spp., P. aeruginosa, E. coli, Streptococcus spp., S. pneumoniae, Enterococcus spp., and Staphylococcus spp. all had a sensitivity and specificity of 100%. The probe specific for S. aureus showed eight discrepancies, resulting in a sensitivity of 100% and a specificity of 93%. These data showed high agreement between conventional testing and our novel real-time PCR assay. Furthermore, this assay significantly reduced the time needed for identification. In conclusion, using pathogen-specific probes offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.
The use of rifampin as an adjunct in biofilm-associated infections is based on the ability to penetrate into biofilms and a presumed activity against dormant bacteria. Yet, its efficacy remains contradictory, and rifampin-resistant strains frequently emerge during therapy. Therefore, the efficacy against rifampin-susceptible and isogenic rifampin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) strains was evaluated. Biofilms were generated under static conditions using MSSA with various genetic backgrounds. Oxacillin alone or with rifampin at various concentrations was subsequently added, and after 24 h biomass and viable cell counts were determined. Upon rifampin addition, interstrain variations in viable count change, ranging from a tendency toward antagonism to synergy, were observed among all strains tested, irrespective of the genetic background of the strain. Similar variations were observed in changes in biomass. The decrease in viable count upon rifampin addition was negatively correlated to formation of large amounts of biomass, since strains embedded by more biomass showed a diminished reduction in viable count. Rifampin (1 μg/ml) as adjunct to oxacillin achieved greater reductions in biomass produced by most rifampin-susceptible isolates, ranging from 17 to 54%, compared to 4% for oxacillin alone. In contrast, rifampin had no additional value in reduction of biomass of isogenic rifampin-resistant mutants. At subinhibitory concentrations of rifampin (0.008 μg/ml), none of the strains tested yielded an extra reduction in biomass that was ≥40%. In conclusion, the effects of rifampin as adjunct on biomass and viable count were unpredictable, and the use of rifampin against biofilm containing rifampin-resistant strains seems unwarranted.
The present study assessed malaria RDT kits for adequate and correct packaging, design and labelling of boxes and components. Information inserts were studied for readability and accuracy of information.
Criteria for packaging, design, labelling and information were compiled from Directive 98/79 of the European Community (EC), relevant World Health Organization (WHO) documents and studies on end-users' performance of RDTs. Typography and readability level (Flesch-Kincaid grade level) were assessed.
Forty-two RDT kits from 22 manufacturers were assessed, 35 of which had evidence of good manufacturing practice according to available information (i.e. CE-label affixed or inclusion in the WHO list of ISO13485:2003 certified manufacturers). Shortcomings in devices were (i) insufficient place for writing sample identification (n = 40) and (ii) ambiguous labelling of the reading window (n = 6). Buffer vial labels were lacking essential information (n = 24) or were of poor quality (n = 16). Information inserts had elevated readability levels (median Flesch Kincaid grade 8.9, range 7.1 - 12.9) and user-unfriendly typography (median font size 8, range 5 - 10). Inadequacies included (i) no referral to biosafety (n = 18), (ii) critical differences between depicted and real devices (n = 8), (iii) figures with unrealistic colours (n = 4), (iv) incomplete information about RDT line interpretations (n = 31) and no data on test characteristics (n = 8). Other problems included (i) kit names that referred to Plasmodium vivax although targeting a pan-species Plasmodium antigen (n = 4), (ii) not stating the identity of the pan-species antigen (n = 2) and (iii) slight but numerous differences in names displayed on boxes, device packages and information inserts. Three CE labelled RDT kits produced outside the EC had no authorized representative affixed and the shape and relative dimensions of the CE symbol affixed did not comply with the Directive 98/79/EC. Overall, RDTs with evidence of GMP scored better compared to those without but inadequacies were observed in both groups.
Overall, malaria RDTs showed shortcomings in quality of construction, design and labelling of boxes, device packages, devices and buffers. Information inserts were difficult to read and lacked relevant information.
The present study evaluated the SD Bioline Malaria Ag 05FK40 (SDFK40), a three-band RDT detecting Plasmodium falciparum-specific parasite lactate dehydrogenase (Pf-pLDH) and pan Plasmodium-specific pLDH (pan-pLDH), in a reference setting.
The SDFK40 was retrospectively and prospectively tested against a panel of stored (n = 341) and fresh (n = 181) whole blood samples obtained in international travelers suspected of malaria, representing the four Plasmodium species as well as Plasmodium negative samples, and compared to microscopy and PCR results. The prospective panel was run together with OptiMAL (Pf-pLDH/pan-pLDH) and SDFK60 (histidine-rich protein-2 (HRP-2)/pan-pLDH).
Overall sensitivities for P. falciparum tested retrospectively and prospectively were 67.9% and 78.8%, reaching 100% and 94.6% at parasite densities >1,000/μl. Sensitivity at parasite densities ≤ 100/μl was 9.1%. Overall sensitivities for Plasmodium vivax and Plasmodium ovale were 86.7% and 80.0% (retrospectively) and 92.9% and 76.9% (prospectively), reaching 94.7% for both species (retrospective panel) at parasite densities >500/μl. Sensitivity for Plasmodium malariae was 21.4%. Species mismatch occurred in 0.7% of samples (3/411) and was limited to non-falciparum species erroneously identified as P. falciparum. None of the Plasmodium negative samples in the retrospective panel reacted positive. Compared to OptiMAL and SDFK60, SDFK40 showed lower sensitivities for P. falciparum, but better detection of P. ovale. Inter-observer agreement and test reproducibility were excellent, but lot-to-lot variability was observed for pan-pLDH results in case of P. falciparum.
SDFK40 performance was poor at low (≤ 100/μl) parasite densities, precluding its use as the only diagnostic tool for malaria diagnosis. SDFK40 performed excellent for P. falciparum samples at high (>1,000/μl) parasite densities as well as for detection of P. vivax and P. ovale at parasite densities >500/μl.
Clara cell protein 10 (CC-10) has been associated with inflammatory and infectious pulmonary diseases. This study evaluates CC-10 concentrations in bronchoalveolar lavage (BAL) fluid as a potential marker of ventilator-associated pneumonia (VAP).
Between January 2003 and December 2007, BAL fluid samples obtained from critically ill patients at the intensive care unit of the Maastricht University Medical Centre clinically suspected of having VAP were included. Patients were divided into two groups: (1) microbiologically confirmed VAP (the VAP group) and (2) microbiologically unconfirmed VAP (the non-VAP group). The concentration of CC-10 was measured by means of a commercially available enzyme-linked immunosorbent assay kit, and retrospective analysis was performed. Areas under the curve of receiver operating characteristic curves were calculated for CC-10 concentrations.
A total of 196 patients (122 men, 74 women) were included. A total of 79 (40%) of 196 cases of suspected VAP were microbiologically confirmed. The median CC-10 concentration in the VAP group was 3,019 ng/mL (range, 282 to 65,546 ng/mL) versus 2,504 ng/mL (range, 62 to 30,240 ng/mL) in the non-VAP group (P = 0.03). There was no significant difference in CC-10 concentrations between patients treated with or without corticosteroids (P = 0.26) or antibiotic therapy (P = 0.9). The CC-10 concentration did not differ significantly between patients with Gram-positive versus Gram-negative bacteria that caused the VAP (P = 0.06). However, CC-10 concentrations did differ significantly between the late-onset VAP group and the non-VAP group.
The CC-10 concentration in BAL fluid yielded low diagnostic accuracy in confirming the presence of VAP.
Malaria rapid diagnostic tests (RDTs) are increasingly used as a tool for the diagnosis of malaria, both in endemic and in non-endemic settings. The present study reports the results of an external quality assessment (EQA) session on RDTs in a non-endemic setting.
After validation of antigen stability during shipment at room temperature, three clinical samples and a questionnaire were sent to clinical laboratories in Belgium and the Grand Duchy of Luxembourg using malaria RDTs. Participants were asked to report the results of the RDTs as observations (visibility of the RDT control and test lines) and interpretations (report as formulated to the clinician). In addition, participants were invited to fill in a questionnaire on the place of RDTs in the diagnostic strategy of malaria.
A total of 128/133 (96.2%) of clinical laboratories using RDTs participated. Six three-band and one four-band RDT brands were used. Analytical errors were rare and included (i) not recognizing invalid RDT results (1.6%) and (ii) missing the diagnosis of Plasmodium falciparum (0.8%). Minor errors were related to RDT test result interpretation and included (i) reporting "RDT positive" without species identification in the case of P. falciparum and non-falciparum species (16.9% and 6.5% respectively) and (ii) adding incorrect comments to the report (3.2%). Some of these errors were related to incorrect RDT package insert instructions such as (i) not reporting the possibility of mixed species infection in the case of P. falciparum and Plasmodium vivax (35.5% and 18.5% respectively) and (ii) the interpretation of P. vivax instead of non-falciparum species at the presence of a pan-species antigen line (4.0%). According to the questionnaire, 48.8% of participants processed ≤20 requests for malaria diagnosis in 2009. During opening hours, 93.6% of 125 participants used RDTs as an adjunct to microscopy but outside opening hours, nearly one third of 113 participants relied on RDTs as the primary (4.4%) or the single tool (25.7%) for malaria diagnosis.
In this non-endemic setting, errors in RDT performance were mainly related to RDT test line interpretations, partly due to incorrect package insert instructions. The reliance on RDTs as the primary or the single tool for the diagnosis of malaria outside opening hours is of concern and should be avoided.
Two novel preanalysis sample treatment tools were evaluated in combination with four DNA extraction kits for the selective isolation of bacterial DNA from whole blood. The combination of performing a preanalysis sample treatment and using a larger sample volume increased the detection limit to 50 CFU per ml.
Along with angioplasty, autologus vein grafts are commonly used for artery bypass grafting in patients with advanced arterial stenosis and drug-resistant angina pectoris. Although initially a successful procedure, long-term functionality is limited due to proliferation and migration of smooth muscle cells. Like in atherosclerosis, common chronic infections caused by viruses and bacteria may contribute to this process of vein graft failure. Here we investigated the possible role of Chlamydia pneumoniae (Cpn) in the pathogenesis of venous graft failure in an experimental animal model. In 2 groups (n = 10 rats/group), an epigastric vein-to-common femoral artery interposition graft was placed. Immediately thereafter, rats were infected with Cpn (5*108 IFU) or injected with control solutions. Rats were sacrificed three weeks after surgery and the grafts were harvested for morphometrical and immunohistochemical analysis.
Cpn administration immediately after vein grafting resulted in a significant increase in medial cross-sectional area, wall thickness and total wall area. There were no significant differences in T-cell or macrophage influx. Likewise, although positive immunostaining for both HSP60 and CRP could be detected, no differences were found between groups. Based on the observation that the number of cells/μm2 was also not altered, we conclude that Cpn infection stimulates smooth muscle cell proliferation by hereunto unknown molecular mechanisms, resulting in a significant increase in intimal hyperplasia.
In conclusion, in a well defined animal model we present here for the first time evidence for a role of Chlamydia pneumoniae in the process of venous graft failure.
Neuroinflammation, initiated by cerebral infection, is increasingly postulated as an aetiological factor in neurodegenerative diseases such as Alzheimer’s disease (AD). We investigated whether Chlamydia pneumoniae (Cpn) infection results in extracellular aggregation of amyloid beta (Aβ) in BALB/c mice. At 1 week post intranasal infection (p.i.), Cpn DNA was detected predominantly in the olfactory bulbs by PCR, whereas brains at 1 and 3 months p.i. were Cpn negative. At 1 and 3 months p.i., extracellular Aβ immunoreactivity was detected in the brain of Cpn-infected mice but also in the brain of mock-infected mice and mice that were neither Cpn infected nor mock infected. However, these extracellular Aβ aggregates showed morphological differences compared to extracellular Aβ aggregates detected in the brain of transgenic APP751SL/PS1M146L mice. These data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD-like Aβ plaques in the brain of BALB/c mice, as suggested before. However, future studies are required to resolve these differences and to investigate whether Cpn is indeed an etiological factor in AD pathogenesis.
Neuroinflammation; Chlamydia pneumoniae; Amyloid-beta
Rat cytomegalovirus (RCMV) is a β-herpesvirus with a 230-kbp genome containing over 167 open reading frames (ORFs). RCMV gene expression is tightly regulated in cultured cells, occurring in three distinct kinetic classes (immediate early, early, and late). However, the extent of viral-gene expression in vivo and its relationship to the in vitro expression are unknown. In this study, we used RCMV-specific DNA microarrays to investigate the viral transcriptional profiles in cultured, RCMV-infected endothelial cells, fibroblasts, and aortic smooth muscle cells and to compare these profiles to those found in tissues from RCMV-infected rat heart transplant recipients. In cultured cells, RCMV expresses approximately 95% of the known viral ORFs with few differences between cell types. By contrast, in vivo viral-gene expression in tissues from rat heart allograft recipients is highly restricted. In the tissues studied, a total of 80 viral genes expressing levels twice above background (5,000 to 10,000 copies per μg total RNA) were detected. In each tissue type, there were a number of genes expressed exclusively in that tissue. Although viral mRNA and genomic DNA levels were lower in the spleen than in submandibular glands, the number of individual viral genes expressed was higher in the spleen (60 versus 41). This finding suggests that the number of viral genes expressed is specific to a given tissue and is not dependent upon the viral load or viral mRNA levels. Our results demonstrate that the profiles, as well as the amplitude, of viral-gene expression are tissue specific and are dramatically different from those in infected cultured cells, indicating that RCMV gene expression in vitro does not reflect viral-gene expression in vivo.
Epstein-Barr virus (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis and Burkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesviruses. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has hitherto escaped attention, yet BILF1-like genes are conserved among all known lymphocryptovirus species, suggesting that they play a pivotal role in viral infection. To determine the function of EBV BILF1, the activity of this gene and its products was studied. BILF1-specific mRNA was detected in various EBV-positive cell types and found to be expressed predominantly during the immediate early and early phases of infection in vitro. Interestingly, in COS-7 cells transfected with BILF1 expression constructs, a decrease in forskolin-induced CRE-mediated transcription was measured, as well as an increase in NF-κB-mediated transcription. In contrast, CRE-mediated transcription was increased in EBV-positive Burkitt's lymphoma cells as well as EBV-positive lymphoblastoid B cells transfected with BILF1, whereas NF-κB-mediated transcription levels remained unaffected in these cells. All observed activities were sensitive to treatment with pertussis toxin, indicating that the BILF1-encoded protein mediates these activities by coupling to G proteins of the Gi/o class. Finally, reduced levels of phosphorylated RNA-dependent antiviral protein kinase were observed in COS-7 and Burkitt's lymphoma cells transfected with BILF1. Neither of the observed effects required a ligand to interact with the BILF1 gene product, suggesting that BILF1 encodes a constitutively active GPCR capable of modulating various intracellular signaling pathways.
Background: As one of the natural perturbants, infection with cytomegalovirus (CMV) is believed to play a role in the development of Type I diabetes. Using the DP-BB rat model for autoimmune diabetes, we here report about possible mechanisms responsible for R(at)CMV-induced accelerated onset of diabetes.
Methods: Rats were i.p. infected with 2 × 106 plaque forming units (pfu) RCMV and followed for diabetes development. Presence of RCMV antigens and DNA was analyzed by immunohistochemistry and PCR on pancreatic tissue and isolated islets. The effect of viral infection on peritoneal macrophages (pMΦ) and diabetes development was studied by analyzing numbers of pMΦ, virus permissiveness and by depletion of this subset by peritoneal lavage.
Results: RCMV accelerated onset of diabetes without infecting pancreatic islets. Immunohistochemistry and PCR on pancreas and isolated islets indicated that islets are non-permissive for RCMV. Infection results in an influx of pMΦ 1 day p.i. of which ~0.05% showed signs of reproductive infection. Depletion of pMΦ on days 1-3 p.i. completely counteracted the accelerating effect of RCMV.
Interpretation: RCMV accelerates onset of diabetes without infecting pancreatic islets. pMΦ might function as an carriage to disseminate virus to the pancreas where they enhance activation of autoreactive T cells resulting in accelerated onset of diabetes.
Background: Viral infections are thought to play a role in the development of autoimmune diseases like type 1 diabetes. In this study we investigated the effect of Rat Cytomegalovirus (RCMV) infection on cellular immunity in a well-defined animal model for diabetes, the Biobreeding (BB) rat.
Methods: Diabetes prone (DP)- and Diabetes resistant (DR)-BB rats were infected with 2 × 106 plaque forming units (pfu) RCMV. Diabetes development was monitored by frequent blood-glucose analysis. Effects of RCMV on CD4+, CD8+ and Vβ-TCR+ T-cell subsets were measured in vivo, and in vitro after restimulation with RCMV-infected fibroblasts. Proliferative capacity was determined by 3H-Thymidine incorporation.
Results: RCMV-infection resulted in a significant acceleration of diabetes onset in DP-BB rats ( p=0.003). Percentages CD4+ and CD8+ T-cells were not affected in vivo. In vitro, RCMV-restimulation resulted in a decreased CD4+/CD8+ blastoid T-cell ratio compared to ConA ( p=0.00028). Furthermore, RCMV-restimulation resulted in a strong RCMV-specific proliferation, which comprises about 50% of the response triggered by ConA. Vβ-TCR percentages did not change upon RCMV-infection or RCMV-restimulation.
Interpretation: RCMV-restimulation of splenic T-cells in vitro resulted in a strong RCMV-specific proliferation, probably also including autoreactive T-cells. In vivo, this polyclonal response might be involved in the observed accelerated diabetes development in DP-BB rats upon RCMV-infection.
Cytomegalovirus (CMV) infections have been shown to dramatically affect solid organ transplant graft survival in both human and animal models. Recently, it was demonstrated that rat CMV (RCMV) infection accelerates the development of transplant vascular sclerosis (TVS) in both rat heart and small bowel graft transplants. However, the mechanisms involved in this process are still unclear. In the present study, we determined the kinetics of RCMV-accelerated TVS in a rat heart transplant model. Acute RCMV infection enhances the development of TVS in rat heart allografts, and this process is initiated between 21 and 24 days posttransplantation. The virus is consistently detected in the heart grafts from day 7 until day 35 posttransplantation but is rarely found at the time of graft rejection (day 45 posttransplantation). Grafts from RCMV-infected recipients had upregulation of chemokine expression compared to uninfected controls, and the timing of this increased expression paralleled that of RCMV-accelerated neointimal formation. In addition, graft vessels from RCMV-infected grafts demonstrate the increased infiltration of T cells and macrophages during periods of highest chemokine expression. These results suggest that CMV-induced acceleration of TVS involves the increased graft vascular infiltration of inflammatory cells through enhanced chemokine expression.
The rat cytomegalovirus (RCMV) R33 gene is conserved among all betaherpesviruses and encodes a protein (pR33) that shows sequence similarity with chemokine-binding G protein-coupled receptors (GPCRs). Previously, the physiological significance of the R33 gene was demonstrated by the finding that an RCMV strain with R33 deleted is severely attenuated in vivo and is unable to either enter or replicate in the salivary glands of infected rats. Here, we report that RCMV pR33 is expressed as a functional GPCR that signals in an agonist-independent manner in both COS-7 and Rat2 cells. Transient expression of pR33 in COS-7 cells results in constitutive activation of phospholipase C (PLC) due to coupling to G proteins of the Gq class. Interestingly, PLC activation is partially inhibited by cotransfection with Gα-transducin subunits, which indicates the involvement of Gβγ as well as Gα subunits in pR33-mediated signaling. Surprisingly, PLC activation is also partially inhibited by addition of pertussis toxin (PTX), suggesting that pR33 activates not only Gq but also Gi/0 proteins. The constitutive activation of Gi/0 proteins by pR33 is further demonstrated by the PTX-sensitive decrease of CRE-mediated transcription and the PTX-sensitive increase of both NF-κB- and SRE-mediated transcription. In contrast to its homolog of human herpesvirus 6B (pU12), pR33 does not bind RANTES.
We have determined the complete genome sequence of the Maastricht strain of rat cytomegalovirus (RCMV). The RCMV genome has a length of 229,896 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. RCMV was found to have counterparts of all but one of the open reading frames (ORFs) that are conserved between murine CMV (MCMV) and human CMV (HCMV). Like HCMV, RCMV lacks homologs of the genes belonging to the MCMV m02 glycoprotein gene family. However, RCMV contains 15 ORFs with homology to members of the MCMV m145 glycoprotein gene family. Four ORFs are predicted to encode homologs of host proteins; R33 and R78 both putatively encode G protein-coupled receptors, whereas r144 and r131 encode homologs of major histocompatibility class I heavy chains and CC chemokines, respectively. An intriguing feature of the RCMV genome is the presence of an ORF, r127, with similarity to the rep gene of parvoviruses as well as ORF U94 of human herpesvirus 6A (HHV-6A) and HHV-6B. Counterparts of these ORFs have not been found in the other sequenced herpesviruses.