Tick-borne fever caused by Anaplasma phagocytophilum in Germany

2012 ◽  
Vol 40 (02) ◽  
pp. 101-106 ◽  
Author(s):  
M. Nieder ◽  
C. Silaghi ◽  
D. Hamel ◽  
K. Pfister ◽  
R. Schmäschke ◽  
...  
Keyword(s):  
Real Time ◽  
Milk Production ◽  
Real Time Pcr ◽  
Anaplasma Phagocytophilum ◽  
Clinical Findings ◽  
Immunofluorescence Assay ◽  
High Fever ◽  
West Germany ◽  
Nasal Discharge ◽  
North West

SummaryFour cows from North-West Germany have been diagnosed with tickborne fever (TBF) based on the demonstration of morulae in neutrophilic granulocytes in their blood smears, positive signals in real-time PCR specific for Anaplasma phagocytophilum using DNA extracted from their buffy coats, and demonstration of specific antibodies in their sera using a commercially available immunofluorescence assay. Clinical findings included high fever, decreased milk production, lower limb edema with stiff walking, eye and nasal discharge, and depression. These signs developed about a week after the animals had been brought to the pasture for the first time in their life. All cows recovered after 5–15 days, although DNA of A. phagocytophilum could be detected by real-time PCR up to 6 weeks after onset of the disease. Considering the known prevalences of A. phagocytophilum in ticks in Germany and its detection in dogs and horses, we think that underdiagnosing of TBE in cattle is highly likely. Therefore TBF should be taken into account as differential diagnosis in case of high fever and/or a sudden decrease in milk production in pastured animals.

scholarly journals Laboratory diagnosis of 37 cases of Bartonella endocarditis based on enzyme immunoassay and real-time PCR

2021 ◽  
Author(s):  
Lev Shapira ◽  
Michal Rasis ◽  
Inbal Binsky Ehrenreich ◽  
Yasmin Maor ◽  
Eugene A. Katchman ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Real Time ◽  
Real Time Pcr ◽  
Heart Valves ◽  
Q Fever ◽  
Laboratory Diagnosis ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Complete Agreement ◽  
Pcr Assay

Bartonella spp., mostly B. quintana and B. henselae, are a common cause of culture-negative endocarditis. Serology, using immunofluorescence assay (IFA) and PCR performed on cardiac tissues are the mainstays of diagnosis. We developed an enzyme immunoassay (EIA) and a novel multiplex real-time PCR assay, utilizing Bartonella genus-specific, B. henselae-specific and B. quintana-specific SimpleProbe probes, for diagnosis of Bartonella endocarditis. We aimed to evaluate the performance of these assays. Thirty-seven patients with definite endocarditis, 18 with B. henselae, 18 with B. quintana and one with B. koehlerae were studied. Diagnosis was confirmed by conventional PCR and DNA sequencing of surgical cardiac specimens. Similarly to IFA, anti-Bartonella IgG titers ≥1:800 were found in 94% of patients by EIA; cross-reactivity between B. henselae and B. quintana precluded species-specific serodiagnosis, and frequent (41%) but low-titer cross-reactivity between Coxiella burnetii antibodies and B. henselae antigen was found in patients with Q fever endocarditis. Low-titer (1:100) cross-reactivity was uncommonly found also in patients with brucellosis and culture-positive endocarditis, particularly Enterococcus faecalis endocarditis. Real-time PCR performed on explanted heart valves/vegetations was in complete agreement with results of sequence-based diagnosis with characteristic melting curves. The genus-specific probe identified five additional endocarditis-associated Bartonella spp. at the genus level. In conclusion, EIA coupled with a novel real-time PCR assay can play an important role in Bartonella endocarditis diagnosis and expand the diagnostic arsenal at the disposal of the clinical microbiologist. Since serology remains a major diagnostic tool, recognizing its pitfalls is essential to avoid incorrect diagnosis.

scholarly journals Seroprevalence of Borrelia burgdorferi in Stray Dogs from Southern Italy

2020 ◽  
Vol 8 (11) ◽  
pp. 1688 ◽  
Author(s):  
Paola Galluzzo ◽  
Francesca Grippi ◽  
Santina Di Bella ◽  
Francesco Santangelo ◽  
Sonia Sciortino ◽  
...  
Keyword(s):  
Risk Factors ◽  
Borrelia Burgdorferi ◽  
Real Time ◽  
Real Time Pcr ◽  
Coat Color ◽  
Bacterial Pathogen ◽  
Immunofluorescence Assay ◽  
Stray Dogs ◽  
Ixodes Ticks ◽  
Potential Risk Factors

Borrelia burgdorferi is a bacterial pathogen transmitted by Ixodes ticks and is responsible for Lyme disease in both humans and dogs. The aim of this work was to evaluate B. burgdorferi diffusion among stray dogs in Palermo (Sicily, Italy) by serological methods in order to study the risk factors associated with the infection. Serum and blood samples of 316 dogs were collected from a shelter in Palermo, and were analyzed for the presence of antibodies against B. burgdorferi by indirect immunofluorescence assay (IFA), and of the ospA gene by real-time PCR, respectively. Seventeen sera (5.4%) were positive for the antibodies via IFA and one blood (0.3%) for ospA via real time PCR. On the basis of serological results, the evaluation of the potential risk factors (sex, age, breed and coat color) was carried out. The multivariate analysis indicated that male sex is a factor significantly associated with B. burgdorferi seropositivity. This study confirms that male dogs have a higher risk of developing the disease than females, and represents the first investigation on the spread of B. burgdorferi among stray dogs in Sicily.

scholarly journals Development and Validation of aPneumocystis jiroveciiReal-time Polymerase Chain Reaction Assay for Diagnosis ofPneumocystisPneumonia

2015 ◽  
Vol 26 (5) ◽  
pp. 263-267 ◽  
Author(s):  
Deirdre L Church ◽  
Anshula Ambasta ◽  
Amanda Wilmer ◽  
Holly Williscroft ◽  
Gordon Ritchie ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Pathogenic Fungus ◽  
Immunofluorescence Assay ◽  
Immunocompromised Patients ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Predictive Values ◽  
Sensitivity Specificity

BACKGROUND:Pneumocystis jirovecii(PJ), a pathogenic fungus, causes severe interstitialPneumocystispneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients.

scholarly journals New reports of Australian cutaneous leishmaniasis in Northern Australian macropods

2009 ◽  
Vol 137 (10) ◽  
pp. 1516-1520 ◽  
Author(s):  
A. DOUGALL ◽  
C. SHILTON ◽  
J. LOW CHOY ◽  
B. ALEXANDER ◽  
S. WALTON
Keyword(s):  
Life Cycle ◽  
Cutaneous Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Findings ◽  
Northern Territory ◽  
Causative Organism ◽  
The World ◽  
Species Specific ◽  
Unique Species

SUMMARYCutaneous leishmaniasis caused by various species of Leishmania is a significant zoonotic disease in many parts of the world. We describe the first cases of Australian cutaneous leishmaniasis in eight northern wallaroos, one black wallaroo and two agile wallabies from the Northern Territory of Australia. Diagnosis was made through a combination of gross appearance of lesions, cytology, histology, direct culture, serology and a species-specific real-time PCR. The causative organism was found to be the same unique species of Leishmania previously identified in red kangaroos. These clinical findings provide further evidence for the continuous transmission of the Australian Leishmania species and its presence highlights the importance of continued monitoring and research into the life-cycle of this parasite.

scholarly journals Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244023
Author(s):  
Darunee Chotiprasitsakul ◽  
Pataraporn Pewloungsawat ◽  
Chavachol Setthaudom ◽  
Pitak Santanirand ◽  
Prapaporn Pornsuriyasak
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
Real World ◽  
Real Time Pcr ◽  
Pneumocystis Pneumonia ◽  
Immunofluorescence Assay ◽  
Cohen’S Kappa ◽  
Cohen's Kappa ◽  
Highly Sensitive ◽  
Sensitivity Specificity

Background PCR is more sensitive than immunofluorescence assay (IFA) for detection of Pneumocystis jirovecii. However, PCR cannot always distinguish infection from colonization. This study aimed to compare the performance of real-time PCR and IFA for diagnosis of P. jirovecii pneumonia (PJP) in a real-world clinical setting. Methods A retrospective cohort study was conducted at a 1,300-bed hospital between April 2017 and December 2018. Patients whose respiratory sample (bronchoalveolar lavage or sputum) were tested by both Pneumocystis PCR and IFA were included. Diagnosis of PJP was classified based on multicomponent criteria. Sensitivity, specificity, 95% confidence intervals (CI), and Cohen's kappa coefficient were calculated. Results There were 222 eligible patients. The sensitivity and specificity of PCR was 91.9% (95% CI, 84.0%–96.7%) and 89.7% (95% CI, 83.3%–94.3%), respectively. The sensitivity and specificity of IFA was 7.0% (95% CI, 2.6%–14.6%) and 99.2% (95% CI, 95.6%–100.0%), respectively. The percent agreement between PCR and IFA was 56.7% (Cohen's kappa -0.02). Among discordant PCR-positive and IFA-negative samples, 78% were collected after PJP treatment. Clinical management would have changed in 14% of patients using diagnostic information, mainly based on PCR results. Conclusions PCR is highly sensitive compared with IFA for detection of PJP. Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.

scholarly journals Multiplex Real-Time PCR for Detection of Anaplasma phagocytophilum and Borrelia burgdorferi

2004 ◽  
Vol 42 (7) ◽  
pp. 3164-3168 ◽  
Author(s):  
J. W. Courtney ◽  
L. M. Kostelnik ◽  
N. S. Zeidner ◽  
R. F. Massung
Keyword(s):  
Borrelia Burgdorferi ◽  
Real Time ◽  
Real Time Pcr ◽  
Anaplasma Phagocytophilum

scholarly journals MiR-145 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells through targeting FoxO1

2017 ◽  
Vol 243 (4) ◽  
pp. 386-393 ◽  
Author(s):  
Wei Hao ◽  
Hongzhi Liu ◽  
Lugang Zhou ◽  
Yujie Sun ◽  
Hao Su ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Immunofluorescence Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Alp Activity ◽  
Gene Assay ◽  
Before And After

In this study, we aimed to investigate the expression of miR-145 before and after hASCs osteogenic differentiation. We also intended to explore the influence of the target relationship between miR-145 and FoxO1 on osteogenic differentiation. Dual-luciferase reporter gene assay and real-time PCR were used to confirm the target relationship between miR-145 and FoxO1. Furthermore, the modulatory effects of miR-145 and FoxO1 on hASCs osteoinductive differentiation were measured by real-time PCR , Western blot, ALP staining, ARS staining, and cell immunofluorescence assay. After osteogenic differentiation, miR-145 was gradually down-regulated, while FoxO1 was up-regulated in hASCs. MiR-145 could directly target FoxO1 3′UTR. FoxO1 was negatively regulated by miR-145. After osteoinductive differentiation, BSP, Ocn, and OPN expression was lowered with the overexpression of miR-145 or the knockdown of FoxO1. Furthermore, ALP and ARS staining assay results showed weakened ALP activity and extracellular matrix calcification. When overexpressing miR-145 and FoxO1 simultaneously, no obvious change in ALP activity and extracellular matrix calcification was seen. MiR-145 could suppress hASCs osteoinductive differentiation by suppressing FoxO1 directly. Impact statement Researching on ASCs was a promising strategy to study osteogenic differentiation. The regulatory role of miR-145 on hASCs osteogenic differentiation remained partially explored. Our study revealed a novel mechanism of the osteogenic differentiation process and suggested that miR-145 and its target gene FoxO1 may be potential targets for the therapy of human osteogenic-related disorders.

scholarly journals Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis

2014 ◽  
Vol 2014 ◽  
pp. 1-4 ◽  
Author(s):  
Mariana R. Pereira ◽  
Fabiana Rocha-Silva ◽  
Cidiane Graciele-Melo ◽  
Camila R. Lafuente ◽  
Telcia Magalhães ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Findings ◽  
Sybr Green ◽  
Polymerase Chain ◽  
Belo Horizonte ◽  
Green System ◽  
Pcr Techniques ◽  
Positive Results

The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome ofLeishmaniaspecies in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n=12) presented positive results for serology and 79% (n=15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient’s blood.

scholarly journals 744. Clinical Performance of Real-time PCR in the Diagnosis of Pneumocystis jirovecii Pneumonia compared with Immunofluorescence Assay

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S420-S420
Author(s):  
Cristina Veintimilla ◽  
Ana Alvarez-Uria ◽  
Pablo Martin-Rabadan ◽  
Luis Alcala ◽  
Patricia Muñoz ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Subunit ◽  
Immunofluorescence Assay ◽  
Pneumocystis Jirovecii ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Rrna Gene ◽  
Sample Type ◽  
Sensitivity Specificity ◽  
Pcr Targeting

Abstract Background The laboratory diagnosis of Pneumocystis jirovecii pneumonia (PJP) has been traditionally based on microscopy techniques, which have suboptimal sensitivity and depends on the experience and skills of the microbiologist. Molecular detection assays based in PCR (Polymerase chain reaction) could improve sensitivity. Our aim was to evaluate the utility of real-time PCR in the diagnosis of PJP compared with IFA (Immunofluorescence assay) performed in different respiratory samples of patients with PJP suspicion for routine use in a clinical laboratory setting. Methods From September 2015 to April 2018, we studied by a real-time PCR targeting the large subunit of rRNA gene of P. jirovecii (PJ-PCR RealCycler PJIR kit Progenie Molecular) and Immunofluorescence assay (MONOFLUO P. carinii IFA BioRad) in all respiratory samples received for microbiological diagnosis of PJP. The definite clinical diagnosis of PJP was established by infectious disease physicians considering symptoms, radiological and laboratory findings. Results Overall, 302 samples were included (182 bronchoalveolar lavage, 67 sputum, 53 tracheal aspirates). PJ-PCR was positive in 51 (16.9%) and IFA in 11 (3.6%) of the patients with PJP. There were not IFA positive/PCR negative samples. Sensitivity, specificity, PPV and NPV for IFA were 26% (95%CI 15.9-39.6%), 100% (95%CI 98.5-100%), 100% (95% CI 77.2-100%) and 87.2% (95% CI 82.6-90.6%). Whereas, sensitivity, specificity, PPV and NPV for PCR was 92% (95%CI 81.2-96.8%), 98% (95% CI 95.4-99.2%), 90.2% (95% CI 79.0-95.7%) and 98.4% (95% CI 96.0-99.4%). PJ-PCR had sensitivity > 80% and specificity > 90% in all type of samples included. A definitive diagnosis of PJP was considered in 50 (16.6%) patients, including 4 (1.3%) cases with negative PJ-PCR. Five cases (9.8%) with positive PJ-PCR were considered as colonization. Conclusion P. jirovecii PCR improves the sensitivity and NPV of PJP diagnosis respecting to IFA, regardless of respiratory sample type. Our results suggest that Microbiology laboratories should use PCR techniques to diagnose PJP better than IFA. Disclosures All Authors: No reported disclosures

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