Low prevalence of Bartonella henselae infections in Norwegian domestic and feral cats

Bartonella henselae is the causative agent of cat scratch disease (CSD). This clinical entity is very rarely encountered in human medical practice in Norway. B. henselae infections including bacteraemia in cats have been frequently reported. The objective of the present study was to investigate the seroprevalence rate and the degree of B. henselae bacteraemia in Norwegian domestic and feral cats. One hundred cats investigated at a small animal veterinary practice in the middle of Norway were included in the study. Blood collected in Isolator blood-lysis tubes and lysates of erythrocytes after freezing and thawing were cultured. PCR analysis of whole blood was also performed. Serology was performed by indirect fluorescence assay (IFA) and enzyme immunoassay (EIA) using immobilised B. henselae Houston-1 strain as antigen. None of the 100 cats investigated was found to be bacteraemic. All 100 cats were seronegative when analysed by IFA; one cat was positive by EIA. The discrepancy between IFA and EIA of this particular cat is probably due to cross-reactive antibodies. Contrary to findings reported from several geographic regions, B. henselae infections in Norwegian cats appear to be virtually absent. This in turn may explain why CSD has not been reported in human medical practice in Norway.     

Bartonellosis: light and shadows in diagnostic and therapeutic issues

Cat-scratch disease involves a prolonged and/or complicated course, and lymph node drainage is usually required. Culture and molecular techniques often yield negative results, but immunofluorescence assays may give early information, and elevated antibodies may persist for months. Cat-scratch disease should be suspected in patients with prominent swelling of lymph nodes draining from the upper limbs, limited systemic involvement, and typical epidemiological-clinical features. The temporal antibody response during the sub-acute course remains unknown. Although biomolecular assays are available, the time between onset and investigation is an obstacle to positive results. The role of surgical debridement and the unpredictable activity of antimicrobial agents warrant further investigation.     

IgM to Bartonella henselae in cat-scratch disease and during acute Epstein-Barr virus infection

The diagnostic value of IgM to Bartonella henselae was evaluated in 20 children with cat-scratch disease (CSD) and controls consisting of 20 blood donors and 20 children with enlarged lymph nodes without CSD by two indirect immunofluorescence assays (IFA). One was based on B. henselae cocultivated with Vero cells (host cell-associated IFA), and the other on B. henselae grown on agar (host cell-free IFA). With the host cell-associated IFA, 18 of 20 children with CSD revealed IgM, whereas only 14 did so with the host cell-free IFA. Sera of two blood donors as well as sera from three children with enlarged lymph nodes without CSD showed also positive IgM to cell-associated B. henselae. This study reveals that the IFA applied had sensitivities of 70 – 90% and specificities of 87.5 – 100% for detecting IgM to B. henselae. Additionally, 20 patients with IgM to Epstein-Barr virus (EBV) capsid antigen were tested for IgM to B. henselae. Sera of 16 and 9 of these patients revealed IgM to B. henselae with the host cell-associated and the host cell-free IFA, respectively. Using Western blot these sera were demonstrated to react against linearized proteins of Vero cells and of B. henselae. Thus, since acute EBV infection may substantially reduce the specificity of B. henselae-specific IgM tests, we conclude that diagnosis of CSD should be confirmed by a significant IgG titer to B. henselae or by detection of this pathogen. Received: 7 May 1997     

Quantification of Chlamydia pneumoniae in cultured human macrophages and HL cells: comparison of real‐time PCR,immunofluorescence and ELISA methods

Poikonen K, Lajunen T, Silvennoinen‐Kassinen S, Leinonen M, Saikku P. Quantification of Chlamydia pneumoniae in cultured human macrophages and HL cells: comparison of real‐time PCR, immunofluorescence and ELISA methods. APMIS 2010; 118: 45–8. Chlamydia pneumoniae is an intracellular gram‐negative bacterium, which replicates only in eukaryotic cells. Quantification of C. pneumoniae in cell culture is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally, this has been performed by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary, and multiple inclusions are also seen. Therefore, methods are needed to quantify exact amounts of C. pneumoniae in cells. Here, we describe a new method based on the real‐time PCR quantification of chlamydial genomes adjusted to the number of human genomes in cultures. In human epithelial (HL) cell cultures, the C. pneumoniae inclusion numbers and the ratio of C. pneumonia genomes/human genome (Cpn/Hum) correlated significantly (r = 0.978, p < 0.001); thus with HL cells, both methods are usable. However, in macrophage cultures, the correlation was weaker (r = 0.133, p = 0.036) and we recommend PCR quantification for exact measurements.     

Detection of Bartonella henselae and Bartonella quintana by a simple and rapid procedure using broad-range PCR amplification and direct single-strand sequencing of part of the 16S rRNA gene

Objective: To detect directly Bartonella henselae and Bartonella quintana using culture-independent, molecular techniques, and to evaluate a simple and rapid procedure that allows uncultivable bacteria to be detected in usually sterile clinical specimens in a diagnostic laboratory.
Method: From four clinical specimens proven to contain B. henselae (n=3) or B. quintana (n=1) DNA, part of the 16S rRNA gene was amplified using the polymerase chain reaction (PCR) and broad-range bacterial primers followed by reamplification and direct, single-strand sequencing. The partial 16S rRNA sequences were compared to reference sequences in databases.
Results: Similarities between sequences derived from clinical samples and those of B. henselae and B. quintana , respectively, were in the range 98.7-100%, indicating a strong association to the genus Bartonella. Intraspecies sequence variations within the B. henselae sequences were observed.
Conclusions: The method described is a rapid, sensitive and reliable tool to generate partial 16S rRNA sequences of B. henselae and B. quintana directly from normally sterile clinical specimens. It is compatible with adequate prevention of contamination as is needed for diagnostic purposes, and it possesses the potential to detect other pathogens, including those as yet unknown.     

Small Indian mongooses and masked palm civets serve as new reservoirs of Bartonella henselae and potential sources of infection for humans

The prevalence and genetic properties of Bartonella species were investigated in small Indian mongooses and masked palm civets in Japan. Bartonella henselae, the causative agent of cat-scratch disease (CSD) was isolated from 15.9% (10/63) of the mongooses and 2.0% (1/50) of the masked palm civets, respectively. The bacteraemic level ranged from 3.0 × 10¹ to 8.9 × 10³ CFU/mL in mongooses and was 7.0 × 10³ CFU/mL in the masked palm civet. Multispacer typing (MST) analysis based on nine intergenic spacers resulted in the detection of five MST genotypes (MSTs 8, 14, 37, 58 and 59) for the isolates, which grouped in lineage 1 with MST genotypes of isolates from all CSD patients and most of the cats in Japan. It was also found that MST14 from the mongoose strains was the predominant genotype of cat and human strains. This is the first report on the isolation of B. henselae from small Indian mongooses and masked palm civets. The data obtained in the present study suggest that these animals serve as new reservoirs for B. henselae, and may play a role as potential sources of human infection.     

Performance characteristics and clinical utility of a hybrid ELISA for detection of ANA.

To investigate the clinical utility of a newly developed hybrid ELISA for antinuclear antibodies (ANA), a cross-sectional study of patients admitted to the Section of Rheumatology was initiated. The ELISA was compared to indirect immunofluorescence (IIF) on HEp-2 cells. Accuracy of tests was analyzed using receiver-operating characteristic methodology (ROC). In addition, diagnostic sensitivity, specificity and predictive values were calculated for each assay. Results from the ROC analysis showed a slightly superior accuracy for IIF as compared to ELISA. Furthermore, IIF showed higher diagnostic sensitivity and positive predictive value for all combinations of patients and reference populations. This was due to enhanced detection by IIF, in contrast to ELISA, of diagnostically useful antibodies. IIF detected 87.4% and ELISA detected 84.2% of sera with antibodies against extractable nuclear antigens (ENA). In addition, IIF detected diagnostically important antibodies that are not included among the anti-ENA. The hybrid ELISA either lacks or does not contain the relevant antigens in sufficient amount. Inclusion of these antigens may further enhance the performance characteristics of the ELISA.     

An enzyme-linked immunosorbent assay for antistriational antibodies associated with myasthenia gravis and thymoma: comparison with indirect immunofluorescence

Autoantibodies to the striations of skeletal muscle (AStrA) detected by immunofluorescence are useful in the diagnosis of a thymoma associated with myasthenia gravis (MG). With the intention of developing a better method, an enzyme-linked immunosorbent assay (ELISA) has been evaluated in 147 MG patients and 200 healthy controls. An additional 107 patients with various autoimmune diseases and autoantibodies were also tested. With a crude actomyosin preparation, the ELISA gave similar results to immunofluorescence, viz. positives in 42% of MG patients, but in all with a histologically proven thymoma. Less than 1% of the healthy controls were positive but false positives were found in patients with liver disease and anti-smooth muscle antibodies. After treatment of rheumatoid arthritis with D-penicillamine the titre of AStrA may rise. The ELISA was shown to be sensitive and reproducible, but immunofluorescence is a more practical method of distinguishing between the different categories of anti-muscle antibodies. ELISA should prove particularly useful for quantitation and sequential monitoring.     

The evaluation of a quantitative enzyme-linked immunosorbent assay (ELISA) for anti-Aspergillus fumigatus IgG

A standard enzyme-linked immunosorbent assay method for anti-Aspergillus fumigatus IgG in human serum was modified to produce a quantitative assay. The resulting assay was reproducible and capable of separating individual precipitin line groups and provided a means of monitoring the variation in antibody levels over long periods in patients with pulmonary aspergillosis.     

检测抗人疱疹病毒6型IgG的间接免疫荧光试验的建立及其应用

目的 :建立检测血清中人疱疹病毒 6型 (HHV 6 )IgG的间接免疫荧光试验 (IFA)。方法 :用HHV 6国内分离株感染人脐带血单个核细胞制备抗原片 ,建立检测HHV 6IgG的IFA方法 ,并用于育龄期妇女血清流行病学调查。结果 :建立的IFA具有特异性。对 116份育龄期妇女血清标本检测表明 ,HHV 6IgG的阳性率为 72 .4 % ,几何平均滴度 (GMT)为 1∶6 1;在孕妇和正常未孕妇女之间 ,以及不同孕期的孕妇之间 ,HHV 6IgG的阳性率和GMT均无差异 (P >0 .0 5 )。结论 :建立了具有特异性的IFA法 ,可用于对育龄妇女HHV 6感染率的流行病学调查     

The phagocytosis capacity assay: a new radiometric phagocytosis test for clinical use

A radiometric technique is described for measuring phagocytosis which does not require separation of extra- and intracellular microorganisms. Following phagocytosis of optimally opsonized yeast cells (Saccharomyces cerevisiae) by mononuclear cells (MNC) or granulocytes, yeast cells remaining extracellular are labeled with [⁷⁵Se]L-selenomethionine. To measure the phagocytic capacity of the cell population investigated, 1–4×10⁶ leukocytes are incubated with 5, 10, 20, 30 and 40×10⁶ yeast cells/ml for testing. Depending on the actual number of phagocytes in the cell populations investigated, different optimal yeast cell concentrations may be determined. Provided measurements are carried out at these optima even moderate effects of therapy can be detected. In a preliminary study of 22 patients with Hodgkin's disease it was shown, that the phagocytic activity of the MNC fraction was higher, but the activity of granulocytes lower, than that of the healthy controls. After combined chemotherapy the above mentioned values returned to normal. This simple, exact method is recommended for clinical use.     

Identification of Bartonella species in rodents, shrews and cats in Denmark: detection of two B. henselae variants, one in cats and the other in the long-tailed field mouse

Small mammals and stray cats were trapped in two areas of North Zealand, Denmark, and their blood cultured for hemotrophic bacteria. Bacterial isolates were recovered in pure culture and subjected to 16S rDNA gene sequencing. Bartonella species were isolated from five mammalian species: B. grahamii from Microtus agrestis (field vole) and Apodemus flavicollis (yellow-necked field mouse); B. taylorii from M. agrestis, A. flavicollis and A. sylvaticus (long-tailed field mouse); B. tribocorum from A. flavicollis; B. vinsonii subsp. vinsonii from M. agrestis and A. sylvaticus; and B. birtlesii from Sorex vulgaris (common shrew). In addition, two variant types of B. henselae were identified: variant I was recovered from three specimens of A. sylvaticus, and B. henselae variant II from 11 cats; in each case this was the only B. henselae variant found. No Bartonella species was isolated from Clethrionomys glareolus (bank vole) or Micromys minutus (harvest mouse). These results suggest that B. henselae occurs in two animal reservoirs in this region, one of variant I in A. sylvaticus, which may be transmitted between mice by the tick Ixodes ricinus, and another of variant II in cats, which may be transmitted by the cat flea (Ctenocephalides felis). To our knowledge, this is the first report of the occurrence of B. henselae and B. tribocorum in Apodemus mice.     

Clinical evaluation of the NS1 antigen‐capture ELISA for early diagnosis of dengue virus infection in Brazil

The fact that the diagnosis of infection with dengue virus is usually made by detecting IgM antibodies during the convalescent phase of the disease interferes with disease management and, consequently, with reducing mortality rates. This study evaluated the sensitivity and specificity of detection of NS1 in samples of patients suspected of acute dengue virus infection in Brazil. The results were used to institute treatment and the sensitivity and specificity of detection of NS1 were compared to the results of detection of IgM, virus isolation, and RT‐PCR. Detection of NS1 yielded better results than RT‐PCR and virus isolation. When considering IgM detection and RT‐PCR positive results as “gold standards,” the sensitivity and specificity of the NS1 assay were 95.9% and 81.1%, respectively. All patients enrolled in the study were treated promptly and had an uneventful course of the disease. The detection of NS1 provided better results than the diagnostic techniques used currently during the acute phase of disease (RT‐PCR and virus isolation). Detection of NS1 is an important tool for the diagnosis of acute dengue infection, particularly in highly endemic areas, allowing for rapid treatment of patients and reduction of disease burden. J. Med. Virol. 82:1400–1405, 2010. © 2010 Wiley‐Liss, Inc.     

Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections.

A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.     

Herpes simplex virus detection by ELISA: effect of enzyme amplification, nature of lesion sampled and specimen treatment

The relative sensitivity of two enzyme detection procedures was investigated in a simultaneous "monoclonal" ELISA for herpes simplex virus (HSV). A cyclical enzyme amplified detection system with alkaline phosphatase, rather than horse-radish peroxidase and a conventional chromogenic substrate, gave an increase in absolute sensitivity and a 20 to 30% increase in the detection of HSV in routine isolation-positive genital specimens collected in transport medium. The HSV detection rate, with both procedures, was shown to vary with the site and clinical stage of lesion sampled; it was highest with penile vesicular lesions. Direct extraction of the swab specimen in a small volume of diluent further increased the sensitivity of antigen detection giving positive and negative predictive values of 100 and 96% respectively. The overall sensitivity of HSV detection was equivalent to that obtained by isolation in cell culture. The amplified ELISA offers an alternative, rapid, simple, non-culture technique for routine HSV diagnosis that does not rely upon retention of virus viability.     

Diagnosis of human coronavirus infection by immunofluorescence: method and application to respiratory disease in hospitalized children.

Rabbit antisera were prepared against coronavirus strains 229E and OC43 and used successfully to detect viral antigen in epithelial cells shed from the nasopharynx of symptomatic volunteers who had received coronavirus inocula three to four days before. The same serologic reagents were applied to nasopharyngeal secretion cells obtained from 106 infants and children hospitalized with respiratory tract disease and apparently not infected with conventional respiratory viruses. No coronavirus infections were detected by this method. It appears that coronavirus OC43 or 229E infections were not common in children in Tyneside hospitals during the period of study. However, fluorescence is a useful method for detection of coronavirus infections in symptomatic human subjects.     

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr≈30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8–3.3 and 3.0–3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose–response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r² of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319±237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.     

The dynamic change of antibody index against Covid-19 is a powerful diagnostic tool for the early phase of the infection and salvage PCR assay errors

BackgroundCurrently, PCR assay is a golden standard for diagnosis of Covid-19. However, it needs nasopharyngeal swabs, expensive instruments and expertise. It even causes PCR errors.MethodsWe validated the antibody assay (Roche) in 36 followed patients and 1879 controls (medical staffs).ResultsOf 1879 medical staffs, only two (0.11%) were positive by Cut off Index (COI; 1.0) (mean ± SD, 0.094 ± 0.047). Thirty six patients were composed of three groups; Group A,4 from Diamond Princess cruise ship, Group B, 2 infected in Africa, and Group C, 30 infected in Japan. PCR assays were conducted at outside laboratories before and repeated in house after hospitalized. Of 36 at admission, positive antibody was seen in 4/4 from the ship, 0/2 from Africa, and 5/30 from Japan. Two from Africa showed the increase of COI and became positive on days 8 and 13. Thirty Japanese was divided in two groups, e.g., 23 showed dynamic increase of COI up to 84.4 within 3 days while active virus replication present (Group C). In remaining 7 (7/30, 23%) (Group C'), no rise of antibody nor positive in house PCR assays, indicative of false positive results of PCR at the beginning.ConclusionThis antibody testing has a wide dynamic ranges of COI and, thus, could be utilized in the early infection phase. This may also compliment and even help to avoid possible PCR errors. Therefore, this can serve as a powerful diagnostic tool, needed in the frontline of the clinic and hospitals.