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An immunohistochemical and polymerase chain reaction evaluation of feline plasmacytic pododermatitis.

Sections of 14 skin biopsies of cats with plasmacytic pododermatitis and a clinical follow-up of 12-36 months were stained with a polyclonal anti-Mycobacterium bovis (Bacille Calmette-Guerin = BCG) antibody cross-reactive to a broad spectrum of fungi and bacteria. All sections were negative for organisms within the actual footpad tissue with the anti-BCG antibody stains. Polymerase chain reaction (PCR) assays that amplify the DNA of Bartonella spp., Ehrlichia spp., Anaplasma phagocytophilum, Chlamydophila felis, Mycoplasma spp., Toxoplasma gondii, and feline herpesvirus 1 (FHV-1) were applied to tissue digests. DNA of those pathogens assessed was not amplified from tissue.

Animals↗

Surveillance of Egyptian fleas for agents of public health significance: Anaplasma, Bartonella, Coxiella, Ehrlichia, Rickettsia, and Yersinia pestis.

Serologic surveys in Egypt have documented human and animal exposure to vector-borne bacterial pathogens, but the presence and distribution of these agents in arthropods has not been determined. Between July 2002 and July 2003, fleas were collected from 221 mammals trapped in 17 cities throughout Egypt. A total of 987 fleas were collected, representing four species (Ctenocephalides felis, Echidnophaga gallinacea, Leptopsylla segnis, and Xenopsylla cheopis); 899 of these fleas were X. cheopis from rats (Rattus spp.). Fleas were tested for DNA from Anaplasma spp., Bartonella spp., Coxiella burnetii, Ehrlichia spp., Rickettsia spp., and Yersinia pestis. Rickettsia typhi, the agent of murine typhus, was detected in X. cheopis and L. segnis from rats from nine cities. A spotted-fever group Rickettsia sp. similar to "RF2125" was detected in E. gallinacea, and two unidentified spotted fever group Rickettsia were detected in two X. cheopis. Novel Bartonella genotypes were detected in X. cheopis and L. segnis from three cities. Coxiella burnetii was detected in two fleas. Anaplasma, Ehrlichia, and Y. pestis were not detected.

Animals↗

In search of encephalitis etiologies: diagnostic challenges in the California Encephalitis Project, 1998-2000.

The California Encephalitis Project was initiated in June 1998 to identify the causes and characterize the clinical and epidemiologic features of encephalitis in California. Testing for >or=13 agents, including herpesviruses, enteroviruses, arboviruses, Bartonella species, Chlamydia species, and Mycoplasma pneumoniae, was performed at the Viral and Rickettsial Disease Laboratory (Richmond, California). Epidemiologic and clinical information collected for each case guided further testing. From June 1998 through December 2000, 334 patients who met our case definition of encephalitis were enrolled. A confirmed or probable viral agent of encephalitis was found in 31 cases (9%), a bacterial agent was found in 9 cases (3%), and a parasitic agent was found in 2 cases (1%). A possible etiology was identified in 41 cases (12%). A noninfectious etiology was identified in 32 cases (10%), and a nonencephalitis infection was identified in 11 (3%). Despite extensive testing and evaluation, the etiology of 208 cases (62%) remained unexplained.

California↗

The identification of two protective DNA vaccines from a panel of five plasmid constructs encoding Brucella melitensis 16M genes.

Five candidate genes from the Brucella melitensis 16M genome were selected. Eukaryotic expression plasmids encoding these antigens were constructed and expression was verified in vitro from transfected Cos7 cells. Each vaccine was assessed for protective efficacy in a BALB/c mouse brucellosis infection model. From these experiments two protective DNA vaccines were identified: p-omp25 and p-ialB. The Omp25 antigen (BMEI1249) has previously been studied in terms of Brucella virulence, serodiagnosis and as a protective antigen. However, this study represents the first report of a significant protective effect achieved against B. melitensis 16M challenge using the Omp25 antigen in a DNA vaccine approach. The other protective vaccine identified in this study was p-ialB. The ialB candidate (BMEI1584) was selected based upon its' putative function as an invasion protein which was assigned due to shared identity with the invasion protein B (ialB) of Bartonella bacilliformis. This candidate has not previously been investigated with regard to Brucella virulence or pathogenesis. This study is the first report to identify the Brucella invasion protein B (BMEI1584) as a novel protective antigen for brucellosis.

Acid Anhydride Hydrolases↗

High prevalence of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in patients with cardiovascular disease and in middle-aged blood donors.

Nested polymerase chain reaction (nPCR) demonstrated the presence of Chlamydia pneumoniae-specific DNA in peripheral blood mononuclear cells (PBMC). PBMC samples were obtained from 103 consecutive patients (62 male, 41 female) aged 22-85 years (mean, 64) admitted for coronary angiography because of suspected coronary heart disease and from 52 blood donors (43 male, 9 female) aged 40-64 years (mean, 49). Of the 101 evaluable patients, 60 (59%) were identified by nPCR assay as C. pneumoniae DNA carriers; C. pneumoniae-specific microimmunofluorescence (MIF) serology confirmed exposure to the bacterium in 57 (95%) of the 60 nPCR-positive patients. Among the 52 blood donors, the nPCR assay identified 24 (46%) C. pneumoniae DNA carriers, all of whom were positive by C. pneumoniae-specific serology. Thirty-two patients (32%) and 23 blood donors (44%) were MIF antibody-positive but repeatedly nPCR-negative; Bartonella henselae- or Bartonella quintana-specific antibodies were not detected among any of these subjects. In this study, C. pneumoniae DNA was common in PBMC of patients with coronary heart disease and in middle-aged blood donors.

Adult↗

Causes of febrile illnesses after a tick bite in Slovenian children.

BACKGROUND: To establish the etiology in Slovenian children with febrile illnesses occurring after a tick bite. METHODS: Eighty-six febrile patients younger than 15 years referred to our institution in 2001 with a history of a tick bite within 6 weeks before onset of the illness were included in this prospective study. Acute and convalescent serum samples were tested for the presence of antibodies to tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Rickettsia conorii, Babesia microti, Bartonella henselae, Bartonella quintana and Francisella tularensis. Cerebrospinal fluid was investigated in patients in whom meningeal involvement was clinically suspected. Blood and/or cerebrospinal fluid from the patients were cultured in modified Kelly-Pettenkofer medium. PCR was performed to detect ribosomal DNA of A. phagocytophilum and E. chaffeensis. RESULTS: Of 86 patients 33 (38%) were excluded because a well-defined febrile illness not associated with tick bite was established. Tick-borne illness was diagnosed in 28 (53%) of the 53 remaining patients. The most common diagnosis was tick-borne encephalitis (64%), followed by Lyme borreliosis (46%), human monocytic ehrlichiosis and human granulocytic ehrlichiosis (serologic evidence of infection in 9 and 4%, respectively). In 6 (21%) patients there was evidence for infection with more than 1 tick-borne agent. CONCLUSIONS: Tick-borne illness was established in 53% of the patients younger than 15 years presenting with febrile illness occurring within 6 weeks after a tick bite. The most common identified illnesses were tick-borne encephalitis and Lyme borreliosis.

Adolescent↗

[Microbial diagnosis with PCR will become clinically beneficial with a faster analysis].

PCR was introduced in 1985 by Mullis and was immediately recognized as a valuable tool in biomedical research and was awarded the Nobel Prize in 1993. Two culture-negative meningitis cases are described where Haemophilus influenzae and Neisseria meningitidis were found by 16SRNA-PCR. The modern real time PCR technology using fluorescent probes (hybridization probes, lightup probes, molecular beacons etc) for detection of the PCR-product or on DNA microarray chips, is under development for routine use. Multiplex technology can be used to simultaneously detect multiple microorganisms as well as resistance genes. Using super-convection with ultracentrifugation high-speed PCR, results can be obtained in 10 minutes and the amplificate can also be analyzed by DNA-sequencing to achieve species identification as well as detection of resistance gene mutations. The technique has mainly been applied to viruses, but is now slowly adapted to bacteria, fungi, protozoa and helminths. PCR is especially well suited for slow growing bacteria like Mycobacteria, fastidious organisms like Bartonella and contagious agents like tularemia, but also for malaria and fungi, where the advantages in sensitivity and speed can be exploited. The limit for application to routine analysis will depend on the development of simple and fast procedures for nucleic acid extraction, as well as interpretation of the PCR analysis per se, since highly efficient thermocyclers now are on the markets.

Adult↗

New pathogens, and diseases old and new. I) Afipia felis and Rochalimaea. II) Parvovirus B 19. III) herpesvirus 6.

The paper describes events that in the last fifteen years, have led to the identification of the aetiological agents of three widely known diseases: cat scratch disease, erythema infectiosum and exanthem subitum. The particular features of Afipia felis and Rochalimaea, Parvovirus B 19 and Herpesvirus 6 are presented. The paternity of new diseases (i.e. bacillary angiomatosis, bacillary peliosis hepatitis, LES-like syndrome, chronic fatigue syndrome, petechial glove and sock syndrome, etc.) has also been attributed to some of these pathogens as has the paternity of some older ones (i.e. aplastic crisis, erythroblastosis fetalis, trench fever, hepatitis, opportunistic infection, etc.). It has been argued that the same pathogen can cause different diseases depending on the immunogenic state of the subject. To date, persisting difficulties in isolating the pathogen or differentiating between latent or active infection, still in some cases raises doubts concerning the attribution of the disease to a specific agent. New immunological or molecular techniques, allowing the direct detection of in vivo replication, are still needed in order to establish a sure connection between some of these agents and some of these diseases. Progress here will both give more accurate data about the epidemiology of some diseases and allow us to apply more appropriate treatment and prevention techniques.

Bartonella henselae↗

Guidelines for the diagnosis of tick-borne bacterial diseases in Europe.

Ticks are obligate haematophagous acarines that parasitise every class of vertebrate (including man) and have a worldwide distribution. An increasing awareness of tick-borne diseases among clinicians and scientific researchers has led to the recent description of a number of emerging tick-borne bacterial diseases. Since the identification of Borrelia burgdorferi as the agent of Lyme disease in 1982, 11 tick-borne human bacterial pathogens have been described in Europe. Aetiological diagnosis of tick-transmitted diseases is often difficult and relies on specialised laboratories using very specific tools. Interpretation of laboratory data is very important in order to establish the diagnosis. These guidelines aim to help clinicians and microbiologists in diagnosing infection transmitted by tick bites and to provide the scientific and medical community with a better understanding of these infectious diseases.

Animals↗

[Cat scratch disease with posterior segment involvement].

PURPOSE: To describe the clinical characteristics of patients with cat scratch disease during the last 2 years. METHODS: Clinical characteristics and anterior and posterior segment manifestations were reviewed in five patients who were serologically diagnosed as having cat scratch disease. RESULTS: Four women and one man were examined. Their ages ranged from 7 to 60 years. Each patient had a markedly elevated serum anti-Bartonella henselae antibody titer. Visual symptoms developed 2 weeks or less after the onset of systemic symptoms. Lymphadenopathy was detected in one of five patients. Neuroretinitis was found in 4 patients, and papillitis in 1 patient. Seven eyes showed retinochoroidal exudates. Anterior uveitis was observed in three eyes. Four patients received systemic corticosteroids. CONCLUSION: The ocular manifestations of cat scratch disease include neuroretinitis, papillitis, retinochoroidal exudates, and anterior uveitis.

Adult↗

Comparative molecular and microbiologic diagnosis of bacterial endocarditis.

Sequencing of 16S rDNA, and of sodAint and rpoBint in some cases, was applied to DNA from heart valves of 46 patients (36 with definite and 10 with possible endocarditis). Sequence-based identifications were compared with those obtained with conventional methods. Among the 36 definite cases, 30 had positive blood cultures and 6 had negative cultures. Among the 30 positive cases, sequencing of 16S rDNA permitted identification of species (18), genus (8), or neither (4); sodAint and rpoBint sequencing was necessary for species identification in 8 cases. Species identifications were identical in only 61.5%, when conventional techniques and DNA sequencing were used. In five of the six blood culture-negative endocarditis cases, sequencing identified Bartonella quintana (3), B. henselae (1), and Streptococcus gallolyticus (1). Our results demonstrate a clear benefit of molecular identification, particularly in cases of blood culture-negative endocarditis and of possible endocarditis, to confirm or invalidate the diagnosis. Moreover, in 19.4% of the definite cases, the improvement in species identification by sequencing led to improved patient management.

Adult↗

Bacillary angiomatosis.

The acquired immunodeficiency syndrome and other causes of immunosuppression have ushered in a variety of opportunistic infections. One of these is bacillary angiomatosis, a vasoproliferative lesion whose principal causative agent is Rochalimaea henselae. Bacillary angiomatosis, while preponderantly a cutaneous affliction, can be systemic, including involvement of the head and neck mucous membranes. Molecular technology and epidemiologic studies used to identify the bacterial agent of bacillary angiomatosis have also uncovered R henselae as the organism responsible for most cases of cat-scratch disease. Why the same organism promotes two different histopathologic lesions, as seen in bacillary angiomatosis and cat-scratch disease, is unknown.

Angiomatosis, Bacillary↗

Detection of selected fastidious bacteria.

The intent of this article is to describe the optimal methods for culture recovery of 7 fastidious bacteria: Legionella species, Brucella species, Francisella tularensis, Leptospira species, Borrelia burgdorferi, Bartonella species, and Bordetella species. These organisms share much in common beyond the fact that their genus names all end in the letter "a." Culture recovery of these organisms, even from adequate clinical specimens, is logistically demanding, often costly, and lacking in both timeliness and sensitivity. In addition, there is generally no need to recover culture isolates on which to perform antimicrobial susceptibility tests because these 7 bacteria are nearly uniformly susceptible to specific, clinically useful antimicrobial agents and because, for some of them, susceptibility tests of proven reliability have not yet been devised. Perhaps for these reasons, alternative, more rapid, direct diagnostic approaches have been developed that are based on either immunochemical or nucleic-acid detection methods. These methods have generally served to supplant culture as a primary diagnostic modality. Situations exist, however, in which culture may be desirable, if not necessary, to establish a definitive diagnosis of infection with these 7 organisms. This review attempts to summarize how best to proceed in those cases.

Bacteriological Techniques↗

Impact of a molecular approach to improve the microbiological diagnosis of infective heart valve endocarditis.

BACKGROUND: Even today, infective endocarditis (IE) remains a severe and potentially fatal disease demanding sophisticated diagnostic strategies for detection of the causative microorganisms. Despite the use of appropriate laboratory techniques, classic microbiological diagnostics are characterized by a high rate of negative results. METHODS AND RESULTS: Broad-range polymerase chain reaction (PCR) targeting bacterial and fungal rDNA followed by direct sequencing was applied to excised heart valves (n=52) collected from 51 patients with suspected infectious endocarditis and from 16 patients without any signs of IE during an 18-month period. The sensitivity, specificity, and the positive and negative predictive values for the bacterial broad-range PCR were 41.2%, 100.0%, 100.0%, and 34.8%, respectively, compared with 7.8%, 93.7%, 80.0%, and 24.2% for culture and 11.8%, 100.0%, 100.0%, and 26.2% for Gram staining. Without exception, database analyses allowed identification up to the (sub)species level comprising streptococcal (n=13), staphylococcal (n=4), enterococcal (n=2), and other signature sequences such as Bartonella quintana and Nocardia paucivorans. Fungal ribosomal sequences were not amplified. All valve tissues of the reference group were negative for both PCR and conventional methods, except one sample that was contaminated by molds. CONCLUSIONS: Culture-independent molecular methods substantially improve the diagnostic outcome of microbiological examination of excised heart valves. Importantly, this was true not only for fastidious, slow-growing, and/or nonculturable microorganisms but also for easy-to-culture pathogens such as streptococci and staphylococci. Both patient management and empiric antibiotic therapy of IE are likely to benefit from improved knowledge of the spectrum of pathogens now causing IE.

Adolescent↗

Do plant and human pathogens have a common pathogenicity strategy?

Recently, a novel 'two-step' model of pathogenicity has been described that suggests host-cell-derived vasculoproliferative factors play a crucial role in the pathogenesis of bacillary angiomatosis, a disease caused by the human pathogenic bacterium Bartonella henselae. The resulting proliferation of endothelial cells could be interpreted as bacterial pathogens triggering the promotion of their own habitat: the host cell. Similar disease mechanisms are well known in the plant pathogen Agrobacterium tumefaciens, which causes crown gall disease. There are notable similarities between the pathogenicity of A. tumefaciens leading to tumourous disease in plants and to the B. henselae-triggered proliferation of endothelial cells in humans. Here, we hypothesize that this pathogenicity strategy might be common to several bacterial species in different hosts owing to shared pathogenicity factors.

Agrobacterium tumefaciens↗

Detection of mip-like sequences and Mip-related proteins within the family Rickettsiaceae.

The Mip surface protein, a prokaryotic analog of the FK506-binding proteins, enhances the ability of Legionella pneumophila to infect macrophages and protozoa. Using mip-specific probes and low-stringency Southern hybridizations, we have detected DNA sequences homologous to mip within Coxiella burnetii and Rochalimaea quintana. Using specific anti-Mip antisera and immunoblot analysis, we also detected Mip-related proteins within these bacteria as well as within Rickettsia and Ehrlichia species. These data suggest that Mip-related proteins have broad significance for host-parasite interactions. However, they also indicate that care must be exercised when using mip probes or anti-Mip antibodies for the detection of Legionella organisms in water or clinical samples.

Bacterial Proteins↗

Evaluation of an extended blood culture protocol to isolate fastidious organisms from patients with AIDS.

Recent reports of fastidious pathogens suggest the need for special blood cultures for immunocompromised patients. Blood cultures from 45 human immunodeficiency virus (HIV)-infected patients with unexplained fever (> or = 38.0 degrees C) and CD4 counts of < 125 cells per mm3 were collected into a vacuum tube with sodium polyanetholsulfonate, an Isolator tube, and BACTEC aerobic and anaerobic bottles. Blood from the sodium polyanethosulfonate tube was inoculated into BACTEC 13A bottles, which were read weekly for 16 weeks. Isolator sediment was divided among eight agar media, including four sheep blood agar media: chocolate agar, brain heart infusion blood agar, heart infusion blood agar, and brucella blood agar. Other agar plates included Sabouraud's, buffered charcoal-yeast extract, Middlebrook 7H11 (M7H11) with hemoglobin, and M7H11 with mycobactin J. Incubation conditions included air and CO2-enriched aerobic, microaerophilic, and anaerobic atmospheres. Aerobic BACTEC broths received an acridine orange stain on day 8 and were subcultured at 2, 4, and 8 weeks. Anaerobic BACTEC bottles were subcultured at 4 weeks. All solid media, including subcultures, were incubated for 8 weeks, providing a total of 16 weeks of incubation for each specimen. Clinically significant isolates included eight Mycobacterium avium complex isolates and one each of Bartonella henselae, Bartonella quintana, Shigella flexneri, Klebsiella oxytoca, and Cryptococcus neoformans. All isolates were detected with commercially available media and, with the exception of Bartonella spp., were recovered within incubation times routinely used in most clinical laboratories.

Acquired Immunodeficiency Syndrome↗

Baccilary Angiomatosis: Negative Results Using Normal Balb/c and Balb/c Nude Mice.

Baccilary angiomatosis has recently been described as a disease that can spread systematically and that is potentially fatal. It is caused by Bartonella henselae and B. quintana, and presents as especially pronounced signs and symptoms in patients suffering from acquired immunodeficiency syndrome (AIDS). To clarify the pathogenesis of the disease and to try to define the relationships among baccilary angiomatosis, cat scratch disease and Carrión's bartonellosis, the authors of this study have attempted to develop an experimental model using mice that were immunocompetent as well as those that had their cellular immunity genetically compromised. A known concentration of B. henselae was inoculated intradermally in Balb/c an isogenic mice or an athymic group of the same lineage. Blood samples were taken on days 0, 3, 7, 10, 14, 28, and 60 after inoculation for indirect immunofluorescence antibody testing. On the 21st and 60th day, one animal from each group was sacrificed and a post mortem carried out including histological evaluation of the liver, spleen, lymph nodes, skin and other organs. Hemocultures of the sacrificed animals were collected. All results of serologic response, cultures and histologic examination were negative. The authors discuss the methodology, especially the use of isogenic animals of the same lineage in B. henselae infection, with and without immunodeficiency, and the resources for the negative results of histopathology, serology and cultures.

Journal Article↗