Comparison of molecular methods for Bartonella henselae detection in blood donors.

The Bartonella genus consists of neglected pathogens associated with potentially transfusional-transmitted and fatal human diseases. We aimed to evaluate Bartonella sp. prevalence in 500 blood donors and compare the results with the data already published about these samples. We used molecular diagnostic methods to detect Bartonella sp.-DNA from blood and liquid culture samples: (A) conventional PCR for two gene regions, the ITS targeting the genus Bartonella and the specific gltA Bartonella henselae; (B) nested PCR for the ftsZ gene and (C) qualitative real-time PCR for the gltA gene, both B. henselae specific. We obtained 30/500 (6%) DNA detections from the blood samples; 77/500 (15.4%) DNA detections from liquid culture samples and five (1%) samples had DNA detection from both. In total, we detected B. henselae DNA from 102/500 (20.4%) donors. The samples used in this study had already been submitted for Bartonella sp.-DNA detection using only a conventional PCR in liquid culture. Sixteen samples (3.2%) were positive previously, and from these 16 samples, 13 were negative in the new investigation. We concluded that the use of liquid culture combined with different molecular tests increases the possibility of detecting Bartonella sp.-DNA, but the tests do not avoid false-negative results. More than a fifth of blood donors had at least one PCR that detected Bartonella sp.-DNA among the eight molecular reactions performed now (four reactions in whole blood and four in liquid culture). Seven percent had B. henselae-DNA detection for two or more distinct regions. Considering the results obtained previously, the DNA of Bartonella spp. was detected or the agent isolated in 23% of analyzed blood donors. The results establish that the low bacteremia and the fastidious characteristics of the bacterium are challenges to laboratory diagnosis and can make it difficult to confirm the infection in patients with bartonelloses.

Risk Factors for Bartonella species Infection in Blood Donors from Southeast Brazil.

Bacteria from the genus Bartonella are emerging blood-borne bacteria, capable of causing long-lasting infection in marine and terrestrial mammals, including humans. Bartonella are generally well adapted to their main host, causing persistent infection without clinical manifestation. However, these organisms may cause severe disease in natural or accidental hosts. In humans, Bartonella species have been detected from sick patients presented with diverse disease manifestations, including cat scratch disease, trench fever, bacillary angiomatosis, endocarditis, polyarthritis, or granulomatous inflammatory disease. However, with the advances in diagnostic methods, subclinical bloodstream infection in humans has been reported, with the potential for transmission through blood transfusion been recently investigated by our group. The objective of this study was to determine the risk factors associated with Bartonella species infection in asymptomatic blood donors presented at a major blood bank in Southeastern Brazil. Five hundred blood donors were randomly enrolled and tested for Bartonella species infection by specialized blood cultured coupled with high-sensitive PCR assays. Epidemiological questionnaires were designed to cover major potential risk factors, such as age, gender, ethnicity, contact with companion animals, livestock, or wild animals, bites from insects or animal, economical status, among other factors. Based on multivariate logistic regression, bloodstream infection with B. henselae or B. clarridgeiae was associated with cat contact (adjusted OR: 3.4, 95% CI: 1.1-9.6) or history of tick bite (adjusted OR: 3.7, 95% CI: 1.3-13.4). These risk factors should be considered during donor screening, as bacteremia by these Bartonella species may not be detected by traditional laboratory screening methods, and it may be transmitted by blood transfusion.

Bartonella spp. bacteremia in blood donors from Campinas, Brazil.

Bartonella species are blood-borne, re-emerging organisms, capable of causing prolonged infection with diverse disease manifestations, from asymptomatic bacteremia to chronic debilitating disease and death. This pathogen can survive for over a month in stored blood. However, its prevalence among blood donors is unknown, and screening of blood supplies for this pathogen is not routinely performed. We investigated Bartonella spp. prevalence in 500 blood donors from Campinas, Brazil, based on a cross-sectional design. Blood samples were inoculated into an enrichment liquid growth medium and sub-inoculated onto blood agar. Liquid culture samples and Gram-negative isolates were tested using a genus specific ITS PCR with amplicons sequenced for species identification. Bartonella henselae and Bartonella quintana antibodies were assayed by indirect immunofluorescence. B. henselae was isolated from six donors (1.2%). Sixteen donors (3.2%) were Bartonella-PCR positive after culture in liquid or on solid media, with 15 donors infected with B. henselae and one donor infected with Bartonella clarridgeiae. Antibodies against B. henselae or B. quintana were found in 16% and 32% of 500 blood donors, respectively. Serology was not associated with infection, with only three of 16 Bartonella-infected subjects seropositive for B. henselae or B. quintana. Bartonella DNA was present in the bloodstream of approximately one out of 30 donors from a major blood bank in South America. Negative serology does not rule out Bartonella spp. infection in healthy subjects. Using a combination of liquid and solid cultures, PCR, and DNA sequencing, this study documents for the first time that Bartonella spp. bacteremia occurs in asymptomatic blood donors. Our findings support further evaluation of Bartonella spp. transmission which can occur through blood transfusions.

The new noninvasive occlusion spectroscopy hemoglobin measurement method: a reliable and easy anemia screening test for blood donors.

BACKGROUND: The tests used for anemia screening in blood donors are based on fingerstick samples, leading to discomfort and complaints. The aim of this study was to analyze the feasibility of occlusion spectroscopy method in blood banks and to compare the method with fingerstick hemoglobinometer and hemoglobin (Hb) determination on an automatic blood analyzer. STUDY DESIGN AND METHODS: The study enrolled 205 consecutive volunteer blood donors. Samples were collected by fingerstick and venous punction to determine Hb level by a Hemocue Hb201+ (Hb-F) and automatic blood analyzer (Hb-V) and compare to the noninvasive Hb determination by occlusion spectroscopy using NBM200 system (Hb-NI). The percentage errors of Hb-F and Hb-NI of all donors as well as stratified by sex, weight, and age levels were compared to Hb-V as reference values using Wilcoxon signed rank test. RESULTS: The results obtained with Hb-F showed significant errors (p<0.001) in the general group as well as when stratified by sex, weight, and age groups, above values obtained with Hb-V. Hb-NI showed significant errors only in females (p=0.026) and weight level of 61 to 70kg (p=0.034), below Hb-V values. CONCLUSIONS: Hb-NI seems to be a good method in terms of precision and feasibility for anemia screening of blood donors as well as being much more comfortable for donors.

Fetal genotyping for platelets antigens: a precise tool for alloimmune thrombocytopenia: case report and literature review.

INTRODUCTION: Maternal-fetal alloimmune thrombocytopenia complicates about 0.1% of all pregnancies and is associated with major fetal and neonatal morbidity and mortality, especially spontaneous central nervous system bleeding leading to death and neurological handicaps. Successful prevention and treatment depend on the identification of at-risk possible carriers of anti-platelet antibodies. CASE REPORT: We report a case of a mother with a previous child that developed neonatal hemorrhage; HPA-5b anti-platelet antibodies were detected post-natally. During the next pregnancy, fetal genotyping confirmed the presence of HPA-5b antigen; she was treated with weekly intravenous human immunoglobulin and oral prednisone. Pregnancy evolved without remarkable features and a full-term baby was delivered, with normal platelet counts. CONCLUSION: Fetal alloimmune thrombocytopenia is a potentially lethal condition, but early detection and prevention lead to successful outcome in most cases.

Bartonellosis as cause of death after red blood cell unit transfusion.

The authors present the case of a young man with aplastic anemia who went into shock and died after several red blood cell unit transfusions. Immunohematological studies did not show any abnormality and blood cultures from patients and blood bags were negative. The ultrastructural findings, allied with current scientific knowledge, permitted the diagnosis of Bartonella sp. infection. In face of this diagnosis, two possibilities should be considered: the first one is that the patient was already infected by the bacteria before the last RBC unit transfusion. The pathogen could be involved in aplastic anemia etiology and in the failure to recover hemoglobin levels, in spite of the transfusions. The second possibility is that the RBC unit was contaminated with a Bartonella sp., which would have led to a state of shock, causing the death of the patient.

Bartonella henselae infects human erythrocytes.

Bartonella henselae, a facultative intracellular bacterium, has been known as the agent of cat scratch disease, bacillary angiomatosis, peliosis hepatis, endocarditis, and bacteremic syndrome in humans. Bartonella species can cause intraerythrocytic infections and have been isolated from the bloodstream of patients by several methods. It was demonstrated that B. bacilliformis and B. quintana infect human endothelial cells and human erythrocytes and B. henselae infects erythrocytes of cats. The aim of this study was to investigate through transmission electron microscopy whether B. henselae infects mature human erythrocytes. One red blood cell (RBC) unit received an experimentally standard strain of B. henselae. Blood aliquots were collected from the infected unit immediately after inoculation, at 30 min and 1, 5, 10, and 72 h for ultrastructural evaluation. B. henselae was seen adhering to human erythrocytes 10 h after inoculation and inside the erythrocyte after 72 h. This study demonstrates that B. henselae adheres to and invades mature human erythrocytes. The results favor the possibility that erythrocytes can serve as a primary target in Bartonella spp. infections. From this observation, further studies are warranted to prevent Bartonella spp. transfusional transmission.

Oral involvement in chronic graft versus host disease: a prospective study of 19 Brazilian patients.

Chronic graft versus host disease (cGVHD) is the most common late complication of allogeneic bone marrow transplantation. The oral cavity is the most common site of cGVHD involvement. This study sought to investigate the incidence of oral cGVHD, as well as the disease's impact on a patient's quality of life and the kind of lesions that resulted. Nineteen patients with cGVHD received a medical and dental evaluation; 18 (94.7%) had oral lesions. Nine patients (47.3%) demonstrated xerostomia and 6 (35.2%) demonstrated dysphagia. Six patients (35.2%) had a lichenoid clinical form of cGVHD in the oral cavity, 6 (35.2%) had an atrophic-ulcerative clinical form, 3 (17.6%) had a hyperceratotic clinical form, and 2 (10.5%) had mixed forms. The results demonstrated predominance of lichenoid and ulcerative-atrophic forms with similar incidence of these lesions. No factor that could contribute to the severity of cGVHD oral lesions was found.

The relationship between ABO groups and subgroups, factor VIII and von Willebrand factor.

The aim of this study was to correlate ABO groups with plasma levels of factor VIII (FVIII), von Willebrand factor (VWF:Ag), and ristocetin cofactor (VWF:RCo). Serological and molecular tests defined blood groups from 114 donors (10 AA, 10 BB, 10 AB, 10 AO1, 10 BO1,16 O1O1, 20 A2O1, 20 A2B, 4 A3O1, 3 AxO1, and 1 BelO1). The levels of VWF:Ag, FVIII and VWF:RCo observed in rare subgroups (A3O1, AxO1, BelO1) were similar to the values found in the O1O1 group. However, levels of these factors were significantly higher in A2O1 donors than in O1O1 donors (VWF:Ag p=0.01; FVIII p=0.04; VWF:RCo p<0.001). Strong correlations were demonstrated between plasma levels of VWF:Ag and FVIII (R=0.77; p=0.001) and between VWF:Ag and VWF:RCo (R=0.75; p=0.001).

Investigation of red blood cell antigens with highly fluorescent and stable semiconductor quantum dots.

We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A2+, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A2+ erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.

Human herpesvirus 6 in oral fluids from healthy individuals.

BACKGROUND: Human herpesvirus 6 (HHV6) is the etiologic agent of exanthem subitum. The virus is latent in salivary glands and saliva is the main form of viral transmission. The objective of this study was to assess HHV6 incidence in the fluids from healthy individuals using a standardised technique for collecting and extracting viral DNA from gingival crevicular fluid, whole saliva and parotid gland saliva. DESIGN: Samples of oral fluids and peripheral blood were collected from 28 blood donors and HHV6 was detected using PCR assay. Parotid gland saliva and gingival crevicular fluid were collected by endodontic paper cones in order to not contaminate these fluids with whole saliva. RESULTS: Of the 28 donors, 20 (71.4%) presented positive results in at least one of the three oral fluids researched. Whole saliva was positive in 19 (67.8%) volunteers, while only four (14.2%) samples of gingival crevicular fluid and four of parotid gland saliva proved to be positive. CONCLUSIONS: The results suggest that HHV6 is present in the saliva of a large proportion of the healthy adult population. The use of endodontic paper cones for oral fluid collection and viral extraction was efficient, simple, cheap and painless. In spite of, the small number of cases studied it was possible to demonstrate that neither gingival crevicular fluid nor parotid gland saliva were the principal source of HHV6 in whole saliva.