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.

Topical use and systemic action of green and roasted coffee oils and ground oils in a cutaneous incision model in rats (Rattus norvegicus albinus).

INTRODUCTION: Wounds are a common health problem. Coffee is widely consumed and its oil contains essential fatty acids. We evaluated the local (skin) and systemic effects associated with the topical use of coffee oils in rats. METHODS: Punch skin wounds (6 mm) incisions were generated on the backs of 75 rats. Saline (SS), mineral oil (MO), green coffee oil (GCO), roasted coffee oil (RCO), green coffee ground oil (GCGO) or roasted coffee ground oil (RCGO) were topically applied to the wounds. Healing was evaluated by visual and histological/morphometric optical microscopy examination; second harmonics generation (SHG) microscopy, wound tissue q-PCR (values in fold-change) and blood serum (ELISA, values in pg/mL). RESULTS: RCO treated animals presented faster wound healing (0.986 vs. 0.422), higher mRNA expression of IGF-1 (2.78 vs. 1.00, p = 0.01), IL-6 (10.72 vs. 1.00, p = 0.001) and IL-23 (4.10 vs. 1.2, p = 0.05) in early stages of wound healing; higher IL-12 (3.32 vs. 1.00, p = 0.05) in the later stages; and lower serum levels of IFN-γ (11.97 vs. 196.45, p = 0.01). GCO treatment led to higher mRNA expression of IL-6 (day 2: 7.94 vs. 1.00, p = 0.001 and day 4: 6.90 vs. 1.00, p = 0.01) and IL-23 (7.93 vs. 1.20, p = 0.001) in the early stages. The RCO treatment also produced higher serum IFN-α levels throughout the experiment (day 2: 52.53 vs. 21.20; day 4: 46.98 vs.21.56; day 10: 83.61 vs. 25.69, p = 0.05) and lower levels of IL-4 (day 4: 0.9 vs.13.36, p = 0.01), adiponectin (day 10: 8,367.47 vs. 16,526.38, p = 0.001) and IFN-γ (day 4: 43.03 vs.196.45, p = 0.05). The SHG analysis showed a higher collagen density in the RCO and GCO treatments (p = 0.05). CONCLUSION: Topical treatment with coffee oils led to systemic actions and faster wound healing in rats. Further studies should be performed are necessary to assess the safety of topical vegetal oil use for skin lesions.

Acute and Late Bartonella henselae Murine Model Infection.

Bartonella spp. are fastidious gram-negative neglected bacilli with worldwide distribution. They are able to cause intraerythrocytic and potentially fatal infection. Cats and dogs are reservoirs of some species of these agents. Blood-sucking arthropods are potential vectors. Our aim was to evaluate the blood, skin, liver, and spleen in BALB/c mice by using molecular tests and confocal microscopy to demonstrate Bartonella henselae infection in the bloodstream and organs after 4 and 21 days of intraperitoneally injected bacterial suspension. We demonstrate that the occurrence of infection in organs precedes the detectable infection in blood. Therefore, late manifestation in blood may be another challenge in early detection and diagnosis of B. henselae infection.

Bartonella henselae transmission by blood transfusion in mice.

BACKGROUND: Bartonella spp. are neglected fastidious Gram-negative bacilli. We isolated Bartonella henselae from 1.2% of 500 studied blood donors and demonstrated that the bacteria remain viable in red blood cell units after 35 days of experimental infection. Now, we aim to evaluate the possibility of B. henselae transmission by blood transfusion in a mouse model. STUDY DESIGN AND METHODS: Eight BALB/c mice were intraperitoneal inoculated with a 30 µL of suspension with 10(4) CFU/mL of B. henselae and a second group of eight mice were inoculated with saline solution and used as control. After 96 hours of inoculation, the animals were euthanized. We collected blood and tissue samples from skin, liver, and spleen. Thirty microliters of blood from four Bartonella-inoculated animals were transfused into a new group (n = 4). Another group received blood from the control animals. B. henselae infection was investigated by conventional and nested polymerase chain reaction (PCR). RESULTS: Blood samples from all 24 mice were negative by molecular tests though half of the tissue samples were positive by nested PCR in the intraperitoneal Bartonella-investigated animals. Tissues from two of the four mice that received blood transfusions from Bartonella-inoculated animals were also nested PCR positives. CONCLUSIONS: Transmission of B. henselae by transfusion is possible in mice even when donor animals have undetectable bloodstream infection. The impact of human Bartonella sp. transmission through blood transfusion recipients must be evaluated.

Bartonella henselae initial infection of mature human erythrocytes observed in real time using bacterial endogenous fluorescence.

Bartonella henselae is a causative agent of anemia, cat scratch disease, bacillary angiomatosis, recurrent fever, hepatitis, endocarditis, chronic lymphadenopathy, joint and neurological disorders. B. henselae are intra-erythrocytic bacteria. The goal of this study was to visualize the B. henselae invasion into enucleated human red blood cells in real time using bacterium endogenous fluorescence. We took advantage of the unique fluorescence emission spectral profile of the bacteria. We used a linear unmixing approach to separate the fluorescence emission spectra of human erythrocytes from native B. henselae when excited at 488nm. Human blood samples were inoculated with B. henselae and incubated for 60 hours. 3-D live images were captured at select intervals using multi-photon laser scanning microscopy. Uninfected blood samples were also analyzed. This study revealed bacteria entering mature erythrocytes over a 60 hour time period.

Association between prehypertension, metabolic and inflammatory markers, decreased adiponectin and enhanced insulinemia in obese subjects.

BACKGROUND: Obesity is associated with development of the cardiorenal metabolic syndrome, which is a constellation of risk factors, such as insulin resistance, inflammatory response, dyslipidemia, and high blood pressure that predispose affected individuals to well-characterized medical conditions such as diabetes, cardiovascular and kidney chronic disease. The study was designed to establish relationship between metabolic and inflammatory disorder, renal sodium retention and enhanced blood pressure in a group of obese subjects compared with age-matched, lean volunteers. METHODS: The study was performed after 14 h overnight fast after and before OGTT in 13 lean (BMI 22.92 ± 2.03 kg/m(2)) and, 27 obese (BMI 36.15 ± 3.84 kg/m(2)) volunteers. Assessment of HOMA-IR and QUICKI index were calculated and circulating concentrations of TNF-α, IL-6 and C-reactive protein, measured by immunoassay. RESULTS: THE STUDY SHOWS THAT A HYPERINSULINEMIC (HI: 10.85 ± 4.09 µg/ml) subgroup of well-characterized metabolic syndrome bearers-obese subjects show higher glycemic and elevated blood pressure levels when compared to lean and normoinsulinemic (NI: 5.51 ± 1.18 µg/ml, P < 0.027) subjects. Here, the combination of hyperinsulinemia, higher HOMA-IR (HI: 2.19 ± 0.70 (n = 12) vs. LS: 0.83 ± 0.23 (n = 12) and NI: 0.98 ± 0.22 (n = 15), P < 0.0001) associated with lower QUICKI in HI obese when compared with LS and NI volunteers (P < 0.0001), suggests the occurrence of insulin resistance and a defect in insulin-stimulated peripheral action. Otherwise, the adiponectin measured in basal period was significantly enhanced in NI subjects when compared to HI groups (P < 0.04). The report also showed a similar insulin-mediated reduction of post-proximal urinary sodium excretion in lean (LS: 9.41 ± 0.68% vs. 6.38 ± 0.92%, P = 0.086), and normoinsulinemic (NI: 8.41 ± 0.72% vs. 5.66 ± 0.53%, P = 0.0025) and hyperinsulinemic obese subjects (HI: 8.82 ± 0.98% vs. 6.32 ± 0.67%, P = 0.0264), after oral glucose load, despite elevated insulinemic levels in hyperinsulinemic obeses. CONCLUSION: In conclusion, this study highlights the importance of adiponectin levels and dysfunctional inflammatory modulation associated with hyperinsulinemia and peripheral insulin resistance, high blood pressure, and renal dysfunction in a particular subgroup of obeses.

Development of hypertension in a pyelonephritis-induced model: the effect of salt intake and inability of renal sodium handling.

The role of the kidney in the control of blood pressure has been convincingly demonstrated by several studies. Recent evidence has suggested that subtle acquired tubulointerstitial injury may cause a defect in sodium excretion function, thus leading to salt-sensitive hypertension. There are no reports, however, examining the effect of experimental chronic pyelonephritis on renal sodium handling and arterial pressure. Thus, to examine the influence of salt intake and unilateral nephrectomy, unanesthetized, unrestrained rats were randomly assigned to one of two separate groups: sham-operated rats (CO) or chronic unilateral pyelonephritic rats (CP). After twenty one days, the pyelonephritic group was subdivided in two: one subgroup continued with water intake (CPw), while the other was changed to 0.9% NaCl intake (CPs), like the control group (COs). After seven days, all rats were submitted to unilateral nephrectomy of the left normal kidney. Data presented herein show that chronic pyelonephritis produced an increase in mean arterial pressure (CO: 121.4 +/- 1.0 mmHg to CP: 127.0 +/- 0.9 mmHg, p = 0.000) that was enhanced by saline ingestion (COs: 121.6 +/- 1.4 mmHg; CPw: 127.0 +/- 1.8 mmHg; CPs: 132.1 +/- 1.2 mmHg, p = 0.000) and further aggravated by unilateral nephrectomy (CO: 125.2 +/- 2.6 mmHg; CPw: 127.5 +/- 0.9 mmHg; CPs: 139.2 +/- 1.1 mmHg, p = 0.000). Unchanged blood pressure measurements (120.2 +/- 2.3 mmHg) were observed beyond 21 days in control rats maintained on water regimen when compared with saline-drinking groups. These changes in mean arterial pressure were observed despite an increased fractional sodium excretion in the CPs group compared to the other groups before uninephrectomy (COs: 0.125 +/- 0.025%; CPw: 0.045 +/- 0.013%; CPs: 0.292 +/- 0.046%; p = 0.000), as compared to CPw after uninephrectomy (COs: 0.249 +/- 0.077%; CPw: 0.062 +/- 0.011%; CPs: 0.363 +/- 0.195%, p = 0.019). In addition, it was shown that daily liquid intake was higher in CPs than in CPw but similar to COs, both before uninephrectomy (COs: 42.8 +/- 2.6 ml/d; CPw: 34.3 +/- 3.5 ml/d; CPs: 51.8 +/- 3.7 ml/d, p = 0.006) and after uninephrectomy (COs: 40.9 +/- 5.5 ml/d; CPw: 33.8 +/- 1.4 ml/d; CPs: 53.0 +/- 3.5 ml/d, p = 0.004). The current data suggest that chronic pyelonephritis promotes an inability of renal tubules to handle sodium excretion when exposed to sodium overload and aggravated by uninephrectomy, thus constituting a model for salt-sensitive hypertension.

Importance of anesthesia for the genesis of neurogenic pulmonary edema in spinal cord injury.

There are reports describing both provocation and inhibition of neurogenic pulmonary edema by anesthetic drugs. Therefore, we compared the effect of two types of anesthesia on the formation of neurogenic pulmonary edema in rats with balloon-induced acute spinal cord injury. Animals with sham procedure (group 1) were anesthesized by intraperitoneal sodium pentobarbital. In the experimental groups, rats were submitted to acute spinal cord lesion by insufflations of a balloon in the epidural space at T8 for 1 min (group 3 under i.p. sodium pentobarbital and group 2 under i.p. xylazine-ketamine anesthesia). In rats with pentobarbital anesthesia, systolic blood pressure doubled the baseline value during compression, whereas this effect was less pronounced in the ketamine-xylazine group. The pulmonary index (100 x wet lung weight/body weight) was 0.395 (+/-0.018) in sham-operated rats, rose to 0.499 (+/-0.060) in group 2, and was maximum under pentobarbital anesthesia (0.639+/-0.14; p=0.0018). Histologic examination of the spinal cord showed parenchymal ruptures and acute hemorrhage. Comparison of the pulmonary index with histologic slides of lung parenchyma revealed that relevant intra-alveolar edema occurred only for index values above 0.55. On electron microscopy, endothelial alterations, and damage of the alveolar lining cells were found. Our study indicates that neurogenic pulmonary edema caused by spinal cord injury is less pronounced in rats under xylazine-ketamine anesthesia, when compared with pentobarbital.