Procedure for spotted fever group Rickettsia isolation from limited clinical blood specimens.

BACKGROUND: Current isolation techniques for spotted fever group Rickettsia from clinical samples are laborious and are limited to tissue, blood and blood derivatives with volumes ideally greater than 1 mL. We validated the use of simplified methodologies for spotted fever group Rickettsia culture isolation that overcome sample volume limitations and provide utility in clinical diagnostics and research studies. METHODOLOGY/PRINCIPAL FINDINGS: A modified cell culture method is evaluated for the isolation of Rickettsia ssp. from human diagnostic samples. Culture sampling method, culture platform, and growth phase analysis were evaluated to determine best practices for optimal culture isolation conditions. Rickettsial isolates (R. conorii, R. rickettsii, and R. parkeri) were grown in Vero E6 cells over a course of 5 to 7 days at low inoculum treatments (~40 bacterial copies) to standardize the sampling strategy at a copy number reflective of the bacteremia in acute diagnostic samples. This methodology was verified using small volumes (50 µL) of 25 unprocessed clinical whole blood, plasma, and serum samples from acute samples of patients suspected of having Rocky Mountain Spotted Fever, of which 10 were previously confirmed positive via the PanR8 qPCR assay, 13 had no detectable Rickettsia DNA by the PanR8 qPCR assay, and 2 were not previously tested; these samples resulted in the cultivation of 7 new R. rickettsii isolates. CONCLUSIONS/SIGNIFICANCE: We observed that rickettsial isolate growth in culture is reproducibly identified by real-time PCR testing of culture media within 72 hours after inoculation. Additionally, specimen sedimentation prior to isolation to remove red blood cells was found to decrease the amount of total organism available in the inoculum. A small volume culture method was established focusing on comparative qPCR detection rather than bacterial visualization, taking significantly shorter time to detect, and requiring less manipulation compared to traditional clinical isolate culture methods.

Acute febrile illness among outpatients seeking health care in Bangladeshi hospitals prior to the COVID-19 pandemic.

Understanding the distribution of pathogens causing acute febrile illness (AFI) is important for clinical management of patients in resource-poor settings. We evaluated the proportion of AFI caused by specific pathogens among outpatients in Bangladesh. During May 2019-March 2020, physicians screened patients aged ≥2 years in outpatient departments of four tertiary level public hospitals. We randomly enrolled patients having measured fever (≥100.4°F) during assessment with onset within the past 14 days. Blood and urine samples were tested at icddr,b through rapid diagnostic tests, bacterial culture, and polymerase chain reaction (PCR). Acute and convalescent samples were sent to the Centers for Disease Control and Prevention (USA) for Rickettsia and Orientia (R/O) and Leptospira tests. Among 690 patients, 69 (10%) had enteric fever (Salmonella enterica serotype Typhi orSalmonella enterica serotype Paratyphi), 51 (7.4%) Escherichia coli, and 28 (4.1%) dengue detected. Of the 441 patients tested for R/O, 39 (8.8%) had rickettsioses. We found 7 (2%) Leptospira cases among the 403 AFI patients tested. Nine patients (1%) were hospitalized, and none died. The highest proportion of enteric fever (15%, 36/231) and rickettsioses (14%, 25/182) was in Rajshahi. Dhaka had the most dengue cases (68%, 19/28). R/O affected older children and young adults (IQR 8-23 years) and was detected more frequently in the 21-25 years age-group (17%, 12/70). R/O was more likely to be found in patients in Rajshahi region than in Sylhet (aOR 2.49, 95% CI 0.85-7.32) between July and December (aOR 2.01, 1.01-5.23), and who had a history of recent animal entry inside their house than not (aOR 2.0, 0.93-4.3). Gram-negative Enterobacteriaceae were the most common bacterial infections, and dengue was the most common viral infection among AFI patients in Bangladeshi hospitals, though there was geographic variability. These results can help guide empiric outpatient AFI management.

Antibody Titers Reactive With Rickettsia rickettsii in Blood Donors and Implications for Surveillance of Spotted Fever Rickettsiosis in the United States.

BACKGROUND: Since 2000, the reported prevalence of tick-borne spotted fever rickettsiosis has increased considerably. We compared the level of antibody reactivity among healthy blood donors from 2 widely separated regions of the United States and evaluated the impact of antibody prevalence on public health surveillance in one of these regions. METHODS: Donor serum samples were evaluated by indirect immunofluorescence antibody assay to identify immunoglobulin G (IgG) antibodies reactive with Rickettsia rickettsii. The Georgia Department of Public Health (GDPH) analyzed characteristics of cases from 2016 surveillance data to evaluate the utility of laboratory surveillance for case assessment. RESULTS: Of the Georgia donors (n = 1493), 11.1% demonstrated antibody titers reactive with R. rickettsii at titers ≥64, whereas 6.3% of donors from Oregon and Washington (n = 1511) were seropositive. Most seropositive donors had a titer of 64; only 3.1% (n = 93) of all donors had titers ≥128. During 2016, GDPH interviewed 243 seropositive case patients; only 28% (n = 69) met inclusion criteria in the national case definition for spotted fever rickettsiosis. CONCLUSIONS: These findings suggest that a single IgG antibody titer is an unreliable measure of diagnosis and could inaccurately affect surveillance estimates that define magnitude and clinical characteristics of Rocky Mountain spotted fever and other spotted fever rickettsioses.

African Tick Bite Fever Treated Successfully With Rifampin in a Patient With Doxycycline Intolerance.

African tick bite fever is the most commonly encountered travel-associated rickettsiosis, occurring in as many as 5% of travelers returning from rural subequatorial Africa. This case report illustrates that rifampin represents an effective alternative to doxycycline for treatment of African tick bite fever in some selective situations.

Notes from the Field: Rickettsia parkeri Rickettsiosis - Georgia, 2012-2014.

During 2012-2014, five cases of Rickettsia parkeri rickettsiosis were identified by a single urgent care practice in Georgia, located approximately 40 miles southwest of Atlanta. Symptom onset occurred during June-October, and all patients had a known tick bite. Patients ranged in age from 27 to 72 years (median = 53 years), and all were male. The most commonly reported initial signs were erythema (n = 3) and swelling (n = 2) at the site of the bite. Two patients reported fever and a third patient reported a rash and lymphadenopathy without fever. Other symptoms included myalgia (n = 3), chills (n = 3), fatigue (n = 2), arthralgia (n = 2), and headache (n = 2). Eschar biopsy specimens were collected from each patient using a 4-mm or 5-mm punch and placed in 10% neutral buffered formalin or sterile saline. These specimens were tested by immunohistochemical (IHC) stains, quantitative polymerase chain reaction (qPCR) assays, or cell culture isolation to determine if there was evidence of infection with a Rickettsia species (1). IHC evidence of spotted fever group rickettsiae was found in the eschar biopsy specimens in all five cases. In four cases, the biopsy specimens were also positive for R. parkeri by qPCR. The fifth case (specimen positive only by IHC testing) was considered a probable R. parkeri case based on clinical signs and symptoms. R. parkeri was grown in cell culture from one specimen from which isolation was attempted. All patients were treated with oral doxycycline (100 mg twice daily) for a minimum of 10 days, and all recovered.

Rickettsia parkeri Rickettsiosis, Arizona, USA.

In the United States, all previously reported cases of Rickettsia parkeri rickettsiosis have been linked to transmission by the Gulf Coast tick (Amblyomma maculatum). Here we describe 1 confirmed and 1 probable case of R. parkeri rickettsiosis acquired in a mountainous region of southern Arizona, well beyond the recognized geographic range of A. maculatum ticks. The likely vector for these 2 infections was identified as the Amblyomma triste tick, a Neotropical species only recently recognized in the United States. Identification of R. parkeri rickettsiosis in southern Arizona demonstrates a need for local ecologic and epidemiologic assessments to better understand geographic distribution and define public health risk. Education and outreach aimed at persons recreating or working in this region of southern Arizona would improve awareness and promote prevention of tickborne rickettsioses.

Ischemia-reperfusion increases transfection efficiency of intracoronary adenovirus type 5 in pig heart in situ.

Efficiency of intracoronary (IC) adenoviral vector transfection is impaired by the vascular endothelium. Ischemia and substances that increase vascular permeability (sodium nitroprusside, nitroglycerin) may augment adenoviral vector transfection efficiency (TE). We tested whether TE of adenoviral vector following IC infusion is improved by nitrates or by ischemia. Fluoroscopically guided angioplasty balloon catheters occluded the coronary artery in Yorkshire pigs and delivered adenoviral type 5 vector encoding the luciferase gene (Ad5Luc, 10(11) viral particles). TE (luciferase activity) was minimal and was not augmented by IC co-administration of 50 µg/min sodium nitroprusside to nonischemic myocardium. Two (but not one) 3-min episodes of occlusion tended to increase luciferase activity (p=0.06), and luciferase activity was further increased by IC co-administration of nitroglycerin (p<0.001). After 75 min of coronary artery occlusion, luciferase activity was greater than with shorter periods of ischemia, and was significantly greater in the ischemia-reperfused zone compared to the border zone 3 and 14 days after infusion; there was no transfection in nonischemic myocardium. IC delivery of Ad5Luc into post-ischemic myocardium caused no local inflammation or hemodynamic instability. We conclude that the uptake of IC Ad5 to ischemic reperfused myocardium validates use of IC Ad5 delivery protocols in future human gene therapy trials in patients following myocardial ischemia.

Selective ß2-adrenoreceptor stimulation attenuates myocardial cell death and preserves cardiac function after ischemia-reperfusion injury.

OBJECTIVE: ß(2)-adrenoreceptor activation has been shown to protect cardiac myocytes from cell death. We hypothesized that acute ß(2)-adrenoreceptor stimulation, using arformoterol (ARF), would attenuate myocardial ischemia/reperfusion (R) injury via NO synthase activation and cause a subsequent increase in NO bioavailability. METHODS AND RESULTS: Male C57BL/6J and endothelial NO synthase (eNOS) knockout mice were subjected to 45 minutes of myocardial ischemia and 24 hours of R. ARF or vehicle was administered 5 minutes before R. Serum troponin-I was measured, and infarct size per area-at-risk was evaluated at 24 hours of R. Echocardiography was performed at baseline and 2 weeks after R. Myocardial cAMP, protein kinase A, eNOS/Akt phosphorylation status, and NO metabolite levels were assayed. ARF (1 µg/kg) reduced infarct size per area-at-risk by 53.1% (P<0.001 versus vehicle) and significantly reduced troponin-I levels (P<0.001 versus vehicle). Ejection fraction was significantly preserved in ARF-treated hearts compared with vehicle hearts at 2 weeks of R. Serum cAMP and nuclear protein kinase A C-α increased 5 and 15 minutes after ARF injection, respectively (P<0.01). ARF increased Akt phosphorylation at Thr(308) (P<0.001) and Ser(473) (P<0.01), and eNOS phosphorylation at Ser(1177) (P<0.01). ARF treatment increased heart nitrosothiol levels (P<0.001) at 15 min after injection. ARF failed to reduce infarct size in eNOS(-/-) mice. CONCLUSIONS: Our results indicate that ß(2)-adrenoreceptor stimulation activates cAMP, protein kinase A, Akt, and eNOS and augments NO bioavailability. Activation of this prosurvival signaling pathway attenuates myocardial cell death and preserves cardiac function after ischemia/reperfusion.

The polysulfide diallyl trisulfide protects the ischemic myocardium by preservation of endogenous hydrogen sulfide and increasing nitric oxide bioavailability.

Diallyl trisulfide (DATS), a polysulfide constituent found in garlic oil, is capable of the release of hydrogen sulfide (H(2)S). H(2)S is a known cardioprotective agent that protects the heart via antioxidant, antiapoptotic, anti-inflammatory, and mitochondrial actions. Here, we investigated DATS as a stable donor of H(2)S during myocardial ischemia-reperfusion (MI/R) injury in vivo. We investigated endogenous H(2)S levels, infarct size, postischemic left ventricular function, mitochondrial respiration and coupling, endothelial nitric oxide (NO) synthase (eNOS) activation, and nuclear E2-related factor (Nrf2) translocation after DATS treatment. Mice were anesthetized and subjected to a surgical model of MI/R injury with and without DATS treatment (200 µg/kg). Both circulating and myocardial H(2)S levels were determined using chemiluminescent gas chromatography. Infarct size was measured after 45 min of ischemia and 24 h of reperfusion. Troponin I release was measured at 2, 4, and 24 h after reperfusion. Cardiac function was measured at baseline and 72 h after reperfusion by echocardiography. Cardiac mitochondria were isolated after MI/R, and mitochondrial respiration was investigated. NO metabolites, eNOS phosphorylation, and Nrf2 translocation were determined 30 min and 2 h after DATS administration. Myocardial H(2)S levels markedly decreased after I/R injury but were rescued by DATS treatment (P < 0.05). DATS administration significantly reduced infarct size per area at risk and per left ventricular area compared with control (P < 0.001) as well as circulating troponin I levels at 4 and 24 h (P < 0.05). Myocardial contractile function was significantly better in DATS-treated hearts compared with vehicle treatment (P < 0.05) 72 h after reperfusion. DATS reduced mitochondrial respiration in a concentration-dependent manner and significantly improved mitochondrial coupling after reperfusion (P < 0.01). DATS activated eNOS (P < 0.05) and increased NO metabolites (P < 0.05). DATS did not appear to significantly induce the Nrf2 pathway. Taken together, these data suggest that DATS is a donor of H(2)S that can be used as a cardioprotective agent to treat MI/R injury.

Beta3-adrenoreceptor stimulation ameliorates myocardial ischemia-reperfusion injury via endothelial nitric oxide synthase and neuronal nitric oxide synthase activation.

OBJECTIVES: This paper examined whether nebivolol protects the heart via nitric oxide (NO) synthase and NO-dependent signaling in an in vivo model of acute myocardial infarction. BACKGROUND: Beta(3)-adrenergic receptor (AR) activation promotes endothelial nitric oxide synthase (eNOS) activity and NO bioavailability. We hypothesized that specific beta(3)-AR agonists would attenuate myocardial ischemia-reperfusion (MI/R) injury via eNOS activation and increased NO bioavailability. METHODS: Mice were subjected to 45 min of myocardial ischemia in vivo followed by 24 h of reperfusion (R). Nebivolol (500 ng/kg), CL 316243 (1 µg/kg), BRL-37344 (1 µg/kg), or vehicle (VEH) was administered at the time of R. Myocardial area-at-risk (AAR) and infarct size (INF)/AAR was measured at 24 h of R. Cardiac tissue and plasma were collected to evaluate eNOS phosphorylation, neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase expression, and nitrite and nitrosothiol levels. RESULTS: Nebivolol (500 ng/kg) reduced INF/AAR by 37% (p < 0.001 vs. VEH) and serum troponin-I levels from 41 ± 4 ng/ml to 25 ± 4 ng/ml (p < 0.05 vs. VEH). CL 316243 and BRL-37344 reduced INF by 39% and 42%, respectively (p < 0.001 vs. VEH). Nebivolol and CL 316243 increased eNOS phosphorylation at Ser-1177 (p < 0.05 vs. VEH) and increased nitrite and total nitrosylated protein levels. Nebivolol and CL 316243 significantly increased myocardial nNOS expression. Nebivolol failed to reduce INF after MI/R in beta(3)-AR (-/-), eNOS(-/-), and in nNOS(-/-) mice. CONCLUSIONS: Our results indicate that beta(3)-AR agonists protect against MI/R injury. Furthermore, the cardioprotective effects of beta(3)-AR agonists are mediated by rapid eNOS and nNOS activation and increased NO bioavailability.

Exercise protects against myocardial ischemia-reperfusion injury via stimulation of ß(3)-adrenergic receptors and increased nitric oxide signaling: role of nitrite and nitrosothiols.

RATIONALE: Exercise training confers sustainable protection against ischemia-reperfusion injury in animal models and has been associated with improved survival following a heart attack in humans. It is still unclear how exercise protects the heart, but it is apparent that endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) play a role. OBJECTIVE: To determine the role of ß(3)-adrenergic receptors (ß(3)-ARs), eNOS activation, and NO metabolites (nitrite and nitrosothiols) in the sustained cardioprotective effects of exercise. METHODS AND RESULTS: Here we show that voluntary exercise reduces myocardial injury in mice following a 4-week training period and that these protective effects can be sustained for at least 1 week following the cessation of the training. The sustained cardioprotective effects of exercise are mediated by alterations in the phosphorylation status of eNOS (increase in serine 1177 and decrease in threonine 495), leading to an increase in NO generation and storage of NO metabolites (nitrite and nitrosothiols) in the heart. Further evidence revealed that the alterations in eNOS phosphorylation status and NO generation were mediated by ß(3)-AR stimulation and that in response to exercise a deficiency of ß(3)-ARs leads to an exacerbation of myocardial infarction following ischemia-reperfusion injury. CONCLUSIONS: Our findings clearly demonstrate that exercise protects the heart against myocardial ischemia-reperfusion injury by stimulation of ß(3)-ARs and increased cardiac storage of nitric oxide metabolites (ie, nitrite and nitrosothiols).