Michael J. Hausmann

A rapid direct fluorescent assay for cell-free DNA quantification in biological fluids

Background Circulating cell-free DNA (CFD) levels may be elevated in trauma, stroke, sepsis, pre-eclampsia and cancer. Owing to the complex and expensive methodology, detection of CFD has hitherto been confined to research laboratories. This study presents a simple, inexpensive and accurate test for CFD. Methods Using the commercial fluorescent SYBR® Gold stain, biological fluids were directly assayed for CFD without prior DNA extraction and amplification. Stain was added to the sample in 96-well plates (final stain dilution: 1:10,000) and fluorescence was read by a fluorometer (excitation wavelength 488 nm, emission wavelength 535 nm). Results The assay was validated with serum, whole blood, urine and supernatant of cell cultures. Specificity and linearity were demonstrated over a wide range of concentrations; the results correlated with the conventional quantitative polymerase chain reaction assay of β-globin (R 2 = 0.9987, P < 0.001). The assay was not affected by exposure of whole blood or serum to room temperature for four or 24 h, respectively. Intra and day-to-day coefficients of variation (16–4.8% and 31–8%, respectively; depending on DNA level) compared well with published data describing more work-intensive tests. The limit of quantitation (170 ng/mL) was below the mean DNA level in a cohort of normal individuals (471 [203] ng/mL). Finally, free DNA in supernatant of cell cultures after cell lysis accurately reflected cell number (R 2 = 0.974, P < 0.0001). Conclusions The direct SYBR® Gold assay proved to be an accurate and simple technique for measuring CFD in biological fluids.

Sporadic culture-negative peritonitis in peritoneal dialysis patients--absence of endotoxin in dialysate.

Background: Indiscriminate use of broad-spectrum antibiotics in peritonitis may have either unwanted side effects or contribute to the development of antibiotic resistance. It is tempting to use broad-spectrum antibiotics in cases of culture-negative peritonitis. This study examines whether Gram-negative agents have to be considered in the management of culture-negative peritonitis. Gram-negative agents are manifested by endotoxin easily detected by the Limulus amebocyte lysate (LAL) test. Methods: 138 episodes of Gram-negative and culture-negative peritonitis have been retrospectively analyzed; episodes of Gram-negative peritonitis were controls. Correlation between LAL and culture results was compared between the two groups. The LAL test was performed using a commercial kit by incubating a mixture of dialysate effluent and LAL reagent at 37°C. Development of a stable solid clot was considered positive. Results: In controls, 80 out of 117 Gram-negative peritonitis were LAL positive (68%). None of the 21 culture-negative episodes was LAL positive. In 7 recurrences of Gram-negative peritonitis, the LAL test turned from negative to positive but in none of the recurrences of culture-negative peritonitis. The difference in correlation was highly significant. Conclusions: Gram-negative organisms do not seem to be involved in sporadic culture-negative peritonitis. In episodes of peritonitis in which bacteriologic cultures stay negative for 48 h, initial coverage of Gram-negative organisms may be dropped.

Aspergillus Peritonitis in Continuous Ambulatory Peritoneal Dialysis Patients

Aspergillus peritonitis is a rare and serious cause of peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients. We report 3 cases of aspergillus peritonitis in CAPD which were successfully treated by catheter removal and amphotericin. Two of the 3 patients returned temporarily to CAPD, but were subsequently transferred to hemodialysis because of membrane failure. A novel finding in 2 of the 3 cases was a positive Limulus amebocyte lysate test, despite negative bacterial cultures. We discuss the possible relevance of this finding to the diagnosis of aspergillus infections and emphasize the importance of early catheter removal for successful treatment of this condition.

Colchicine-sensitive nephrotic syndrome due to AA amyloidosis

A 28-year-old women is presented who was evaluated for a new-onset postpartum nephrotic syndrome with normal renal function. Histological diagnosis was AA amyloidosis but no underlying disease has been diagnosed despite extensive workup. Complete remission was achieved with colchicine. Upon discontinuation of colchicines, the patient’s nephrotic syndrome flared up but completely responded to reinstitution of colchicine therapy. Remission of this patient’s nephrotic syndrome is thus not attributable to resolution of any “idiopathic” primary disease.

Short- and Long-Term Comparative Study of Anistreplase Versus Streptokinase in Acute Myocardial Infarction

Streptokinase is well established as an effective thrombolytic. Anistreplase, a new thrombolytic drug, is a complex of streptokinase and acylated human plas minogen that can be administered by intravenous bolus and activates plasmino gen at the clot site. Although both streptokinase and anistreplase are effected in treating myocardial infarction (MI), they have different pharmacologic proper ties. This study was designed to identify short- and long-term differences in their clinical effectiveness, safety in use, and survival rates in patients with acute MI. One hundred ten successive patients under seventy years of age admitted within three hours after onset of sustained chest pain suggestive of acute MI were randomized to receive either 30 units of anistreplase intravenously over five minutes or intravenous injection of 750,000 units of streptokinase over thirty to sixty minutes. Reperfusion was achieved in 34 of the 52 (65%) patients treated with anis treplase and in 41 of the 58 (71%) patients treated with streptokinase (p = NS). The two drugs were equally effective in preserving left ventricular ejection frac tion, which was found to be significantly better in patients with anterior wall MI who had achieved reperfusion than it was in those who did not (p < 0.02). One-month, twelve-month, and thirty-six-month survival rates were high (96% to 88%) with no significant difference between the two treatment groups. The authors conclude that the two drugs are equally effective thrombolytic agents but that anistreplase has the advantage that it can be administered as a bolus injection.

Prolonged fever following kidney biopsy: a case report

This case report describes a patient with prolonged fever following a kidney biopsy. Workup disclosed a large perirenal and retroperitoneal hematoma. Neither imaging nor blood cultures supported an infective cause of his fever. Although the patient was initially treated with antibiotics, fever eventually resolved spontaneously. A review of the literature is provided addressing the association of fever with resorption of hematoma. Fever should be added to the list of potential complications of kidney biopsy. A conservative management is advocated.