Vincent A. Spagna

Evaluation of Plasmodium berghei for endotoxin by the Limulus lysate assay.

The presence of endotoxin in the malaria parasite has long been suspected but has not been directly demonstrated by the Limulus amoebocyte lysate (LAL) assay or other assays. Tests for endotoxins have been done on plasma of infected individuals but not on the parasites themselves (Glew and Levin, 1975, Proc. Soc. Exp. Bio. Med. 148: 508-510). Recently, Tubbs (1980, Trans. R. Soc. Trop. Med. Hyg. 74: 121-123) detected endotoxin activity in the plasma of mice infected with Plasmodium berghei and in patients infected with P. falciparum using the LAL assay, and suggested that the endotoxin was derived from either the parasites or the endogenous bacteria in the gut. By assaying intact plasmodia washed free of contaminating host cells and plasma, it is possible to determine whether the plasmodia are the source of the endotoxin. We obtained free P. berghei using ultrasonic energy and tested the plasmodia for endotoxin activity using the LAL assay. The results obtained are the subject of this report. Groups of mice and rats were infected by the intraperitoneal injection of approximately 106 P. berghei parasites, and later the blood was collected aseptically by cardiac puncture when parasitemia reached 40%. The blood was transferred to pyrogen-free, sterile, heparinized tubes and washed three times in sterile, pyrogen-free 0.9% NaCl. Blood from uninfected mice was treated in an identical manner and served as a control. The blood specimens were then sonicated as previously described (Prior and Kreier, 1972, Exp. Parasitol. 32: 239-243). The continuous-flow chamber and sonicator probe tip were rendered pyrogen-free by heating at 160 C overnight. All equipment and solutions were tested for endotoxin contamination with the LAL test before use. Following sonication the specimens were centrifuged to harvest the plasmodia, and then adjusted to 25% packedcell volume. The specimens were lysed by three cycles of freeze-thawing and assayed quantitatively for the presence of endotoxin using the microdilution LAL assay (Prior and Spagna, 1979, J. Clin. Microbiol. 10: 394395). The minimum sensitivity of the Limulus lysate utilized in the microdilution assay (courtesy of Mallinckrodt Inc., St. Louis, Missouri) was 0.06 ng/ml of LPS using the E. coli reference standard (lot EC-2, Bureau of Biologics, Food and Drug Administration). Aliquots of free parasite and control samples were seeded with known quantities of the E. coli endotoxin and assayed. Because certain heat-labile protein substances may give positive LAL test results, portions of the samples were heated at 100 C for 5 min and then tested for endotoxin activity. Parasites and control samples were also cultured on blood agar aerobically and anaerobically to detect any possible bacterial contamination. The results obtained in four experiments using the LAL assay on the lysed parasite suspensions and controls are shown in Table I. A reaction suggestive of endotoxin was ob-

Rapid presumptive diagnosis of gonococcal cervitis by the limulus lysate assay

In an evaluation of the limulus lysate assay (LLA) as a method for detecting gonococcal endotoxin in cervical exudates diluted 1:800, positive LLA results were obtained from 17 of 18 patients (94%) with culture-proved gonococcal cervicitis, and negative results were obtained from 22 of 22 patients (100%) with culture-negative specimens. In vitro tests comparing the sensitivity of the LLA for Neisseria gonorrhoeae and other gram-negative organisms showed the LLA to be more sensitive in detecting N. gonorrhoeae (minimum sensitivity, 10(4) organisms per milliliter) than other commonly encountered urogenital gram-negative bacteria (minimum sensitivity, greater than 10(5) organisms per milliliter). Thus, in preliminary studies involving otherwise healthy women, the LLA appeared to correlate with bacteriologic methods for diagnosing gonococcal cervicitis and may aid in identifying nongonococcal cervicitis. In addition, the LLA was easy to perform, with test results available within an hour after the patients initial examination.

Successful treatment with cefaclor of gonococcal urethritis in men.

Cefaclor, a new orally administered cephalosporin, was evaluated by a randomized trial for effectiveness in the treatment of uncomplicated urethritis due to Neisseria gonorrhoeae in men. Regimens included 2,3, and 4 g of cefaclor, with or without 1 g of orally administered probenecid, as single daily doses for three days. The diagnoses were confirmed by isolation of N. gonorrhoeae; cures or therapeutic failures were determined by follow-up cultures on day 7 after completion of therapy. Sixty-six (73%) of 90 treated patients were evaluable for efficacy. The bacteriologic cure rate was 98% (65/66); one patient treated with 2 g of cefaclor plus probenecid had a positive culture for N. gonorrhoeae on follow-up examination. Adverse reactions consisted of mild nausea in five patients (7%) and vomiting in one patient (1%) who received 3- or 4-g doses. No treatment was discontinued, and no abnormality of screening hematologic tests or enzymes was observed. Thus, cefaclor, given in multiple doses, was highly efficacious for treatment of uncomplicated gonococcal urethritis in men.

Application of the limulus lysate assay in evaluation of disseminated gonorrhea in women.

The limulus lysate assay was utilized as a diagnostic adjunct in the evaluation of three cases of disseminated gonorrhea in women. Although not a specific test for Neisseria gonorrhoeae, the limulus lysate assay, when used with properly diluted endocervical samples, gave results that correlated with conventional diagnostic techniques. If the advantages and limitations of the limulus lysate assay become fully appreciated, it may serve as a useful clinical tool for diagnosis of this syndrome.

Limulus endotoxin assay and meningitis.

Excerpt To the editor: The article by Dr. Neu in the November issue (1) is a concise, well-organized review of the services available to the clinician from the microbiology laboratory. However, we ...