The Limulus amebocyte lysate test as a laboratory aid in the diagnosis of infectious diseases

27 Infectious Diseases Newsletter 7(4) April 1988 sensitive test for the etiology of gastroonteritis. The Norwalk virus, Snow Mountain agent, caliciviruses, and astroviruses are detectable by

assays employing their immunoelectronmicroscopy, RIA, or avidin-biotin techniques. These essays are available as research tools in only a handful of centers around the world. Rapid diagnostic tests and methods for culture are needed, particularlyfor classificationof these ubiquitous agents and development of measures preventive of disease.

Bibliography Caul EO, Appleton H: The electron mi-

croscopical and physical characteristics of small round human fecal viruses. An interim scheme for classification. J Med Virol 9:257-265, 1982. Cubith WD, Blacldow NR, Hermann JE, et al: J Infect Dis 156:806-814, 1987. Greenberg HB, Wyatt RG, Kalica AR, et al: New insights in viralgastroenteritis. Perspect Virol 11:163-184, 1981. Kaplan JE, Gary GW, Baron RC, et al: Epidemiology of Norwalk gastroenteritis and the role of Norwalk virus in outbreaks of acute nonbacterial gastroenteritis. Ann Intern Med 96:756761, 1982. Keswick BH, Satterwhite TK, Johnson PC, et al: Inactivation of Norwalk virus in drinking water by chlorine. Appl Environ Microbiol 50:261-264, 1985. Kurtz JB, Lee TW, Craig JW, et al: As-

The Limulus Amebocyte Lysate Test as a Laboratory Aid in the Diagnosis of Infectious Diseases Michael A. Saubolle Microbiology Section, Department of Pathology Good Samaritan Medical Center Phoenix, Arizona

The observations of Frederik Bang, together with later studies by Jack Levin, on the coagulation properties of blood cells of the horse-shoo crab (Limulus polyphemus) when exposed to Gram-negative bacteria, led to the introduction of a very sensitive in vitro assay for endotoxin. Presently, Limulus amebocyte lysate test methods include the original gelationclot tube test (LAL), a second test miniaturizing the gelatin method while applying it to a glass slide, and a recently introduced chromogenic amebocyte lysate (CLAL) test. The LAL test relies on an endotoxin-initiated activation cascade culminating in the conversion of soluble coagulogen into an insoluble coagulin with the formation of a clot. The test requires a 30-60-minute incubation period and is prone to mechanical disruption. The newer CLAL test utilizes endotoxin initiation of the proteolytic activity of a proonzyme and subsequent cleaving of a chromo-

genic p-nitroanilide group from its peptide carrier, resulting in the production of a yellow color that is proportional in intensity to the concentration of endotoxin. The CLAL test is more sensitive than the original LAL test, requires only a 5-30-minute incubation period, is not prone to mechanical disruption, and as easily adapted to a microtiter system and to spectrophotometric quantitation. The Limulus amebocyte lysate test greatly simplified detection of endotoxin in medical and pharmaceutical products, as well as clinical specimens. Biomedical application of the test is now well entrenched; however, its role in the clinical diagnosis of infectious diseases is more controversial and less popular. Despite lack of enthusiasm for its use in clinical diagnosis, substantial evidence has accumulated supporting its potential use in the diagnosis of certain infections caused by Gram-negative bac© 1988 Elsevier Science Publishing Co., Inc. 0278-2316/88/$0.00 + 2.20

trovirus infection in volunteers. J Med Virol 3:221-230, 1979. Madore lIP, Treanor JJ, Dolin R: Characterization of the Snow Mountain agent of viral gastroenteritis. J Virol 58:487492, 1986. Morse DL, Guzewich JJ, Hanrahan JP, et al: Widespread outbreaks of clam and oyster-associated gastroenteritis: role of Norwalk virus. N Engl J Med 314:678681, 1986. Parrino TA, Schreiber DS, Trier JS, et al: Clinical immunity in acute gastroenteritis caused by Norwalk agent. N Engl J Med 297:86-89, 1977. Wyatt RG, Dolin R, Blacklow NR, et al: Comparison of three agents of acute infectious nonbacterial gastroenteritis by cross-challenge in volunteers. J Infect Dis 129:709-714, 1974.

teria. Clinical applications of the LAL/CLAL tests have included detection of endotoxin in meningitis, bacteduria, gonococcal urethritis and cervicitis, ocular keratitis and endophthalmitis, septic arthritis, peritonitis, intra-amniotic infections, and endotoxemia. Although few in number, clinical assessments of the LAL test for rapid diagnosis of Gram-negative ocular, synovial, and intra-anmiotic infections suggest potential applications. However, recent reports placing the sensitivity of the test at 100% for Gram-negative organisms in effluents from continuous ambulatory peritoneal dialysis have not been substantiated in our experience with 19 cases (semitivity of only 32%). A large number of studies have focused on applying the LAL/CLAL tests to rapid screening of genitourinary specimens for Gram-negative bacteria. Multiple studies, using either dilutions of urine or "time-togelation" techniques, yielded overall sensitivities of approximately 84%-92% and 94%-100% when the counts of Gram-negative bacteria exceeded 100,000/ml of urine. The newer CLAL test with spectrophotometric reading may have enhanced sensitivity and utility in this setting.

28

Infectious Diseases Newsletter 7(4) April 1988

Nevertheless, the sensitivities of all tests fell when there were fewer than 105 bacteria/ml. Thus, the usefulness of the LAL/CLAL tests for helping diagnose urinary tract infections may be limited because bacterial counts between 100 to 10,000 per ml may be clinically significant. For the presumptive diagnosis of gonococcal urethritis, the sensitivity of the LAL test is no better than that of the Gram-stain of urethral exudate. In screening women for gonorrhea, nonspecific factors that confounded the results of earlier studies were avoided in more recent work by standardizing the methods for collecting and processing specimens. While interference by non-gonococcal flora resident in the female genital tract was not eliminated, the specificity of the CLAL test using standard methods was 43%-52%, and the predictive value of a negative test for the absence of gonococci was 100%. The clinical utility of the Limulus amebocyte lysate test in screening genitourinary tract specimens will depend on the definition of the following: the role of the test in such studies, the patient populations being studied, and the prevalence of the specific disease entities under consideration. The usefulness of the LAL test for detecting endotoxemia has been clouded by a myriad of conflicting reports reflecting the influence of nonspecific activators or inhibitors present

in certain body liquids. Although several methods for the removal of such nonspecific factors have been proposed, significant problems still remain that detract from the utility of the test. The application of the CLAL test to blood has increased interest in correcting the problems associated with blood. Thus, recent application of the CLAL test for the detection of endotoxemia resulted in sensitivities and specificities approaching 75%-92% and 91%-98%, respectively. As methods and standards are improved, the CLAL test may become a useful tool in evaluation of certain high risk patients (e.g., with neutropenia or following surgical operations). The most convincing evidence for the utility of the Limulus test has accrued from studies in meningitis. The LAL, LAL microslide, and CLAL methods have all been used to detect endotoxin in cerebrospinal fluid (CSF). In the majority of reports, the overall sensitivity and specificity for detecting endotoxin in the CSF of patients with meningitis approaches 97% and 99%, respectively. However, there is an apparent dichotomy of efficacy in that sensitivities range from greater than 99.5% in communityacquired meningitis (caused by Haemophilus influenzae type b [Hib] and Neisseria meningitidis [Nm]) to sensitivities of 67%-71% and 33% in neonatal or nosocomially-acquired meningitis (caused by Gram-negative

Table 1. Results of cultures, Gram-stains, antigen detection studies, and Limulus tests on CSF from patients with meningitis. Number of CSF positive/number of CSF tested (%) Organism Groups Hib and Nm GNB (other than Hib) Gram-positive Mycobacterial, treponemal, or fungal Viral

Culture

Gram-stain

Antigen

Limulus test

115/127 (91)

86/106 (81)

47/55 (86)

127/127 (100)

-25/37 (71)

-22/28(79)

10/15 (67) 0/47 (0)

15/15 (100) 42/4.4 (96)

m

m

m

0/12 (0) 0/30 (0)

Adapted from SauboUe and Jorgensen (Diagn Microbio Infect Dis 7:177-183, 1987). Antigen: Antigen detection using commerical latex kits Hib and Nm: Haemophilus influenzae, type b, and Neisseria meningitidis

GNB: Gram-negative bacilli

© 1988 Elsevier Science Publishing Co., Inc. 0278-2316/88/$0.00 + 2.20

bacilli [GNB] other than Hib) and in primary parameningeal infections, respectively. The discrepancy may be ascribed to the normally higher bacterial counts, and thus higher concentrations of endotoxin, in CSF from patients with community-acquired meningitis caused by either Hib or Nm. In meningitis with Hib and Nm, we found that the LAL and CLAL tests were more sensitive for detecting endotoxin in the CSF (100%) than cultures (91%), Gram-stain (81%), or antigen detection latex tests (86%). In earlier studies, the sensitivity of the Limulus test was superior to the Gram-stain in a similar group of patients (99.5% and 67%, respectively). The high sensitivity/specificity, relative technical ease, and low cost of the Limulus test (especially using the CLAL method) recommend it for screening prior to performance of rapid antigen detection studies. That is, in patients in whom evaluation of the CSF with antigen detection batteries is planned, the Limulus test might be performed first. Specimens with negative LAL or CLAL tests would require further testing only with Gram-positive bacterial antigen detection reagents, whereas those with positive tests would merit testing only with Hib and Nm reagents. In the latter case, initially unsuccessful antigen detection attempts might be enhanced by concentrating the CSF. Quantitation of endotoxin in the CSF in meningitis may be of value in prognosis and patient management. It has been suggested that initial concentrations of endotoxin exceeding 150 ng/ml are associated with seizures. The Limutus lysate test may provide important leads to making appropriate decisions not only with regard to the manipulation of specimens to optimize yield while minimizing cost, but also to optimize patient care. The test must, however, be used judiciously, with an understanding of its capabilities and limita-

29 Infectious Diseases Newsletter 7(4) April 1988 tions, and in conjunction with other tests.

Bibliography Buller HR, ten Cate JW, Sturk A, et al: Validity of the endotoxin assay in post surgical patients. In: ten Cate JW, BuUer HR, Sturk A, Levin J (eds): Bacterial Endotoxins: Structure, Biomedical Significance, and Detection with the Limulus Amebocyte Lysate

Test. New York, Alan R. Liss, 1985, pp 369-384.

Naehum R, Berzofsky RN: Chromogenic Limulus amebocyte lysate assay for rapid detection of Gram-negative bacteriuria. J Clin Microbiol 21:759-763, 1985.

Prior RB, Spagna VA: Rapid evaluation of gonococeal and nongonoeoceal urethrifts in men with Limulus amoebocyte lysate and a ehromogenic substrate. J Clin Microbiol 17:485-488, 1983. Romero R, Kadar N, Hobbins JC, et al: Infection and labor: the detection of endotoxin in amniotie fluid. Am J Obstet Gynecol 157:815-819, 1987. Saubolle MA, Jorgensen JI-I: Use of the Limulus amebocyte lysate test as a cost-effective screen for Gram-negative agents of meningitis. Diagn Microbiol Infect Dis 7:177-183, 1987.

vealed slight splenomegaly and moderate hepatomegaly without focal lesions and no intraabdominal masses or lymphadenopathy. An echocardiogram was unremarkable. During his first two hospitalizations he defervesced without antimicrobic therapy. During his third hospitalization, he was treated empirically with trimethoprim-sulfamethoxazole (160 mg + 800 mg, PO, 12-hourly), and he defervesced; treatment was continued for 10 days during which he had one episode with fever to 101°F. He continued afebrile for one week after therapy. However, on the morning of referral, he again had the sudden onset of chills, rigors, fever, profuse sweating, nausea, and vomiting. From the time of onset of illness to admission he lost 20 pounds of body weight. He denied cough, sputum production, dysuria, hematuria, diarrhea, abdominal pain, neck pain, or photophobia. He reported no recent travel, denied eating uncooked meat or poorly prepared food, drinking unpurified water, and reported no recent insect bites. On direct questioning, he recalled cleaning ticks off his dogs, but insisted he had never been bitten by ticks. His past medical history was significant for a closed fracture of the right wrist in April, 1987, a history of prostatitis, and trans-sigmoidoscopic removal of a colonic polyp (reported benign by histopathology)

one year before entry. There was no history of medication or allergies. His home is a 100-acre ranch in the Sierra foothills; there are horses, dogs, and cattle on the ranch, as well as indigenous wildlife. Culinary water is taken from a well for the entire family; none of the other family members has been ill. The patient has smoked a pipe for many years, and customarily takes 3-5 highballs each day. On physical examination, he was an obese white male who appeared to be acutely ill with temperature (oral) of 37.2°C, pulse of 108, regular respirations of 24, and non-orthostatic blood pressure of 124/70. His skin was without rash or lesions. There were no abnormalities of the eyes, ears, mouth, oropharynx, lungs, or heart. The abdomen was obese, soft, nontender, bowel sounds were normal, and the liver was 12 cm in span by percussion, and palpable, but nontender, 2 cm below the right costal margin; there was no splenomegaly. The prostate was nontender, and normal in size and consistency. The feces was guaiac negative. All of the extremities were normal. There were no neurologic deficits. The peripheral leukocyte count was 9,800/I.d with 49% mature and 27% band neutrophils, 135 lymphocytes, 11% monocytes, and no eosinophils. The hematocrit was 35%; platelets 219,000/ixl. The urine sediment con-

Dwelle TL, Dunlde LM, Blair L: Correlation of cerebrospinal fluid endotoxinlike activity with clinical and laboratory variables in Gram-negative bacterial meningitis in children. J Clin Mierobiol 25:856-858, 1987.

CASE REPORT A 53-year-old previously healthy white male sheriff who lives on a cattle ranch was admitted to the hospital for evaluation of fever of unknown origin. Five weeks prior to admission, he noted the sudden onset of fever to 102°F, drenching sweats, chills, rigors, intermittent nausea and vomiting, malaise, anorexia, joint pains, and headache. After two days of symptoms, he was hospitalized by the referring physician and blood cultures were drawn. On the second hospital day he spontaneously defervesced and was discharged; the blood cultures were negative. Over the next four weeks he experienced recurrent episodes of fever, drenching sweats, malaise, and anorexia. Each episode lasted 2-5 days, with 4-8 days between episodes; during the afebrile periods, he felt better than when he had fever, but not well enough to return to work. He was again hospitalized during two of the episodes of fever; multiple blood cultures yielded no growth, and there was insignificant seroreactivity by testing for salmonellal and brucellar agglutinins, and for EBV virus. Several complete blood counts (including differentials), multiple blood chemical tests, and thyroid function tests were all normal. His AST and ALT were 36 and 53, respectively, and his sedimentation rate (ESR) was consistently between 40 and 50 ram/hr. An abdominal CT re-

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