Reaction of Bacillus subtilis products with amebocyte lysates of the Japanese horseshoe crab, Tachypleus tridentatus.

Autoclaved aqueous extracts of Candida albicans cells (and the glucans isolated from them) give a positive reaction with a chromogenic substrate combined with amebocyte lysates of the Japanese horseshoe crab, Tachypleus tridentatus (CS-TAL). The extracts and glucans activate the lysate enzyme compound G, which in turn activates clotting enzyme. Activated clotting enzyme causes a positive CS-TAL reaction. C. albicans extracts and glucans react positively with a commercially available, unaltered CS-TAL preparation (Toxicolor), but they give a negative reaction with a CS-TAL from which compound G has been excluded (Endospecy). An autoclaved, sterile preparation of Sabouraud glucose broth used as a control in one experiment gave (like Candida extracts) a positive reaction with Toxicolor and a negative reaction with Endospecy. We found that the peptone powder used to make the Sabouraud glucose broth was contaminated with a strain of Bacillus subtilis. Autoclaved aqueous extracts of culture-grown B. subtilis cells were positive with Toxicolor and negative with Endospecy. This was also the case with two other strains of B. subtilis. Polysaccharides obtained from these extracts gave the same result. Endotoxin activates clotting enzyme through activation of the lysate enzyme compound C, which is present in both Toxicolor and Endospecy. Endotoxin, therefore, reacts with both CS-TAL preparations. Simultaneous assay with Toxicolor and Endospecy distinguishes endotoxin from fungal products, but since products of fungi and B. subtilis both give a positive Toxicolor and a negative Endospecy test, a simultaneous assay cannot differentiate them. However, this does not decrease the clinical value of the simultaneous Toxicolor-Endospecy assay for distinguishing fungal infection from endotoxemia because B. subtilis so rarely causes disease that it can be excluded from clinical consideration.

Reaction of fungal products with amebocyte lysates of the Japanese horseshoe crab, Tachypleus tridentatus.

A commercially available endotoxin assay (CS-TAL) employing a chromogenic peptide and an amebocyte lysate from the Japanese horseshoe crab, Tachypleus tridentatus, gave a positive result with aqueous extracts of all 15 strains of Candida albicans and 1 strain each of Candida tropicalis, Cryptococcus neoformans, and a Mucor species that we tested. Purified glucans prepared from the Candida strains gave the same results. Reconstruction experiments showed that the positive results were not due to contaminating endotoxin. By contrast, assays employing amebocyte lysates of the American horseshoe crab, Limulus polyphemus, were inconsistent. Japanese workers have presented evidence that glucans activate the Tachypleus amebocyte lysate system by acting on an enzyme different from that on which endotoxin acts. Using a Tachypleus lysate preparation (Endospecy; Seikagaku Kogyo, Tokyo, Japan) from which this enzyme was excluded, we demonstrated a 5- to 10-fold drop in reactivity to the aqueous Candida extracts and glucans, whereas reactivity to endotoxin was unchanged. Normal human plasma was shown to decrease the effect of fungal extracts on CS-TAL. This inhibition was completely removed by heating the plasma. Our results suggest that Tachypleus systems may be of use clinically in distinguishing bacterial from fungal infections.

Endotoxin levels in immunocompromised children with fever.

Febrile episodes for which no cause can be found are common in immunocompromised children. We postulated that circulating endotoxin, a known pyrogen, might be responsible for some of these episodes in the absence of documented infection. Plasma endotoxin levels were assayed using a recently developed Limulus amebocyte lysate assay enhanced in sensitivity and objectivity by the addition of a chromogenic substrate. Eighty-seven plasma endotoxin determinations were made in 36 immunocompromised children with fever. Convalescent endotoxin levels and levels in normal children were also obtained. It was concluded that a plasma endotoxin level of 35 pg (0.10 EU)/ml constitutes the upper limit of normal in children. Five children (14%) had elevated endotoxin levels in the course of the febrile episodes, in the absence of bacteremia or clinically diagnosed infection. In each case, the levels returned to normal during convalescence. It is concluded that endotoxemia is a possible cause or contributing cause of unexplained fever in immunocompromised children.

Status of immunity of newborns against Yersinia enterocolitica: a preliminary report.

Out of 221 cord blood specimens assayed by whole cell agglutination for antibodies against Y. enterocolitica serogroup 0:3, 0:5,27, and 0:8, only four showed agglutinins despite the high percentage (33%) of agglutinating antibodies in 151 sera of pregnant women, ranging in titer from 4 to 32. Of 199 different cord sera tested, 25% to 28% showed low-level antibodies against erythrocytes treated with individual heat-extracts from Y. enterocolitica 0:3, 0:5,27, and 0:8. These sera equally agglutinated erythrocytes sensitized with the heat-extract of a randomly selected Escherichia coli strain. Conversely, when anti-yersinia hemagglutinins were absent in cord sera, concomitant cross-reactivity with E. coli was also absent. None of the 199 cord sera with a HA titer greater than or equal to 4 were reactive by whole cell agglutination. Under the test conditions used, CEA (common enterobacterial antigen) could be the main antigenic component to which the Y. enterocolitica hemagglutinins were directed. Nine of the reactive sera from pregnant women were treated with staphylococcal protein A with resultant loss of reactivity. Transplacentally acquired antibody directed primarily against the CEA antigen of Y. enterocolitica may protect the newborn against Y. enterocolitica infection.

Treatment of Salmonella gastroenteritis in infants. The significance of bacteremia.

During 1981, we treated 20 infants, less than 24 months old, for nontyphoid Salmonella (NTSal) gastroenteritis (GE). Blood cultures were obtained in 17 cases, and Salmonella bacteremia was demonstrated in 8 (47%). Of the 13 children 3 to 24 months of age, 7 (54%) had positive blood cultures. One child (8 months old) appeared septic. The patients with bacteremia were treated with parenteral ampicillin. All 20 infants recovered, and no focal infectious complications occurred. The incidence of bacteremia in NTSal GE is highest in children under 2 years of age. Previous reports have shown that the peak incidence occurs among infants less than 3 months of age. An infant with Salmonella bacteremia may be afebrile and show no symptoms of sepsis. In most cases, bacteremia is transient and does not alter the course of NTSal GE, but it may result in life-threatening complications such as septicemia and meningitis. Therefore we believe an infant with NTSal GE under 3 months old should have a blood culture and receive antibiotic treatment.

Penicillin prophylaxis and neonatal streptococcal disease.

Despite a number of efforts no means of preventing neonatal group B beta-hemolytic strep (GBS) infection by maternal treatment or immunization has proved effective. But impetus to a direct approach to the infant has been provided by the serendipitous discovery that routine penicillin prophylaxis for gonococcal ophthalmia may also protect against GBS. Large-scale controlled studies are now under way.