Human cryptosporidiosis: spectrum of disease. Report of six cases and review of the literature.

Although once regarded exclusively as an animal parasite, Cryptosporidium has emerged during the last decade as a cause of diarrhea in humans. Of the 43 cases of human cryptosporidiosis reported, 27 patients in whom either humoral or cell-mediated immune defects were present had chronic protracted diarrhea that was almost invariably unresponsive to therapy and culminated in death. In contrast, 16 patients with intact immune systems had either self-limited disease or were asymptomatic. Animal exposure was almost exclusively recorded in the latter group. During the last six months at University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, gastrointestinal (GI) tract cryptosporidiosis developed in six homosexual men with acquired immune deficiency syndrome (AIDS). The diagnosis of cryptosporidiosis was established histologically in five patients. In four, cryptosporidial oocysts were detected in the stool. In addition to extensive GI tract cryptosporidiosis, two patients had biliary tract involvement. Multiple chemotherapeutic agents failed to control the disease, and five patients eventually died. Cryptosporidiosis should be considered in the differential diagnosis of diarrhea, in animal handlers or in the severely compromised patient, especially one with AIDS, who has chronic protracted diarrhea.

Mycobacterium fortuitum bacteremia in patients with cancer and long-term venous catheters.

Primary bacteremia due to Mycobacterium fortuitum is an uncommon occurrence. Four cases of M. fortuitum bacteremia in patients with cancer, one of whom was neutropenic, are presented. None of the patients had evidence of disseminated disease or endocarditis, and there was no mortality directly associated with this infection. Two patients had polymicrobial sepsis with skin commensal organisms. The infection was related to the use of long-term central venous catheters or recent instrumentation in all patients. M. fortuitum should be added to the growing list of organisms causing catheter-related infections.

Serologic studies in hairy cell leukemia: high prevalence of Epstein-Barr and cytomegalovirus antibodies and absence of human T-cell lymphotrophic viruses antibodies.

Serum from 60 patients with hairy cell leukemia (HCL) were studied for the presence and the titers of antibodies to Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human T-cell lymphotrophic viruses (HTLV). Eighty-three percent of the patients were seropositive for EBV, with a (reciprocal) geometric mean titer (GMT) of 960. Seventy-eight percent of the patients had antibodies to CMV with a GMT of 435. All 21 patients tested for HTLV I and HTLV III were seronegative; only one patient showed detectable antibodies to HTLV II. The potential role of these infections in the physiopathology of HCL is discussed.

Detection by counterimmunoelectrophoresis of anti-Candida precipitins in sera from cancer patients.

Five antigen preparations from yeast-phase cells and hyphalphase cells of Candida albicans were used to detect antibodies in sera from cancer patients by use of counterimmunoelectrophoresis (CIE). Sera from uninfected patients, patients with superficial infections, and patients who had invasive or systemic candidiasis were analyzed for antibodies. A high incidence (27%) of false-negative data was obtained for patients who had systemic candidiasis. Serum titers of all patient groups were below 1:10. Of the antigens tested, cytoplasmic antigen from yeast-phase cells provided the greatest sensitivity and diagnostic value.

Effects of tuftsin on postsplenectomy sepsis.

Tuftsin is a tetrapeptide within the CH2 domain of the IgG immunoglobulin. Enzymatically cleaved from its parent globulin, it increases the phagocytic activity of macrophages, monocytes, and neutrophils by specific receptor mechanisms. In splenectomized hosts the circulating levels of tuftsin are reduced. Postsplenectomy sepsis is due to impaired clearance of intravascular bacteria; it has been postulated that tuftsin deficiency may contribute to this impairment. In this experiment splenectomized DBA/2 mice were subjected to pneumococcal sepsis. The groups of mice treated with tuftsin and those that received autotransplanted splenic tissue had significantly improved survival rates. We conclude that tuftsin deficiency plays a role in postsplenectomy sepsis and that treatment with synthetic tuftsin protects the splenectomized host against pneumococcal septic death.

Semiquantitative cultures of intravascular catheters from cancer patients.

Three-hundred seventy-nine catheter tips were prospectively cultured by both a semiquantitative method and by broth culture, over a 2-month period. One hundred eleven of the catheters were culture-positive in broth, and 47 of these were also culture-positive by the semiquantitative method. Clinical signs of infection were reviewed for the 111 culture-positive catheters and for 50 of the 268 culture-negative catheters. Both culture-positive and culture-negative catheters were infrequently associated with local signs of infection (10% and 12%, respectively). Culture-positive catheters, however, were more likely to be associated with systemic signs of infection than were culture-negative catheters (15% and 2%, respectively). Among the culture-positive catheters, those that yielded greater than or equal to 15 colonies on semiquantitative culture were more likely to be associated with septicemias than were those with less than 15 colonies (22% and 6%, respectively). Nevertheless, there were five catheter-related bacteremias that were associated with catheters which were culture-negative on semiquantitative culture but culture-positive in broth. The proportion of patients with culture-positive and culture-negative catheters who were febrile was similar (30% and 42%, respectively). Semiquantitative cultures of catheters from cancer patients are useful, but the result should be interpreted with some caution.

Use of molecular biological techniques in the diagnostic laboratory for detecting and differentiating fungi.

This review will address the value of nucleic acid amplification techniques used for the laboratory diagnosis of fungal infections. Although detection of all fungi will be considered, the emphasis will be placed on diagnosis of disseminated candidiasis. The diagnosis of most serious life threatening fungal infections in immunosuppressed patients remains a laboratory dilemma. The antemortem diagnosis of disseminated candidiasis, if made at all, is often made just prior to the death of the patient negating any hope of successful medical/surgical intervention. Molecular techniques such as those based on the polymerase chain reaction offer increased sensitivity since, in theory, these techniques require the presence of a single fungal cell. This manuscript will review 1) the current problems related to classical methods of diagnosing life threatening fungal infections; 2) considerations one must address relative to use of nucleic acid-based approaches such as polymerase chain reaction (PCR) for diagnosis of these fungal infections; and 3) status of use of these molecular approaches in the clinical mycology laboratory at the present time.

Purification of an antigenic vaccine protein by selective displacement chromatography.

A recent advance in the state of the art of displacement chromatography has been the development of selective displacement chromatography. In this process, the bioproduct of interest is selectively displaced while impurities with lower retention are eluted in the induced salt gradient and higher retained impurities are desorbed after the breakthrough of the displacer front. In this manuscript, selective displacement chromatography is employed to purify an antigenic vaccine protein (AVP) from an industrial process stream. Displacers were screened and an operating regime plot was employed to establish appropriate conditions for selective displacement. The selective displacement process was successful and resulted in AVP that was equivalent in purity to product obtained at commercial production scale after conventional step gradient chromatography. Methods used to characterize the purified protein include size-exclusion chromatography, SDS-PAGE, isoelectric focusing, N-terminal amino acid sequence analysis, and amino acid composition analysis. This is the first report of the purification of a commercially and pharmaceutically significant protein using selective displacement chromatography and thereby sets the stage for the implementation of selective displacement chromatography for the downstream processing of biologicals.

Disseminated candidiasis caused by four different Candida species.

Fatal disseminated candidiasis developed in a patient with hairy-cell leukemia. Four different Candida species were isolated from multiple sites during hospitalization and from cultures made post mortem.

Use of 111In-labelling to follow tissue distribution of Candida albicans in mice.

Two strains of Candida albicans, a wild type and a derived mutant, were labelled with 111Inoxine. Labelled cells were injected into mice and tissue distribution patterns were determined from 0.5 to 48 h. During the first 4-h post-injection phase, remarkable differences in tissue distribution were observed between the two strains. Radiolabelling of C. albicans with 111Inoxine is shown to be a much more reliable method for determining early tissue distribution patterns in infected animal models than culturing the infected tissue.

Solubilization and characterization of pellet-associated human brain alpha-L-fucosidase activity.

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at -20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.

Radiometric determination of the concentration of amphotericin B in body fluids.

A new assay was developed to determine the concentration of amphotericin B in body fluids. The Bactec radiometric system was used to measure CO(2) production by a test strain, Candida albicans MDA 448, in the presence of amphotericin B. After 5-h incubation, drug concentrations as low as 0.2 mug/ml could be detected. The results are comparable to those of the commonly used agar diffusion assay with Paecilomyces varioti.

Amphotericin B susceptibility testing of yeasts with a bactec radiometric system.

The effect of amphotericin B on CO(2) production was studied using a Bactec 225. A radiometric procedure for yeast susceptibility testing that requires 3 h of incubation was developed. A total of 48 yeast isolates was tested in this system and the results were correlated to tube dilution studies. The drug concentration causing at least a 44% decrease in CO(2) production correlated to the tube dilution minimal inhibitory concentration in 85% of isolates tested and was within one tube dilution of the minimal inhibitory concentration in over 95% of yeasts tested.

Caffeic acid-containing medium for identification of Cryptococcus neoformans.

A new growth medium containing caffeic acid and ferric citrate is described. The pigment produced on this medium is specific for the identification of Cryptococcus neoformans and differentiates it from other cryptococci. The medium is more easily compounded and requires less time for pigment formation than the conventional Guizotia extract media. The medium is stable in the dry form as well as in the prepared form.

Six-hour pigmentation test for the identification of Cryptococcus neoformans.

Cryptococcus neoformans colonies can be identified within 6 h using paper disks containing caffeic acid and ferric citrate. Indentification is based on the development of a dark brown pigment. Saprophytic Cryptococcus species and common clinically isolated yeasts do not develop the brown color. The concentration of the reagents and the method of storage of the impregnated paper disks are critical for the rapid and specific development of the pigment.

Diarrhea due to Plesiomonas shigelloides in cancer patients.

Plesiomonas shigelloides was isolated from the stools of two patients with gastrointestinal malignancies and from one "healthy" patient with diarrhea. No other enteric pathogens were isolated. One patient was neutropenic after antineoplastic chemotherapy. The two cancer patients had crampy abdominal pain and severe diarrhea and required antibiotic therapy for resolution of symptoms. The third patient responded to symptomatic treatment. Patients with gastrointestinal malignancies may be more susceptible to gastroenteritis caused by P. shigelloides than normal individuals, and these infections may be more severe than those seen in normal individuals.

Amphotericin B susceptibility testing of Candida species by flow cytometry.

We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.