Search

Flow cytometry (FC) has the ability to discriminate a variety of cell parameters including cell size and complexity, and fluorescence intensity. As yeast cells or fungal spores germinate they undergo a morphological transformation from round oval shaped cells to elongate filamentous forms. To date, monitoring these events has been performed using microscopic examination. Microscopic examination is a labor intensive process that examines a very small percentage of the total cell population. We have developed a method using FC that is rapid, simple to perform, and reproducible. The major advantages of FC include analysis of a larger number of cells, increased objectivity due to nonselective measurements of all cells in the population studied, and the computer related data analysis capability of the flow cytometer.

We examined the potential risk of tuberculosis transmission if we modified our policy for release of patients from the "airborne precautions" category from three negative acid-fast bacillus (AFB) smears to two, or even one. Over a 4-year period, respiratory cultures from 42 patients grew Mycobacterium tuberculosis. Of these, 36 patients (81%) had a positive AFB smear result on the first submitted specimen. One additional patient (2%) had a first smear-positive finding on the second submitted specimen, and no patients had a first smear-positive result on the third submitted specimen. Respiratory cultures from five patients (12%) grew M. tuberculosis without ever having a positive AFB smear result. These data indicate that in our institution, reducing the number of negative smears required before removal of patients from the airborne precautions category would pose little, if any, increase in the risk of spreading tuberculosis.

Objective: To review the pathophysiology, epidemiology, treatment, and prophylaxis of disseminated Mycobacterium avium complex (MAC) infection in HIV-infected individuals., Data Sources: A MEDLINE (January 1966-July 1997) and AIDSLINE (January 1980-July 1997) search of basic science articles pertinent to the MAC infection in HIV-infected patients., Study Selection and Data Extraction: All articles were considered for possible inclusion in the review. Pertinent information, as judged by the authors, was selected for discussion., Data Synthesis: The organism, epidemiology, and pathophysiology of disseminated MAC are discussed for background. A review of clinical trials for the treatment and prophylaxis of disseminated MAC are presented, along with unresolved issues concerning these topics., Conclusions: The incidence of disseminated MAC has increased dramatically with the AIDS epidemic. The infection can lead to increased morbidity and mortality in HIV-infected patients. Treatment regimens for patients with a positive culture for MAC from a sterile site should include two or more drugs, including clarithromycin. Prophylaxis against disseminated MAC should be considered for patients with a CD4 cell count of less than 50/mm3.

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.

Multiplex strand displacement amplification (mSDA) is capable of amplifying three distinct DNA sequences simultaneously. These include sequences present in most genera of mycobacteria, a sequence specific for Mycobacterium tuberculosis, and an internal control. mSDA was used to detect the presence of these target sequences in 154 (72 positive, 76 negative, and 6 failed) clinical specimens cultured in the mycobacterial growth indicator tube (MGIT) system. A wide variety of specimen types were processed and cultured. Once these cultures were deemed positive by MGIT fluorescence or were deemed negative after 8 weeks of incubation, MGIT culture aliquots were processed for mSDA analyses. A chemiluminescent microwell assay was used to detect the amplified products. The procedure was relatively simple and took less than 6 h to complete. The sensitivity of mSDA for detecting acid-fast bacilli was 96.4% compared to that of MGIT culture. Sensitivity and specificity were 97.2 and 96.1%, respectively, when all clinical criteria were considered. mSDA was shown to be a rapid and effective method for confirming the presence of M. tuberculosis and other mycobacteria in positive MGIT cultures.

We compared the Mycobacteria Growth Indicator Tube (MGIT) system with the BACTEC 460 (B460) and Lowenstein Jensen (LJ) systems for the recovery of mycobacteria (acid-fast bacteria [AFB]) from 1,441 clinical specimens. Excluding 13 isolates of Mycobacterium gordonae, 178 significant AFB isolates were recovered from 113 patients. Isolates (119) of the Mycobacterium avium complex (MAC) accounted for 67% of all isolates, while isolates (30) of the Mycobacterium tuberculosis complex (MTB) accounted for 17% of isolates. The MGIT system recovered 98 (82%) MAC and 27 (90%) MTB isolates, while the B460 system recovered 101 (85%) MAC and 28 (93%) MTB isolates and the LJ system recovered 91 (76%) MAC and 25 (83%) MTB isolates. Overall, the MGIT system recovered 152 isolates of AFB (85.4% sensitivity), and the B460 and LJ systems recovered 151 (84.8% sensitivity) and 137 (76.9% sensitivity) AFB isolates, respectively. The recoveries of AFB with combinations of media were as follows: MGIT + LJ, 93.2%; B460 + LJ, 92.1%; and MGIT + B460, 96.6%. Although the sensitivity of MGIT was equivalent to that of B460, MGIT required a longer incubation (median, 11 days) than did B460 (median, 8 days) to become positive (P < 0.05).

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.

We recently tested serum from a human immunodeficiency virus-infected patient for the presence of cryptococcal antigen using the Meridian latex agglutination (LA) test (Cryptococcal Antigen Latex Agglutination System). Two pronase-treated serum specimens from the patient had LA titers of 80 and 160, but the patient had no evidence of cryptococcal disease. The serum was negative for rheumatoid factor, a well-documented cause of false-positive LA reactions. Seven blood culture supernatants from the patient were also LA positive, but were culture negative for cryptococcus. When the sera and blood culture supernatants were treated with 0.01 M 2-beta-mercaptoethanol (2-ME), the agglutinating activity was ablated. Similar results were seen when the sera were tested by two other commercial LA assays. Serum and cerebrospinal fluid specimens from patients with confirmed cryptococcal disease were treated with 2-ME, and the results were compared with those obtained after pronase (sera) or heat (cerebrospinal fluid) inactivation. The titers were identical (n = 56) or within 1 dilution (n = 3). One hundred serum specimens from human immunodeficiency virus-seropositive patients with no known history of cryptococcal disease were examined to determine the frequency of false-positive reactivity in this patient population. Of this group, three were positive following pronase treatment. One remained positive after 2-ME treatment; the remaining two were negative. These data indicate that 2-ME can be used to eliminate nonspecific reactivity in the LA test without affecting true-positive results.

Pulmonary colonization and infection of patients with cystic fibrosis by Mycobacterium spp. has recently been recognized as a potentially important clinical problem. However, frequent contamination of mycobacterial cultures by pseudomonads has hampered efforts to define the extent of this problem. This study was done to evaluate current techniques and to establish a more efficient method of recovering mycobacteria from respiratory secretions of patients with cystic fibrosis. Decontamination of respiratory specimens (n = 121) with 0.25% N-acetyl-L-cysteine and 1% sodium hydroxide (NALC-NaOH) was associated with a high rate of pseudomonas overgrowth for both Lowenstein-Jensen slants (74%) and BacTec vials supplemented with PANTA (polymyxin B [50 U/ml], amphotericin B [5 micrograms/ml], nalidixic acid [20 micrograms/ml], trimethoprim [5 micrograms/ml], azlocillin [10 micrograms/ml]) (36%). This overgrowth limited recovery of mycobacteria to only 64% (9 of 14) of specimens positive by smear for acid-fast bacilli (AFB). Decontamination of specimens (n = 441) with NALC-NaOH, followed by 5% oxalic acid treatment, resulted in contamination of only 5% of Lowenstein-Jensen slants and 3% of BacTec vials. AFB were recovered from all 90 AFB smear-positive specimens following the use of this decontamination technique. We recommend that respiratory secretions be decontaminated with NALC-NaOH and oxalic acid to decrease the incidence of Pseudomonas aeruginosa overgrowth.

We have developed a method for processing and detecting fungi in clinical specimens using the polymerase chain reaction (PCR) methodology. This PCR amplification of a segment of a ribosomal DNA gene results in a 310 bp product. The gene sequences used as amplimers have been highly conserved throughout the fungal kingdom and positive PCR results have been obtained for all genera and species of fungi tested (n = 42). Neither human nor a variety of pathogenic bacteria (n = 24) gave an amplified product. This PCR method can detect as few as 15 cells of Candida albicans in clinical specimens following a simple processing procedure. The sensitivity of the PCR for detecting other fungi remains to be determined. Analysis of the 310 bp amplified product, following digestion with the restriction enzyme HaeIII, can be used to further characterize the identity of the fungus involved into the following five groups: (i) Candida species and closely related yeasts; (ii) Cryptococci and Trichosporon species; (iii) Aspergilli and clinically related septate molds; (iv) the Zygomycetes; and (v) the dimorphic fungi.

The performance of the Septi-Chek AFB System (Roche) in the isolation of mycobacteria was compared to that of culture on Lowenstein-Jensen (LJ) medium and the Bactec radiometric system. The Septi-Chek AFB system detected a significantly higher number of positive specimens (62/66 versus 47/66 for Bactec and 39/56 for LJ medium) and was more often the only medium in which an isolate was recovered. The average time for detection of isolates was very similar for the Septi-Chek AFB and Bactec systems which were both significantly faster than LJ medium in the majority of isolates.

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.

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.

Pellet-associated human brain alpha-L-fucosidase was solubilized with 0.5% (w/v) Triton X-100 and purified by affinity chromatography on agarose-6-aminohexanoyl-fucosamine resin. The procedure resulted in a 290,000-fold purification, a 58% yield and a final specific activity of 11,500 nmol/min per mg of protein. Isoelectric focusing indicated that all six major isoforms (with pI values between 4.1 and 5.3) present in crude brain pellet preparations were purified by the affinity technique. SDS/PAGE indicated the presence of one subunit (54 kDa) and a minor protein band at 67 kDa, which presumably is a contaminant since it was not immunoreactive on Western blotting. The pH optimum of the brain enzyme and its apparent Km for the synthetic substrate 4-methylumbelliferyl alpha-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver alpha-L-fucosidases were both capable of hydrolysing fucosyl-GM1 ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37 degrees C was found at pH 3.4 for both alpha-L-fucosidases, with no activity at pH values above 4.0.

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.

Organisms belonging to Drechslera species are rare causes of human infection and are considered opportunistic pathogens. Only 10 cases of human infection with these organisms have been reported in the literature; an additional case--that of a healthy young woman with chronic pansinusitis due to Drechslera spicifera causing nasal obstruction and erosion of bone--is described herein. This case was managed successfully by means of surgical debridement and therapy with amphotericin B and ketoconazole. A review of the literature suggests that infections caused by Drechslera species can in fact occur in a healthy host. Involvement of the paranasal sinuses and the central nervous system is common and potentially life-threatening. Appropriate therapy consists of the administration of amphotericin B, either alone or in combination with other antifungal agents, and surgical debridement when indicated.

Necrotizing gas-forming infections in cancer patients present some unique characteristics, such as nontraumatic, spontaneous clostridial gangrene and gangrene involving an ischemic tumor mass. These infections can be rapidly progressive and uniformly fatal without surgical debridement. We review ten cases of gas gangrene seen during an 18-year period. Four were caused by Clostridium species and six by other organisms. Neutropenia was present in seven patients. During the last nine years, Clostridium septicemia occurred in 54 patients; in only two of those patients did gas gangrene ensue.

Agar dilution and Autobac I tests were performed to compare the susceptibility of 1,000 bacterial isolates to cefaclor and cephalothin. The standard Autobac methodology will correctly predict resistance to cefaclor. The test methodology seems to influence in vitro results, since more strains of gram-negative bacteria were susceptible to cefaclor by the Autobac I method than by the agar dilution test.

Two additional cases of systemic mycosis due to Trichosporon cutaneum are reported and are compared with the previously published case of Rivera and Cangir. Both patients (a four-year-old male and a 57-year-old female) had acute leukemia for which they were receiving chemotherapy, and both presented with fever that was unresponsive to conventional antibiotics. Both had positive blood cultures for Trichosporon cutaneum. The disease was further documented in the four-year-old male by renal biopsy and by bone marrow culture; he was treated with apparent success with amphotericin B. However, the 57-year-old female died shortly after the begining of similar treatment, and autopsy demonstrated involvement of the left kidney, spleen, bone marrow, and liver. The organism in both these cases, as well as the case of Rivera and Cangir, exhibited both hyphal and yeastlike forms in tissue sections. We believe that the therapeutic success in the case of the four-year-old male was primarily related to his remission from leukemia.

In disseminated candidiasis, various organs frequently become involved, usually as a consequence of hematogenous spread of the organism. However, involvement of the joints is rare even with dissemination. Nineteen cases of joint involvement have previously been reported in adults and 21 cases in children. The most commonly involved joint has been the knee; in such cases, amphotericin B has been effective in controlling the infection. Five patients with cancer developed septic arthritis due to Candida species at the M. C. Anderson Hospital and Tumor Institute in the past five years. Four of these patients were seen in 1980. Candida albicans was isolated from three patients and Candida tropicalis from two. All five patients had predisposing conditions - e.g., intravenous and/or urinary catheterization, neutropenia, and previous treatment with steroids and antibiotics. The knee was the affected joint in all five. Different modalities of treatment were used, including intravenous miconazole, oral ketoconazole, and systemic and local amphotericin B; adequate levels of these drugs were found in the joint fluid when measured. Infection was cured in two patients. The condition of the third patient improved. The fourth patient died of disseminated disease despite therapy, and the fifth died of malignancy without the benefit of antifungal therapy. For treatment of such infections, the use of an antifungal agent is recommended in addition to frequent evacuation of the joint fluid. Some new compounds may prove useful alternatives to amphotericin B. Arthritis can be resolved even in the presence of unresolved disseminated disease.

To diagnose systemic candidiasis we developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of Candida albicans cytoplasmic antigen in sera of cancer patients. The antigen concentration was evaluated in 59 patients divided into several groups according to clinical, histological, and microbiological findings as well as antimicrobial treatment. All of seven patients with proven disseminated candidiasis had elevated antigen concentrations (mean = 1,200 ng/ml). Among 13 patients with suspected disseminated candidiasis, 9 had elevated antigen concentrations (mean = 1,039 ng/ml). The remaining four patients showed negative antigen (less than 125 ng/ml). In a group of patients with fever of unknown origin, 5 of 17 had high antigen concentrations (mean = 1,220 ng/ml) and the rest were negative. Patients with bacterial infection (6 patients), aspergillosis (3), cryptococcosis (4), coccidioidomycosis (3), and no infection (6), as well as normal healthy adults (31), had antigen concentrations of less than 100 ng/ml. This ELISA is rapid, sensitive, and specific and is a helpful diagnostic tool in the evaluation of candidiasis in cancer patients.

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.

The efficacy of tuftsin, a naturally-occurring immunomodulating peptide, in the prophylaxis of disseminated Candida albicans infections in mice was studied. The intravenous administration of tuftsin caused prolonged survival at all concentrations tested.

alpha-L-Fucosidase was purified from human liver to apparent homogeneity and subjected to exhaustive digestion with Pronase. The resulting glycopeptides were isolated by gel filtration on Sephadex G-50 and further fractionated by Bio-Gel P-4 chromatography. Five glycopeptide fractions were obtained. The structures of the carbohydrate portions of all glycopeptide components were fully characterized by a combination of 500-MHz 1H NMR spectroscopy and carbohydrate composition analysis. Fraction I contained disialyl diantennary glycopeptides of the N-acetyllactosamine type. Fractions II and III contained predominantly mono(sialyl-N-acetyllactosaminyl) diantennary glycopeptides with the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----2) branch attached to alpha(1----3)-linked Man in II and to alpha(1----6)-linked Man in III. The N-acetyllactosamine-type glycopeptides in fractions I to III have a small portion (10-15%) of their Asn-linked GlcNAc residues substituted by additional alpha(1----6)-linked Fuc. Also, a minor portion of the NeuAc residues appeared to be attached to Gal in alpha(2----3) rather than alpha(2----6) linkage. Fraction IV contained a mixture of larger-size oligomannoside-type glycopeptides with a variable number (6 to 9) of Man residues. Smaller-size oligomannoside-type glycopeptides were found in fraction V, containing 3 or 5 Man residues; a small portion (10%) of the Man3GlcNAc2Asn component appeared to contain in addition a Fuc residue in alpha(1----6) linkage to the Asn-bound GlcNAc. The overall ratio of oligomannoside-type to N-acetyllactosamine-type carbohydrate structures was found to be 5:4. This article is the first account of the complete characterization of the oligomannoside-type structures in alpha-L-fucosidase; furthermore, the occurrence in alpha-L-fucosidase of mono(sialyl-N-acetyllactosaminyl) structures, Fuc-containing oligosaccharides, and NeuAc alpha(2----3) linked to Gal are reported for the first time.

During a 10-year period from 1974 to 1984, 19 patients at M. D. Anderson Hospital and Tumor Institute in Houston had invasive infection due to Trichosporon beigelii. Infection was manifested as either a nonspecific febrile illness or pneumonia, and the diagnosis was not suspected before death in 25% of the patients. The majority were neutropenic at the onset of infection, and recovery was directly related to resolution of myelosuppression. The overall mortality rate was 74%. The majority of patients had polymicrobial infections. Twenty-four additional cases reported in the medical literature indicate that this pathogen is an increasingly important cause of mortality in immunosuppressed patients. In both the present series and the cases reported in the literature, the majority of neutropenic patients with disseminated T. beigelii infection died, despite antifungal therapy. Only those patients whose neutropenia resolved following remission of their leukemia recovered.

Bacteremia caused by Aeromonas species occurred in 24 hospitalized patients in a cancer institute during a 13-year period. All but one of these patients had a malignancy (88% had leukemia), and most were receiving chemotherapy for cancer. There was a striking numerical predominance of male patients (82%). Unlike some previously described patients with infections due to this organism, none of these 24 patients had recently been exposed to fresh or salt water or to fish. The source of the infecting organism was thought to be endogenous--i.e., from patients' own gastrointestinal tracts. The clinical presentation of sepsis caused by this organism was nonspecific, except that ecthyma gangrenosum occurred in several patients. The overall mortality rate was 28%. The combination of an aminoglycoside and a cephalosporin is appropriate therapy for bacteremia caused by Aeromonas species.

The present study was conducted to examine the effect of a lipophilic analog of muramyl dipeptide, 6-O-stearoyl-N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine (6-O-S-Abu-MDP), a macrophage activator, on the prophylactic activity of liposomal amphotericin B (L-AmpB) against disseminated candidiasis in mice. Multilamellar vesicles containing AmpB and (6-O-S-Abu)-MDP were prepared by using dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol (7:3 molar ratio). Hale-Stoner mice (6 to 8 weeks old) were injected with 7 X 10(5) CFU of Candida albicans 336 isolated from a patient. Groups of mice were injected intravenously with different doses of L-AmpB and L-(6-O-S-Abu)-MDP, individually or in combination, 2 days before challenge with C. albicans. The mice were injected with a fixed dose of L-AmpB (1.2 mg/kg in 400 mg of lipid per kg) and various doses of L-(6-O-S-Abu)-MDP (0.6, 1.2, 2, and 4 mg/kg in 400 mg of lipid per kg) or vice versa. Other control groups included untreated mice and those receiving empty liposomes (400 mg of lipid per kg), free AmpB (0.6 mg/kg), or free (6-O-S-Abu)-MDP (4 mg/kg). The mice receiving L-AmpB (1.2 mg/kg) plus L-(6-O-S-Abu)-MDP (0.6 to 4.0 mg/kg) survived up to 25 to 30 days as compared with those injected with L-AmpB alone (15 days) or with L-(6-O-S-Abu)-MDP alone (10 to 15 days). All the mice in other control groups died within 7 to 11 days. The kidney cultures of the mice that received L-AmpB (4 mg/kg) plus L-(6-O-S-Abu)-MDP (1.2 mg/kg) were free of C. albicans infection, unlike those injected with L-AmpB. Variance analysis of these findings indicates a synergistic activity between L-AmpB and L-(6-O-S-Abu)-MDP in the prophylaxis of candidiasis.

A patient with chronic myelogenous leukemia in blastic crisis who developed pulmonary and systemic infection with Cunninghamella species is described. The emerging role of this newly recognized pathogen in immunocompromised patients is discussed.

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.

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.

We studied the toxic effects of amphotericin B and gramicidin S, alone and in combination, using free and liposome-encapsulated drugs. In vitro toxic effects of the drugs on human erythrocytes and Candida albicans were determined by measuring leakage of intracellular potassium ions (K+). Liposomal formulations of both drugs greatly reduced K+ leakage from human erythrocytes, whereas liposomal gramicidin S, but not liposomal amphotericin B, prevented K+ leakage from C. albicans. In both free and liposomal forms, the combinations of drugs produced decreased toxicity to erythrocytes compared with the drugs alone. This protective effect was more apparent with liposomal combinations than with free drug combinations. A significant increase in fungal cell toxicity was observed, however, when free and liposomal drug combinations were tested against C. albicans. The results suggest that optimal concentrations of liposomal drug combinations (amphotericin B and gramicidin S) may provide increased toxicity against fungal cells and simultaneously protect mammalian cells.

The BACTEC 225 automated radiometric blood culture system was compared with a conventional blood culture bottle method for its ability to improve the rapid laboratory diagnosis of bacteremia in cancer patients. The BACTEC 225, in combination with routine blind subcultures and smears of radiometrically negative culture vials, detected two thirds, of all positive cultures within 24 h and shortered the detection time generally by 24-48 h. With the recommended growth index setting of 30, radiometrically false-positive findings are rare and are usually due to leukocytosis resulting from infection or leukemia.

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.

Enzyme extraction methods are commonly used to release antigen for Lancefield serogrouping of streptococci. Recently, up to 47% of enterococci have been found to possess antigens cross-reactive with both the group D and the group G grouping reagents. We investigated whether the organisms had recently emerged or whether changes in extraction methods (enzyme extraction) have resulted in our awareness of their existence. We found that approximately 20% of our enterococcal isolates released material cross-reactive with the group G reagents. These organisms have been present for at least 6 years, and probably for many more. In addition, we found that most strains of Listeria monocytogenes also released antigen cross-reactive with the group G reagent. The clinical significance of these findings is also discussed.

A 28 year old male heroin addict developed Candida tropicalis infection of the knee joint in association with candidemia. Assessment of amphotericin B therapy was facilitated by the determination of serum and synovial fluid amphotericin B concentrations using a radiometric bio-assay method. The results indicate that adequate synovial fluid drug levels were achieved with intravenous systemic therapy.

A pseudooutbreak of cryptococcal meningitis was caused by the use of contaminated albumin solution used in the preparation of Papanicolaou-stained slides of cerebrospinal fluid. Organisms were seen in cytocentrifuge preparations, but not in India ink preparations of cerebrospinal fluid specimens. Cryptococcal antigen tests were positive and Cryptococcus neoformans was cultured from the albumin-treated cerebrospinal fluid specimens and from the albumin solution.

Terminal subcultures from 5,354 negative BACTEC blood culture bottles did not significantly improve the detection of positive cultures. Only 15 of the 545 total isolates were recovered from the terminal subcultures. All 15 of these isolates were either considered contaminants or had been previously detected.

Amphotericin B (AmpB) disrupts membrane integrity by binding to sterols in fungal and mammalian cell membranes. The gramicidins, which form pores in all membranes but exhibit poor antifungal activity, are too toxic to mammalian cells to be used systemically. This study demonstrated synergistic antifungal activity of free and liposomal forms of AmpB when combined with the free and liposomal forms of gramicidin S and gramicidin NF against five Candida strains. In vitro erythrocyte lysis was prevented by using the liposomal forms of all drugs tested alone or in combination. Presumably, AmpB increases accessibility of the fungal cell membrane to the gramicidins, while liposome encapsulation decreases the rate of transfer of the drugs to the mammalian cell membrane. Liposome encapsulation of inactive or toxic drugs, used in combination with liposomal AmpB, may give new life to drugs previously believed to be inactive or too toxic for therapeutic consideration.

A case of septic, histoplasmal monoarthritis of the knee in a leukaemic patient is described. Ketoconazole therapy failed to eliminate the infection, but after histoplasmosis was diagnosed prolonged therapy with amphotericin B was curative.

The in vitro activities of liposome-encapsulated amphotericin B and free amphotericin B against Candida albicans 336 were comparable. Amphotericin B concentrations 12-fold and greater than 50-fold higher were required to kill the same organism when cholesterol and ergosterol were incorporated into the liposomes. The addition of cholesterol to liposomes caused a significant increase in the minimal fungicidal concentration of amphotericin B in 7 of 19 other yeast strains tested, whereas ergosterol caused an increase in 18 of the 19 strains.

Bacteremia caused by newly described Campylobacter-like organisms occurred in two immunosuppressed homosexual patients with tuberculosis. Although these organisms grow well in aerobic bottles using a radiometric blood culture system, they are not readily seen in gram-stained smears and are easily missed if routine subculture methods are used. Microscopic examination of wet preparations and subculture to brucella agar base supplemented with 10% sheep blood and incubated in microaerophilic conditions are useful for identification and isolation. The recovery of Campylobacter-like organisms from the blood suggests that these organisms, formerly known only to be associated with proctocolitis or asymptomatic rectal infection in homosexual men, can also cause systemic infection in these 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.