A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis.

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.

5-Fluorocytosine antagonizes the action of sterol biosynthesis inhibitors in Candida glabrata.

The concentration-dependent antagonistic interaction between 5-fluorocytosine and a sterol biosynthesis inhibitor (SBI) was studied using intact cells and cell-free extracts of Candida glabrata. 5-Fluorocytosine promoted incorporation of radioactivity into 4-desmethylsterols (P < 0.01), and enhanced the relative and absolute increases of ergosterol (P < 0.05) in C. glabrata incubated aerobically with an SBI (miconazole or amorolfine). Further aerobic incubation of C. glabrata with combinations of a nucleic acid or protein synthesis inhibitor (rifampicin or chlortetracycline) and an SBI (miconazole) promoted a similar increase in ergosterol biosynthesis. In contrast, 5-fluorocytosine reduced the incorporation of radioactivity into 4,4-dimethylsterols (P < 0.01), but had no obvious effect on the absolute ergosterol level in C. glabrata incubated statically with miconazole. In cell-free extracts of cultures previously incubated with 5-fluorocytosine, ergosterol synthesis was less sensitive to the action of miconazole. Antagonism between 5-fluorocytosine and the SBI is thus mediated by a reversal of inhibition of intracellular ergosterol synthesis. The possible mechanisms underlying antagonism between 5-fluorocytosine and SBIs that inhibit different sites of the sterol biosynthesis pathway, as well as its clinical relevance to combination therapy, are discussed.

Acinetobacter bacteremia in Hong Kong: prospective study and review.

The epidemiological characteristics of 18 patients with acinetobacter bacteremia were analyzed. Patients (mean age, 55.5 years) developed bacteremia after an average of 14.1 days of hospitalization. Fifteen of 16 patients survived bacteremia caused by Acinetobacter baumannii. Cultures of blood from the remaining two patients yielded Acinetobacter lwoffii. Most patients (78%) resided in the general ward, while four patients (22%) were under intensive care. Genotyping by arbitrarily primed polymerase chain reaction analysis and the temporal sequence of isolation were more useful than phenotyping by antimicrobial susceptibility in the determination of the source of bacteremia, and the intravascular catheter was the leading infection source (39% of cases). The possibility of an association of glucose with the pathogenesis of acinetobacter infection was raised.

The effect of antifungal drugs in combination on the growth of Candida glabrata in solid and liquid media.

The effect of drugs in combination on the growth of Candida glabrata was studied in solid medium by demonstration of reduced or enhanced growth, and in liquid medium by determination of interaction indices. Amphotericin B and 5-fluorocytosine showed a synergic effect, while combinations of amphotericin B and miconazole, and miconazole and 5-fluorocytosine exhibited antagonistic effects. In addition, concentration-dependent antagonism was observed amongst combinations of metabolic inhibitors, between inhibitors of sterol biosynthesis (terbinafine, miconazole, ketoconazole, clotrimazole, econazole, fluconazole, itraconazole and amorolfine) and inhibitors of nucleic acid or protein biosynthesis (5-fluorocytosine, 5-fluorouracil, rifampicin and chlortetracycline). This antagonism was strain-specific, occurring with C. glabrata strain 4, Saccharomyces cerevisiae strain 237 and Candida albicans strain 72R, but not with C. albicans strain 6406 or Candida parapsilosis strain 3104.

Identification of acinetobacters on blood agar in presence of D-glucose by unique browning effect.

A positive phenotypic characteristic of glucose-oxidizing acinetobacters was demonstrated with blood agar containing D-glucose. Glucose-oxidizing Acinetobacter baumannii, Acinetobacter genospecies 3, Acinetobacter lwoffii, and Acinetobacter genospecies 13 sensu Tjernberg and Ursing caused a unique brown discoloration of media supplemented with 5% blood (of horse, sheep, or human origin) and an aldose sugar (0.22 M D-glucose, D-galactose, D-mannose, D-xylose, or lactose). The browning effect was not observed when a ketose sugar (D-fructose or sucrose) was substituted for the aldose sugar or under high osmolarity in the presence of mannitol, glycerol, or sodium chloride. Other gram-negative nonfermenters (non-glucose-oxidizing acinetobacters, Pseudomonas aeruginosa, other Pseudomonas spp., Stenotrophomonas maltophilia, Flavobacterium spp., and Moraxella spp.) did not cause similar discoloration. This novel browning effect may serve as an alternative trait for identifying glucose-oxidizing acinetobacters.

Clinical significance of alimentary tract microbes in bone marrow transplant recipients.

A prospective study on the microbes isolated from the alimentary tract in 120 bone marrow transplant (BMT) recipients (1991-1993) was undertaken to define the spectrum of organisms isolated under antimicrobial prophylaxis, their temporal sequence of emergence, and the associated morbidity and mortality. Clostridium difficile (n = 20), isolated in the pre-engraftment and early post-engraftment periods (day 2-45 post-BMT), was the most common microbe recovered from stool of patients with diarrhea. In contrast to previous reports, no significant difference in mortality was observed between patients with and without C. difficile isolated in stool. Two patients had neutropenic ileocecitis with concomitant bacteremia due to Escherichia coli and Klebsiella pneumoniae. One patient was found to have astrovirus gastroenteritis (day 7), and Giardia lamblia was recovered from the stool of another (day -7). Heavy growth of Staphylococcus aureus from direct smear-positive specimens was found from the upper airway of two patients with severe mucositis and complete dysphagia (day 12 and 23). Salmonella spp. of groups B and E were found in the stool of five asymptomatic patients at the time of conditioning. No specific organisms was recovered from the endoscopic brushing of two patients with lower end esophagitis, three patients with upper gastrointestinal bleeding, and three patients with perirectal cellulitis. During the post-engraftment period, five patients had documented cytomegalovirus gastroenterocolitis (days 34-97), one had Mycobacterium chelonae colitis (day 70), and another had nodular gastritis due to Acremonium falciforme (day 270). Overall, only 28% of patients with alimentary tract symptoms/syndrome had specific pathogens isolated from clinical specimens. Differentiation of the causation of alimentary tract symptoms was often difficult because noninfectious complications such as conditioning toxicity, graft-versus-host disease, and its treatment often caused alimentary tract symptoms in addition to predisposed BMT patient to infection. The reluctance of obtaining tissue biopsy for ascertaining the importance of those potential alimentary tract pathogens often dictate the use of empirical treatment.

Distinct genotypic distributions of cytomegalovirus (CMV) envelope glycoprotein in bone marrow and renal transplant recipients with CMV disease.

A prospective study of the spectrum of glycoprotein B (gB) and glycoprotein H (gH) genotypes of cytomegalovirus (CMV) was conducted with five categories of patients: viremic bone marrow-transplant (BMT) recipients who developed CMV disease after BMT (n = 22), viremic BMT recipients without CMV disease (n = 11), viremic renal-transplant recipients who developed CMV disease after transplantation (n = 14), viremic renal-transplant recipients without CMV disease (n = 13), and premature babies with asymptomatic congenital CMV infections (n = 13). Genotypic stability was observed because the gB and gH genotypes of multiple isolates obtained from a single patient were identical. The distribution of gH genotypes in patients of all groups studied were similar. However, there was a unique distribution of the gB genotype in the first category of patients, i.e., BMT recipients with CMV disease, which was distinct from those of all other categories (P < 0.05). CMV isolates from 54% of BMT recipients with CMV disease exhibited gB type 2, while isolates from 46, 50, 69, and 77% of the BMT recipients without CMV disease, renal-transplant recipients with and those without CMV disease, and premature babies with congenital CMV infection, respectively, were of gB type 1. An analysis of the clinical characteristics of BMT recipients with CMV disease indicated that all underwent either an allogeneic or matched, unrelated donor transplant, and half had severe acute graft-versus-host disease (grades 2 to 4). The statistically significant genotypic difference between CMV isolates from BMT recipients with and without CMV disease was not observed between isolates from renal-transplant recipients with and without CMV disease. We speculate that differences in pathogenesis in different patient groups might account for these observations. These findings would also facilitate decision making about the choice of recombinant CMV glycoprotein vaccine required to immunize transplant donors and the subsequent adoptive transfer of immunity to BMT recipients. When the source of CMV DNA required for genotyping was investigated among renal-transplant recipients, direct use of peripheral blood leukocytes was 95% effective compared to the effectiveness of cells obtained from conventional culture of peripheral blood specimens.

Stage-specific manifestation of mold infections in bone marrow transplant recipients: risk factors and clinical significance of positive concentrated smears.

Potassium hydroxide-concentrated smears, prepared from sedimented remains of clinical specimens, were used to distinguish between mold infection and exogenous contamination in fungal culture-positive specimens. This method was applied in the study of 3,857 clinical specimens from 230 bone marrow transplant recipients who were followed up prospectively for infectious complications. Concentrated smears of only 86 (from 21 infected patients) of 149 fungal culture-positive specimens were positive for hyphae; 82 of the strains were Aspergillus species. Concentrated smears of the remaining 63 fungal culture-positive specimens were negative; the strains identified by culture were considered as exogenous contaminants (87% of which were Penicillium species). A stage-specific manifestation of mold infection was observed: 67% of mold infections occurred during acute graft-vs.-host disease (GVHD) a median of 47 days after transplantation, whereas 9% of mold infections occurred as rapidly fatal invasive disease before engraftment. Overall, of the 21 patients with mold infection, 17 (81%) had invasive mold disease, and four (19%) had mold colonization of airways secondary to chronic GVHD after day 100. The significant risk factors for mold infection were total-body irradiation and grade 2-4 acute GVHD. Because of our high mortality rate (82%), the consideration of antimold prophylaxis for such patients may be warranted.

Detection of CMV DNA in bone marrow transplant recipients: plasma versus leucocyte polymerase chain reaction.

AIMS: To compare the nested polymerase chain reaction (PCR) assay for the detection of cytomegalovirus (CMV) DNA in peripheral blood leucocytes and plasma obtained from heparinised blood; to determine the efficiency of various DNA extraction methods to minimise inhibition of plasma PCR and their effect on the sensitivity of plasma PCR; to determine the inhibitory effect of heparin, dextran, and EDTA on the CMV PCR assay. METHODS: 217 heparinised blood specimens from 58 bone marrow transplant patients were processed and the sensitivities and specificities of the PCR assays using peripheral blood leucocytes and plasma (with simple, Instagene, and Geneclean extraction methods) were compared to those of conventional CMV culture. In a separate experiment, dilutions of heparin, dextran, and EDTA were included in PCR assays. RESULTS: The detection of CMV DNA using peripheral blood leucocytes for PCR assay was significantly more sensitive (100%) than when using plasma (60%). Instagene and Geneclean extraction removed inherent inhibition but did not improve the sensitivity of the plasma PCR reaction. Heparin had an inhibitory effect on PCR. CONCLUSIONS: PCR assay using peripheral blood leucocytes is better than plasma for guiding the prescription of ganciclovir to bone marrow transplant patients. Heparin is inhibitory to the plasma PCR reaction.

The epidemiology of acinetobacter infections in Hong Kong.

A retrospective survey was conducted of the characteristics of acinetobacter infections in Hong Kong--seasonal and geographic distributions, frequency of isolation from various body sites, antimicrobial susceptibility and molecular epidemiology. Most (80%) isolates of Acinetobacter spp. belonged to DNA groups 2 (A. baumannii) or 13, as defined by growth at 44 degrees C. An increased isolation rate in summer was related to higher ambient temperatures. The notion that acinetobacters are opportunist nosocomial pathogens was supported by the body site- and ward-specific distributions, which were similar to those of Pseudomonas aeruginosa and in marked contrast to those of coagulase-negative staphylococci and Escherichia coli. Typing of Acinetobacter isolates by arbitrary-primed polymerase chain reaction revealed extensive genotypic polymorphism, suggesting that numerous unrelated strains were circulating between patients. In view of the association with a high incidence of polymicrobial bacteraemia and multiresistance to antibiotics, a careful selection of appropriate antibiotics in combination is necessary for empirical therapy of infections caused by Acinetobacter spp.

Activities of enzymes associated with phosphoenolpyruvate metabolism in the mudskippers, Boleophthalmus boddaerti and Periophthalmodon schlosseri.

1. PK and LDH activities in the muscle of Periophthalmodon schlosseri and Boleophthalmus boddaerti were at least 100-fold higher than their respective activities in the liver. 2. The ratio of PK:PEPCK in liver of B. boddaerti was smaller than that of P. schlosseri. 3. PK:PEPCK ratios in both fishes were intermediate between those of aerobic and anaerobic organisms. 4. MDH activity was higher than other enzymes assayed in the liver of both fishes. 5. The ratios of LDH:MDH in the liver of both mudskippers were comparable to those of anaerobic organisms. 6. AST was at least eight times more active than ALT in the liver of both fishes. 7. In the muscle of these mudskippers, the aspartate content was significantly less than that of alanine. 8. Exposure of these fishes to various experimental conditions led to changes in specific activities of PEPCK, LDH, AST and ALT.