Antifungal Susceptibility and Virulence Attributes of Bloodstream Isolates of <Emphasis Type="Italic">Candida</Emphasis> from Hong Kong and Finland

Candida bloodstream infection has dramatically increased in the last decade due to the growing number of immunocompromised populations worldwide. In this study, we evaluated the antifungal susceptibility profiles and virulence attributes of Candida bloodstream isolates (CBIs) derived from Hong Kong and Finland, information which are vital for devising empirical clinical strategies. Susceptibility testing of a wide range of antifungals including fluconazole, itraconazole, voriconazole, ketoconazole, 5-fluorocytosine, amphotericin B and caspofungin was performed. Haemolytic activity and secretion of proteinase of CBIs were also examined. All CBIs derived from Hong Kong were susceptible to all the antifungals tested whilst some CBIs from Finland were resistant to azoles and caspofungin. C. albicans, C. glabrata and C. tropicalis showed higher haemolytic activity whereas C. parapsilosis and C. guilliermondii were non-haemolytic in general. Proteinase activity of the Finland C. albicans isolates was significantly higher than the Hong Kong isolates. Our data provide a glimpse of the possible evolutionary changes in pathogenic potential of Candida that may be occurring in different regions of the world. Therefore, continuous surveillance and availability of local data should be taken into consideration when treating candidemia patients.     

Is Human Cytomegalovirus Infection Associated with Hypertension? The United States National Health and Nutrition Examination Survey 1999–2002

Purpose

Recent studies have implicated the human cytomegalovirus (HCMV) as a possible pathogen for causing hypertension. We aimed to study the association between HCMV infection and hypertension in the United States National Health and Nutrition Examination Survey (NHANES).

Methods

We analyzed data on 2979 men and 3324 women in the NHANES 1999–2002. We included participants aged 16–49 years who had valid data on HCMV infection and hypertension.

Results

Of the participants, 54.7% had serologic evidence of HCMV infection and 17.5% had hypertension. There were ethnic differences in the prevalence of HCMV infection (P<0.001) and hypertension (P<0.001). The prevalence of both increased with age (P<0.001). Before adjustment, HCMV seropositivity was significantly associated with hypertension in women (OR = 1.63, 95% CI = 1.25–2.13, P = 0.001) but not in men. After adjustment for race/ethnicity, the association between HCMV seropositivity and hypertension in women remained significant (OR = 1.55, 95% CI = 1.20–2.02, P = 0.002). Further adjustment for body mass index, diabetes status and hypercholesterolemia attenuated the association (OR = 1.44, 95% CI = 1.10–1.90, P = 0.010). However, after adjusting for age, the association was no longer significant (OR = 1.24, 95% CI = 0.91–1.67, P = 0.162).

Conclusions

In this nationally representative population-based survey, HCMV seropositivity is associated with hypertension in women in the NHANES population. This association is largely explained by the association of hypertension with age and the increase in past exposure to HCMV with age.     

Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease

The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N₁₂/N₁₆) away from the modified cytosine at the 3′-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-to-back dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N₁₂ cleavage in the same strand and then the second distal N₁₆ cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans.     

The use of new probes and stains for improved assessment of cell viability and extracellular polymeric substances in <Emphasis Type="Italic">Candida albicans</Emphasis> biofilms

Phenotypic and genotypic cell differentiation is considered an important feature that confers enhanced antifungal resistance in candidal biofilms. Particular emphasis has been placed in this context on the viability of biofilm subpopulations, and their heterogeneity with regard to the production of extracellular polymeric substances (EPS). We therefore assessed the utility of two different labeled lectins Erythrina cristagalli (ECA) and Canavalia ensiformis (ConA), for EPS visualization. To evaluate the viability of candidal biofilms, we further studied combination stains, SYTO9 and propidium iodide (PI). The latter combination has been successfully used to assess bacterial, but not fungal, viability although PI alone has been previously used to stain nuclei in fungal cells. Candida albicans biofilms were developed in a rotating disc biofilm reactor and observed in situ using confocal scanning laser microscopy (CSLM). Our data indicate that SYTO9 and PI are reliable vital stains that may be used to investigate C. albicans biofilms. When used together with ConA, the lectin ECA optimized EPS visualization and revealed differential production of this material in mature candidal biofilms. The foregoing probes and stains and the methodology described should help better characterize C. albicans biofilms in terms of cell their viability, and EPS production.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

A new haemagglutinin from the amoebocytes of the horseshoe crab Carcinoscorpius rotundicauda. Purification and role in cellular aggregation.

The present paper describes the purification and function of a haemagglutinin from the amoebocyte lysate of the horseshoe crab Carcinoscorpius rotundicauda. The purified protein consisted of a single subunit of Mr 24 000 and agglutinated human blood-group-A+ erythrocytes. Its haemagglutinin activity was inhibited by purified lysate, coagulogen, but not by sugars. The haemagglutinin differed immunologically and in activity from the sialic-acid-binding lectin carcinoscorpin present in the haemolymph. It caused aggregation of forma-fixed amoebocytes, and on the basis of this observation its role in cell-cell adhesion is proposed. This new haemagglutinin promotes cell-cell aggregation in amoebocytes in a manner that shares some similarities with thrombospondin-mediated platelet aggregation in vertebrates [Jaffe, Leuang, Nachman, Levin & Moseher (1981) Nature (London) 295, 246-248].     

Clinical pharmacology studies with Centchroman.

A pharmacological evaluation of centchroman (3,4-trans-2,2,dimethyl-3-phenyl-4-p-(beta-pyrrolidino ethoxy)-phenyl-7-methoxychroman), a new postcoital contraceptive, in normal healthy human volunteers was performed to determine the maximum tolerated dose and to discover any abnormal toxic effects in humans. The study was carried out for both men and women as a double-blind noncrossover trial in 2 parts: 1) a single dose study (40 volunteers) and 2) a multiple dose study (28 females) for 30 days. Centchroman was well tolerated without significant side effects in single doses up to 320 mg, severalfold higher than the anticipated therapeutic dose. In the multiple dose schedule, the compound was found safe at doses of 60-120 mg/day. however, similar effects were observed in subjects receiving placebo and are not unexpected in a normal population over a 1-month period. Centchroman is presently undergoing clinical trials for postcoital contraceptive efficacy in humans.     

Effects of dietary sugars and saliva and serum on Candida bioflim formation on acrylic surfaces

The effect of two dietary sugars, glucose and galactose, on biofilm formation of the oral fungal pathogen Candida on denture acrylic strips coated with saliva and serum pellicles was examined in vitro using Candida albicans (3 isolates), C. glabrata (2 isolates) and C. tropicalis (2 isolates). The degree of biofilm activity was affected by both the dietary sugar and the nature of the pellicle (ANOVA, p < 0.01). With most isolates the glucose grown yeasts demonstrated significantly more bioflim activity than the galactose grown fungi, in the presence of pellicles (ANOVA, p < 0.01 or P < 0.01). In contrast, one isolate of galactose-grown yeast elicited significantly higher biofilm activity than glucose-grown yeasts on the control strips (ANOVA, p < 0.01). Taken together, these results imply that a saliva or a serum pellicle, and the carbon source in the environment, act a complex manner modulating Candida bioflim formation. This revised version was published online in June 2006 with corrections to the Cover Date.