Alphaflexivirus Genomes in Stony Coral Tissue Loss Disease-Affected, Disease-Exposed, and Disease-Unexposed Coral Colonies in the U.S. Virgin Islands.

Stony coral tissue loss disease (SCTLD) is decimating Caribbean corals. Here, through the metatranscriptomic assembly and annotation of two alphaflexivirus-like strains, we provide genomic evidence of filamentous viruses in SCTLD-affected, -exposed, and -unexposed coral colonies. These data will assist in clarifying the roles of viruses in SCTLD.

Invasive candidiasis in Pakistan: clinical characteristics, species distribution and antifungal susceptibility.

This study reports for the first time, to our knowledge, descriptive epidemiological data for 188 invasive Candida isolates from Pakistan, including species identification and antifungal susceptibility against fluconazole, itraconazole, voriconazole, caspofungin, micafungin, anidulafungin and amphotericin. Risk factors for invasive candidiasis (IC) were determined for 96 patients from Karachi, Pakistan. In adults and neonates, Candida tropicalis (38 and 36 %, respectively) was the most common species, followed in adults by Candida parapsilosis (17.8 %), Candida glabrata (15.9 %) and Candida albicans (12.3 %). C. albicans (21 %) was the second most common in neonates. In children, C. albicans (31.9 %), C. tropicalis (26.4 %) and C. parapsilosis (19.4 %) were the most common. C. albicans IC was significantly associated with paediatric age [crude odds ratio (COR) 3.46, 95 % confidence interval (CI) 1.63-7.32]. Rare species made up 17.5 % of the total isolates studied. Resistance to fluconazole was seen in C. glabrata (15 .0%) and Candida krusei (100 .0%). Only one isolate (C. glabrata) was resistant to all three echinocandins. Low MICs of fluconazole for 98 % (184/188) of isolates tested support its continued use as an empiric therapy for IC. Non-C. albicans IC was associated with the use of ß-lactam inhibitor combinations (COR 3.16, 95 % CI 1.05-9.57). Use of healthcare devices was documented in 85.4 % of IC patients, whilst 75 .0% had been admitted to special care units. Surprisingly, 66.7 % of patients with IC were not obviously immunosuppressed. The high frequency of modifiable risk factors in this population indicates that candidaemia can be reduced with stringent antibiotic and infection control measures. These data will be useful for empiric selection of antifungals in Karachi, and contribute to global assessments of antifungal resistance.

Investigating the behavioural responses of trapped fishes using underwater video surveillance.

An underwater video surveillance system known as TrapCam was used to continuously record (15 ×c. 24 h periods) fish behaviour within and immediately surrounding an experimental fish trap situated in a coral reef ecosystem in the United States Virgin Islands. Of the 100 fishes (18 species, 12 families) trapped, surgeonfishes (Acanthuridae) and snappers (Lutjanidae) were most common. Thirteen distinctively identifiable behaviours were observed for trapped fishes. Species did not differ significantly in the proportion of time allocated to different behaviours (ANOSIM, R = 0·142). Doctorfish Acanthurus chirurgus and grey angelfish Pomacanthus arcuatus allocated the largest proportion of their recorded time to enter and exit the trap. Fishes spent an average of 15 min in the trap before escaping. Sixty-seven per cent of trap approaches consisted of an individual of the same species as one already trapped suggesting that conspecific attraction may have occurred. Fifteen per cent of trapped species were observed with abrasions to the head and 70% were observed approaching the trap corners. The results of this study provide a greater understanding of the behavioural interactions between fishes and traps that can help explain patterns of catch composition, the physical condition of fishes in traps and inform design of gear modifications to optimize by-catch reduction in the trap fishery.

Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR.

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

Diagnosis of acute pulmonary histoplasmosis by antigen detection.

BACKGROUND: Antigen detection, which has proven useful in diagnosis of disseminated histoplasmosis, has not been studied in acute pulmonary histoplasmosis (APH). Because treatment is indicated in most patients with moderately severe or severe APH, antigen detection for rapid diagnosis could be helpful. METHODS: Histoplasma antigen detection was evaluated in 130 patients with APH. RESULTS: Antigenuria was detected in 64.6%, antigenemia in 68.6%, and antibody in 64.3%. If both urine and serum specimens were tested, antigen was detected in 82.8%, of which 45.8% had antigenemia only; and if both antigen and antibody were measured, results were positive in 93.3%, of which antigen only was positive in 35.7%. CONCLUSIONS: Testing for antigenemia, antigenuria, and antibodies using the complement fixation test offers a sensitive, noninvasive method for diagnosis of APH.

Targeted disruption of Aurora A causes abnormal mitotic spindle assembly, chromosome misalignment and embryonic lethality.

Aurora A (also known as STK15/BTAK in humans), a putative oncoprotein naturally overexpressed in many human cancers, is a member of the conserved Aurora protein serine/threonine kinase family that is implicated in the regulation of G(2)-M phases of the cell cycle. In vitro studies utilizing antibody microinjection, siRNA silencing and small molecule inhibitors have indicated that Aurora A functions in early as well as late stages of mitosis. However, due to limitations in specificity of the techniques, exact functional roles of the kinase remain to be clearly elucidated. In order to identify the physiological functions in vivo, we have generated Aurora A null mouse embryos, which show severe defects at 3.5 d.p.c. (days post-coitus) morula/blastocyst stage and lethality before 8.5 d.p.c. Null embryos at 3.5 d.p.c. reveal growth retardation with cells in mitotic disarray manifesting disorganized spindle, misaligned and lagging chromosomes as well as micronucleated cells. These findings provide the first unequivocal genetic evidence for an essential physiological role of Aurora A in normal mitotic spindle assembly, chromosome alignment segregation and maintenance of viability in mammalian embryos.

Posaconazole treatment for Apophysomyces elegans rhino-orbital zygomycosis following trauma for a male with well-controlled diabetes.

We report a case of rhino-orbital zygomycosis in a 43-year-old male with well-controlled diabetes mellitus. The patient initially received liposomal amphotericin B, but the infection continued to progress, so posaconazole treatment was begun and eventually led to the cure of his infection. The causative agent was identified as Apophysomyces elegans, an emerging cause of zygomycosis in immunocompetent hosts.

Candidemia in pediatric outpatients receiving home total parenteral nutrition.

This is a cohort study of pediatric outpatients receiving total parenteral nutrition (TPN) and follow-up care in a Tennessee hospital between January and June 1999. The study was conducted following an increase in the incidence of candidemia. Of 13 children receiving home TPN, five had candidemia; three were due to Candida parapsilosis. Case patients were more likely to have an underlying hematologic disease (P = 0.02) as well as previous history of fungemia (P = 0.02). Two case patients had successive candidemia episodes 3 months apart; karyotypes and RAPD profiles of each patient's successive C. parapsilosis isolates were similar. Candida spp. were frequently detected in hand cultures from cohort members (four of 10) and family member caregivers (nine of 11); C parapsilosis was isolated from five caregivers. Our findings underscore the challenges of maintaining stringent infection control practices in the home health care setting and suggest the need for more intensive follow-up and coordination of home TPN therapy among pediatric patients.

Computational simulation of chronic persistent virus infection: factors determining differences in clinical outcome of HHV-6, HIV-1 and HTLV-1 infections including aplastic, hyperplastic and neoplastic responses.

A computational model was recently designed to simulate cellular changes in the T cell immune system. The model was validated by simulating cell changes in viral infections which target the same CD4+ T cell, yet cause either hyperplastic, aplastic or neoplastic responses. Respective case material for comparison was available from human infections with human herpesvirus-6 (HHV-6), human immunodeficiency virus (HIV-1) or human T cell leukemia virus (HTLV-1). Starting with cell values for a healthy human individual, factorial changes that influence the individual course of the various infections were determined by an algorithm search procedure. Such factorial differences determining a clinical course with aplasia, hyperplasia or neoplasia are outlined and further discussed in this paper.

Population-based surveillance and a case-control study of risk factors for endemic lymphocutaneous sporotrichosis in Peru.

Population-based surveillance and a case-control study were conducted in Abancay, Peru, to estimate the burden of disease and to determine risk factors for sporadic lymphocutaneous sporotrichosis (LS). Laboratory records from local hospitals were reviewed for the years of 1997 and 1998, and prospective surveillance was conducted for the period of September 1998 through September 1999. A case-control study was conducted with 2 matched control subjects per case patient. The mean annual incidence was 98 cases per 100,000 persons. Children had an incidence 3 times higher than that for adults and were more likely to have LS lesions on the face and neck. Identified risk factors included owning a cat, playing in crop fields, having a dirt floor in the house, working mainly outdoors, and having a ceiling made of raw wood or conditions associated with a lower socioeconomic status. Decreased environmental exposure, such wearing protective clothing during construction activities for adults or limiting contact with cats and soil for children, and improvements in living spaces may decrease the incidence of LS.

Epidemiology, clinical manifestations, and therapy of infections caused by dematiaceous fungi.

The dematiaceous (brown-pigmented) fungi are a large and heterogenous group of moulds that cause a wide range of diseases including phaeohyphomycosis, chromoblastomycosis, and eumycotic mycetoma. Among the more important human pathogens are Alternaria species, Bipolaris species, Cladophialophora bantiana, Curvularia species, Exophiala species, Fonsecaea pedrosoi, Madurella species, Phialophora species, Scedosporium prolificans, Scytalidium dimidiatum, and Wangiella dermatitidis. These organisms are widespread in the environment, being found in soil, wood, and decomposing plant debris. Cutaneous, subcutaneous, and corneal infections with dematiaceous fungi occur worldwide, but are more common in tropical and subtropical climates. Infection results from traumatic implantation. Most cases occur in immunocompetent individuals. Dematiaceous moulds are also important causes of invasive sinusitis and allergic fungal sinusitis. Infection is thought to follow inhalation. Although cerebral infection is the commonest form of systemic phaeohyphomycosis, other localized deep forms of the disease, such as arthritis, and endocarditis, have been reported. Disseminated infection is uncommon, but its incidence is increasing, particularly among immunocompromised individuals. Scedosporium prolificans is the most frequent cause. A number of dematiaceous fungi are neurotropic, including Cladophialophora bantiana, Ramichloridium mackenziei, and Wangiella dermatitidis. Although cases have occurred in immunocompromised persons, cerebral phaeohyphomycosis is most common in immunocompetent individuals with no obvious risk factors. Most forms of disease caused by dematiaceous fungi require both surgical and medical treatment. Itraconazole is currently the most effective antifungal agent for chromoblastomycosis and subcutaneous phaeohyphomycosis, while ketoconazole remains useful for mycetoma. Extensive surgical debridement combined with amphotericin B treatment is recommended for chronic invasive sinusitis. Long-term treatment with itraconazole has led to improvement or remission in some patients that had failed to respond to amphotericin B. Allergic fungal sinusitis requires surgical removal of impacted mucin combined with postoperative oral corticosteroids. Antifungal treatment is not usually of benefit, but post-operative itraconazole may reduce the need for reoperation. The clinical outcome of cerebral and other deep-seated forms of phaeohyphomycosis is dismal, with long-term survival being reported only when complete surgical resection of discrete lesions is possible. The development of new antifungal agents and combination treatment may help to improve the management of these infections.

Dynamics of infection with human herpesvirus-6 in EBV-negative infectious mononucleosis: data acquisition for computer modeling.

Ten adult patients with active HHV-6 variant A infections and clinical infectious mononucleosis-like disease (IM) were studied over a period of 32 weeks after onset of disease for their viral DNA load, changes in peripheral blood T-lymphocytes and subpopulations and frequency of cell death in peripheral blood cells. The data were collected as the basis for an advanced computer simulation study for which available data in the literature were too varied. Since the exact time of primary infection of the patients was not known and thus no time relationship of viral effects at cellular level were determined, we supplemented such data from separate tissue culture studies using HHV-6 alpha infection of HSB2 cells. Patients with IM demonstrate an increase in-HHV-6 DNA copies from 0 to 8.2 log 10/5 microL blood within 4 weeks return to normal by 16 weeks. Total T-lymphocytes follow infection with a 20-fold increase above normal peaking at 8-10 weeks and then return to normal by 24-28 weeks. Coincidently, less mature lymphoid cells carrying markers for stem cells, thymic cortical and medullary cells increase 8-10-fold indicating an enhanced mobilization of such cells from premature cell compartments. Cell death in peripheral mononuclear cells peaked with 30% at 8 weeks after onset of clinical disease and normalized by 24 weeks. HHV-6 replication in cell culture as determined by antigen expression, electron microscopy and harvest of infectious virus indicated a complete cycle of virus infection and replication of at least 6 days. The presented data compare well with others from the literature and will serve for testing in a computer simulation model, which is the subject of a forthcoming paper.

Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates in the United States: 1992 to 1994 and 1996 to 1998.

The antifungal drug susceptibilities of two collections of Cryptococcus neoformans isolates obtained through active laboratory-based surveillance from 1992 to 1994 (368 isolates) and 1996 to 1998 (364 isolates) were determined. The MICs of fluconazole, itraconazole, and flucytosine were determined by the National Committee for Clinical Laboratory Standards broth microdilution method; amphotericin B MICs were determined by the E-test. Our results showed that the MIC ranges, the MICs at which 50% of isolates are inhibited (MIC(50)s), and the MIC(90)s of these four antifungal agents did not change from 1992 to 1998. In addition, very small numbers of isolates showed elevated MICs suggestive of in vitro resistance. The MICs of amphotericin B were elevated (>or=2 microg/ml) for 2 isolates, and the MICs of flucytosine were elevated (>or=32 microg/ml) for 14 isolates. Among the azoles, the fluconazole MIC was elevated (>or=64 microg/ml) for 8 isolates and the itraconazole MIC (>or=1 microg/ml) was elevated for 45 isolates. Analysis of 172 serial isolates from 71 patients showed little change in the fluconazole MIC over time. For isolates from 58 patients (82% of serial cases) there was either no change or a twofold change in the fluconazole MIC. In contrast, for isolates from seven patients (12% of serial cases) the increase in the MIC was at least fourfold. For isolates from another patient there was a 32-fold decrease in the fluconazole MIC over a 1-month period. We conclude that in vitro resistance to antifungal agents remains uncommon in C. neoformans and has not significantly changed with time during the past decade.

Identification of functionally important amino-terminal arginines of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by alanine scanning mutagenesis.

Treatment of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase with the arginyl reagent phenylglyoxal resulted in complete desensitization to fructose 6-phosphate (F6P) activation, and partial desensitization to pyruvate activation. The enzyme was protected from desensitization by ATP, F6P, pyruvate, and phosphate. Alignment studies revealed that this enzyme contains arginine residues in the amino-terminal region that are relatively conserved in similarly regulated ADP-glucose pyrophosphorylases. To functionally evaluate the role(s) of these arginines, alanine scanning mutagenesis was performed to generate the following enzymes: R5A, R11A, R22A, R25A, R32A, R33A, R45A, and R60A. All of the enzymes, except R60A, were successfully expressed and purified to near homogeneity. Both the R5A and R11A enzymes displayed desensitization to pyruvate, partial activation by F6P, and increased sensitivity to phosphate inhibition. Both the R22A and R25A enzymes exhibited reduced V(max) values in the absence of activators, lower apparent affinities for ATP and F6P, and reduced sensitivities to phosphate. The presence of F6P restored R22A enzyme activity, while the R25A enzyme exhibited only approximately 1.5% of the wild-type activity. The R32A enzyme displayed an approximately 11.5-fold reduced affinity for F6P while exhibiting behavior identical to that of the wild type with respect to pyruvate activation. Both the R33A and R45A enzymes demonstrated a higher activity than the wild-type enzyme in the absence of activators, no response to F6P, partial activation by pyruvate, and desensitization to phosphate inhibition. These altered enzymes were also insensitive to phenylglyoxal. The data demonstrate unique functional roles for these arginines and the presence of separate subsites for the activators.

Feedback control of a biodynamical model of HIV-1.

We describe a continuous differential equation model of the interaction dynamics of HIV-1 and CD4 and CD8 lymphocytes in the human body. We demonstrate several methods of stable control of the HIV-1 population using an external feedback control term that is analogous to the introduction of a therapeutic drug regimen. We also show how the immune system components can be bolstered against the virus through a feedback control approach.

Dynamics of chronic active herpesvirus-6 infection in patients with chronic fatigue syndrome: data acquisition for computer modeling.

Ten adult patients with persistent active HHV-6 variant A infection and clinical chronic fatigue syndrome (CFS) were studied over a period of 24 months after initial clinical diagnosis. CFS was diagnosed according to IIIP-revised CDC-criteria as defined by the CFS Expert Advisory Group to the German Federal Ministry of Health in 1994. Changes in HHV-6 antibody titer, viral DNA load, peripheral blood T lymphocytes and subpopulations, as well as CD4/CD8 cell ratio and cell death (apoptosis) were monitored. Data were collected for comparison with respective changes in acute HHV-6 infection and as a basis for future computer simulation studies. The results showed variable but slightly elevated numbers of HHV-6 DNA copies in the blood of patients with CFS, while PBL (peripheral blood lymphocyte) apoptosis rates were clearly increased. CD4/CD8 cell ratios varied from below 1 up to values as seen in autoimmune disorders. Contrary to acute HHV-6 infection, T lymphocytes do not exhibit the usual response to HHV-6, that is elevation of mature and immature populations suggesting a certain degree of unresponsiveness. The data suggest that persistent low-dose stimulation by HHV-6 may favor imbalanced immune response rather than overt immune deficiency. This hypothesis requires confirmation through additional functional studies.

Dysregulative lymphoma theory revisited; what can we learn from cytokines, CD classes and genes?

Based largely on animal experiments, a dysregulative lymphoma theory was designed some 15 years ago as a basis for computer simulation studies. The basic concept of this theory was that lymphomas arise when persistent immunostimulation coincides with some kind of immune deficiency. The present article reviews exemplary data from human lymphoma cases in an attempt to further support or to reject the hypothesis. T- and B-cell lymphomas according to the REAL classification were reviewed with regard to the functional effects of their CD markers and their ligands, interleukin activities and cytogenetic changes. The results are summarized and further discussed. Essentially in all cases, a combination of enhanced stimulation of lymphoid cells and functional deficiency is identified, thus supporting the general pathogenetic hypothesis of malignant lymphomas. Despite using the most modem lymphoma classification, however, lymphoma entities and theirfunctional changes are so heterogeneous that cases need to be studied individually when it comes to pathogenetic considerations.

Dynamics of active progressive infection with HIV1: data acquisition for computer modeling.

Nineteen adult patients with progressive HIV1 infection, which progressed within 5 years from acute HIV syndrome to final AIDS were studied. Changes in HIV antibody titer, viral RNA load, peripheral T lymphocytes and subpopulations as well as CD4/CD8 cell ratio and cell death (apoptosis) were monitored. The data were collected for comparison with HHV-6 infection, which involves the same cell populations yet patients usually recover, and to serve as a further basis for future computer simulation studies. The results showed progressive increases of viral RNA copies in the patients' plasma even during clinical latency, which correlates with lymphocyte apoptosis and CD4 cell loss. Besides apparent direct CD4 cell destruction, there was indication of a disturbed intrathymic T cell differentiation. Pathologic cell changes in HIV infection continue until final death of the patient and do not return to normal after variable times as in HHV-6 infection. While HHV-6 infection can serve as models for immunostimulation, with or without immune dysregulation in computer simulation studies, HIV infection is a model for immunostimulation with final immune deficiency and cellular aplasia.

Candida dubliniensis fungemia: the first four cases in North America.

We report the first four North American cases of Candida dubliniensis fungemia, including the first isolation of this organism from the bloodstream of an HIV-infected person. All isolates were susceptible in vitro to commonly used antifungal drugs. This report demonstrates that C. dubliniensis can cause bloodstream infection; however, the incidence of disease is not known.