Development of a transformation system for Crinipellis perniciosa,the causal agent of witches' broom in cocoa plants

Protoplasts of the pathogenic plant fungus, Crinipellis perniciosa, were transformed to hygromycin B resistance using the pAN7-1 plasmid, which contains the Escherichia coli hph gene under the control of Aspergillus nidulans regulatory sequences. The pAN7-1 plasmid was introduced by PEG/CaCl(2) treatment. Transformation frequencies of 1.6-2.5 transformants/microg of DNA were achieved. About 54% of the transformants were abortive and 40 analyzed transformants were mitotically stable and showed different hygromycin B resistance levels. The presence of the hph gene was checked by PCR in five transformants and the integration of multiple plasmid copies into different genome sites was observed by Southern analysis. This is the first report of a C. perniciosa transformation system and represents an important step for further research into genetic manipulation of this fungal plant pathogen.     

Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.     

Hantaviruses in Central South America: phylogenetic analysis of the S segment from HPS cases in Paraná, Brazil

We sequenced the complete S segments of hantaviruses detected from 12 HPS patients living in southern of Brazil. Samples were obtained from patients diagnosed in different years, in distinct areas, and with a broad spectrum of clinical signs. Despite these differences, all the S proteins of hantavirus from Paraná were identical, except for one amino acid substitution. Phylogenetic analyses of the complete S segment nucleotide and amino acid sequences indicated that hantaviruses from Paraná form a distinct clade from those circulating in South and North America. Other hantaviruses from Brazil were not placed in the same clade. The Oligoryzomys nigripes-associated strains ITA37 and ITA38 from Paraguay were found to belong to the same clade as the hantaviruses from Paraná. Paraguay and Paraná state are located at the same latitude and some ecosystems are similar in both places. The geographic position and common rodent hosts could explain this phylogenetic relationship.     

Production of transforming growth factor-beta 1 (TGF-beta1) by blood monocytes from patients with different clinical forms of leprosy

In the present study, the concentration of TGF-beta1 secreted by adherent cells isolated from human peripheral blood mononuclear cells (PBMC) and either stimulated with PGL-1 or lipopolysaccharide (LPS) or left unstimulated was determined by ELISA. The cells were isolated from untreated patients with different clinical forms of leprosy and healthy individuals. The adherent cells exhibited spontaneous release of TGF-beta1 in all clinical forms of leprosy and in healthy individuals; however, lepromatous leprosy/borderline leprosy (LL/BL) patients presenting erythema nodosum leprosum (ENL) displayed significantly higher concentrations of TGF-beta1 than either the other patients studied or the controls. These high TGF-beta1 levels were consistently observed when LL/BL ENL cells were stimulated with phenolic glycolipid (PGL-1) or LPS, and even in the absence of a stimulus (P < 0.01). The most significant differences in TGF-beta1 levels were observed when comparing the results in the presence of PGL-1 from ENL with, in order of significance: tuberculoid leprosy (TT) patients (P < 0.001), LL/BL patients without ENL (P < 0.01), healthy individuals (P < 0.01) and borderline-borderline/borderline-tuberculoid (BB/BT) patients with reversal reaction (RR) (P < 0.01). The BB/BT patients produced equivalent levels of TGF-beta1 compared with LL/BL patients without ENL, for all types of stimuli (P > 0.05). In contrast, TT patients produced the lowest levels of TGF-beta1 among all the subjects studied (both patients and healthy controls), especially following PGL-1 stimulation (P < 0.001, and P < 0.05, respectively). In conjunction with our previous data regarding TGF-beta1 expression in dermal lesions, it appears that TGF-beta1 probably plays different roles in leprosy: (i) to mediate a suppressive action locally, associated with the presence of PGL-1, and (ii) to induce proinflammatory effects when secreted systemically by monocytes, thereby acting as a modulatory cytokine in the acute inflammatory reactions of ENL and associated with the Th2 immune response in multibacillary forms of leprosy.     

Leishmania major-Like Antigen for Specific and Sensitive Serodiagnosis of Human and Canine Visceral Leishmaniasis

An antigen (LMS) prepared from Leishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1,822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas' disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas' disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested.     

Comparison of C(18)-carboxypropylbetaine and standard N-acetyl-L-cysteine-NaOH processing of respiratory specimens for increasing tuberculosis smear sensitivity in Brazil

Techniques to improve the sensitivity of smear microscopy would facilitate early tuberculosis (TB) diagnosis and disease control, especially in low-income countries where the positive predictive value is high. C(18)-carboxypropylbetaine (CB-18) is a zwitterionic detergent that helps to compensate for the innate buoyancy of mycobacteria, potentially enhancing recovery by centrifugation. Previous data suggest that CB-18 may increase the sensitivity of smear, culture, and molecular amplification diagnostic testing. The goal of the present study was to evaluate if the sensitivity of the smear technique using light microscopy could be improved by treating respiratory samples with CB-18. In the first phase, respiratory specimens were collected consecutively from patients with suspected pulmonary tuberculosis in a tertiary-care hospital in Rio de Janeiro, Brazil (236 specimens were analyzed). After protocol modifications, another 120 respiratory specimens were evaluated. The standard technique was N-acetyl-L-cysteine with sodium hydroxide (NALC-NaOH) treatment, smear concentration with centrifugation, and Ziehl-Neelsen staining. Culture on L?wenstein-Jensen slants was performed on all specimens for use as the "gold standard." No specimens from patients undergoing active TB treatment were included. The initial protocol for CB-18 processing resulted in a sensitivity of 59.6% and specificity of 96.8% compared to standard processing with a sensitivity of 66.0% and specificity of 96.8%. Using the modified protocol, the sensitivity of CB-18 increased to 71.4% with a specificity of 97.0% versus standard processing with a sensitivity of 61.9% and a specificity of 99.0%. The diagnostic yield of acid-fast bacillus smear with CB-18 in the absence of fluorescence microscopy and PCR compared to standard processing with NALC-NaOH was not significantly different, although the power to detect a difference by the modified assay was low.     

Whole chromosome 17 loss in ovarian cancer

Chromosomal deletions, associated with the loss of normal function of tumour suppressor genes, have been identified in a variety of both familial and sporadic human cancers. Although the molecular pathology of ovarian cancer is not understood, several studies have reported deletions in chromosome 17 in ovarian tumours. We have used 13 restriction site polymorphic, microsatellite, and variable number tandem repeat markers to make a detailed analysis of chromosome 17 deletions in 12 benign and 19 malignant ovarian tumours. Two benign and 11 malignant tumours were informative for at least one marker on each arm of the chromosome. Loss of heterozygosity (LOH) was detected in both arms (by all informative markers) in 5 malignant tumours from four women (three with the disease at FIGO stage la). In a further bilateral ovarian tumour a partial LOH affecting 17q22-q25 was present in one ovary only. By contrast to a number of previous studies, none of the 19 malignant and 12 benign tumours showed ERBB2 (17q12ndash;22) amplification. The data presented show that the loss of a whole copy of chromosome 17 is a frequent and relatively early event in the development of some ovarian cancers. This suggests the possible involvement of multiple chromosome 17 loci in the pathogenesis of ovarian cancer. Equally plausible is that the loss of a whole chromosome copy could be the product of chromosomal instabilities induced by loss of the normal allele of tumour suppressors, such as TP53, located on this chromosome. © 1993 Wiley-Liss, Inc.     

Passive immune hemolysis for detection of heat-labile enterotoxin produced by Escherichia coli isolated from different sources.

Fifty-one strains of Escherichia coli isolated from humans, swine, food, and water and identified as enterotoxinogenic by the Y-1 adrenal cell assay, were examined for heat-labile enterotoxin (LT) production by the passive immune hemolysis test. Cholera antitoxin, anti-choleragenoid and anti-LT were used as antisera. Cholera antitoxin was much more potent than anti-choleragenoid and LT antiserum in the detection of LT-positive strains. All strains isolated from pigs and sausage were negative in tests made with LT antiserum. A few strains isolated from humans, food, and water also gave negative results. These data showed that the passive immune hemolysis test is not as efficient as the Y-1 adrenal cell assay in the detection of enterotoxinogenic E. coli strains.     

A randomised controlled trial to test the feasibility of a collaborative care model for the management of depression in older people

BACKGROUND: Depression is the most common mental health disorder in people aged over 65 years. Late-life depression is associated with chronic illness and disability. AIM: To investigate the feasibility of a collaborative care model for depression in older people in a primary care setting. DESIGN OF STUDY: Randomised controlled trial with 16-weeks follow up. SETTING: A primary care trust in Manchester. METHOD: Participants were 105 people aged 60 years or older who scored 5 or more on the Geriatric Depression Scale; 53 were randomly allocated to an intervention group and 52 to a usual care group. The intervention group received care managed by a community psychiatric nurse who delivered an intervention comprising a facilitated self-help programme with close liaison with primary care professionals and old-age psychiatry according to a defined protocol. The usual care group received usual GP care. A nested qualitative study explored the views of the health professionals and patients regarding the acceptability and effectiveness of the intervention. RESULTS: The main outcome measure was recovery from depression. Patients in the intervention group were less likely to suffer from major depressive disorder at follow up compared with usual care (0.32, 95% confidence = interval = 0.11 to 0.93, P = 0.036). The qualitative component of the study demonstrated the acceptability of the intervention to patients. CONCLUSION: A model of collaborative care for older people with depression, used in a primary care setting with a facilitated self-help intervention is more effective than usual GP care. This study demonstrates that the implementation of a collaborative care model is feasible in UK primary care and that the intervention is effective and acceptable to patients.