Demonstration of different patterns of musculoskeletal, soft tissue and visceral involvement in melioidosis using 99m Tc stannous colloid white cell scanning.

Melioidosis is an infectious disease that can present with multiple foci of disease involvement. Assessment of disease extent can be difficult, especially in musculoskeletal, visceral and soft tissue infection. This study examined the usefulness of white cell scans in this condition. 99mTc stannous colloid white cell scanning was performed in 21 patients with culture-proven melioidosis. Scan results were compared with clinical assessment and correlated with other forms of imaging. White cell scans demonstrated all but one of the clinically apparent sites of musculoskeletal, visceral and other soft tissue infection. Unsuspected disseminated soft tissue lesions were seen in two patients, including femoral node uptake in both, and these patients subsequently presented with relapsing musculoskeletal disease. Unsuspected musculoskeletal disease was found in one patient. Clinically suspected musculoskeletal disease was accurately excluded by white cell scan in another patient. The results of white cell scanning were also examined in disease of other viscera. Renal and prostatic disease were visualized. Unsuspected parotid involvement was found in two patients. Only one of two spinal lesions was visualized. Pulmonary disease was not necessarily associated with abnormal uptake. White cell scanning is a quick and effective way of assessing the extent of musculoskeletal, visceral and soft tissue disease in melioidosis.

Burkholderia pseudomallei virulence: definition, stability and association with clonality.

Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.

Determinants of dengue 2 infection among residents of Charters Towers, Queensland, Australia.

Dengue fever is caused by one of the four serotypes of the dengue virus and is transmitted by the urban mosquito Aedes aegypti. In 1993, the city of Charters Towers in the tropical north of Australia experienced an epidemic caused by the dengue 2 virus. A cross-sectional sample of 1,000 people was assessed for determinants of recent symptomatic dengue infection. After exclusion of people with prior exposure to dengue 2, a study group of 797 persons, including 196 patients with recent infection, were evaluated. Stepwise logistic regression analysis identified four determinants of infection: the presence of a case of dengue fever within two residential blocks (odds ratio (OR) = 3.61, 95% confidence interval (CI) 2.56-5.10), house screening (OR = 0.60, 95% CI 0.40-0.89), the presence of a water tank within two residential blocks (OR = 1.51, 95% CI 1.02-2.22), and the use of knockdown insecticide (OR = 1.75, 95% CI 1.22-2.51). Classification and Regression Tree analysis identified a group of 152 individuals in whom the prevalence of dengue infection was 50%. These people lived within two blocks of a suspected dengue fever case, did not have house screening, and used knockdown sprays. If dengue had not occurred within two residential blocks, there were no additional factors that significantly influenced the prevalence of dengue fever. Control of dengue epidemics should involve attempts to geographically contain the spread of infection, use of house screening, and the removal of mosquito breeding sites such as water tanks.

The 1993 dengue 2 epidemic in North Queensland: a serosurvey and comparison of hemagglutination inhibition with an ELISA.

An epidemic of dengue type 2 infection occurred in North Queensland during 1992 and 1993. A random serosurvey of 1,000 residents of a population that experienced this epidemic only during 1993 was conducted to determine the proportion of the population at risk for secondary infection in the event of another epidemic with a different serotype. The ability of an ELISA to detect prior exposure to the dengue virus was compared with the hemagglutination inhibition assay. Dengue 2 virus plaque-reduction neutralization assays were performed to evaluate the specificity of the antibody response. Antibodies to dengue virus, or closely related flaviviruses, were detected in 61.9%. Seroprevalence increased with age and correlated well with known previous epidemics in the region. The sensitivity and specificity of the ELISA was 99.2% and 96.2%, respectively. An estimated 26% of the population was infected during the 1993 epidemic.

The 1993 dengue 2 epidemic in Charters Towers, North Queensland: clinical features and public health impact.

In 1993 an epidemic caused by dengue virus type 2 occurred in several North Queensland population centres. Charters Towers, estimated population 10,000, had 155 officially notified cases. An analysis of symptoms was undertaken using a random sample of 1000 residents to determine specificity of symptoms, the subclinical infection rate, and to establish the true extent of the epidemic. Retrospective diagnoses of dengue fever were based on the presence of both serum dengue 2 neutralizing antibody and presence of symptoms. An estimated 20% of the population had dengue fever. The rate of subclinical infections in this epidemic was 14.6%. There were no symptoms that were specific for dengue fever. Bleeding occurred more frequently in people who recalled a previous dengue infection during a dengue 1 epidemic 12 years earlier (55.6% vs. 16.8%, P = 0.003). Surveillance for future epidemics should be based on serological and virological confirmation of dengue virus infection amongst symptomatic patient.

Oral delivery of foreign antigens by attenuated Salmonella: consequences of prior exposure to the vector strain.

Several strains of Salmonella have been used as vectors for the delivery of Escherichia coli fimbrial proteins to the gut-associated lymphoid tissue (GALT) of the mouse. Plasmids carrying a complementing thyA+ gene, together with genes specifying synthesis of K88 or K99, were introduced into non-reverting thyA Salmonella mutants. The resulting constructs expressed the foreign pilin protein on their surfaces and, provided the vector was able to colonize the GALT, elicited strong serum responses to K88 or K99. These responses were dramatically impaired however, in recipients with pre-existing immunity to the vector strain. Mice initially infected with Salmonella stanley ca 4, 10 or 20 weeks prior to oral administration of S. stanley-K88 showed greatly reduced serum responses to K88 as determined by ELISA. The hypo-responsiveness seen in vector-primed mice could be largely overcome by changing the serotype of the strain subsequently used to deliver the foreign protein.

Cell surface determinants on typhoid-specific B cells isolated from peripheral blood after oral Ty21a typhoid vaccination in humans.

Antigen specific B cells (ASC) that circulate after oral immunization with the typhoid vaccine Ty21a display cell surface determinants which are potentially involved in B cell differentiation and homing to mucosal sites. These ASC were isolated from peripheral blood after oral Ty21a, and dual labelled for binding of typhoid antigen and expression of various cell surface determinants: alpha 4 integrin (CD49d), CD45RO, CD45RA, L-selectin, CD44 and CD11a. Of particular interest was the finding of CD45RO expression on ASC. A comparison of cell surface determinants on typhoid-specific cells was also made following binding to high endothelial venules on peripheral and mesenteric lymph nodes, and venules in the lamina propria of the small intestine. Generally more typhoid ASC bound to mesenteric compared with peripheral lymph node. More ASC expressing CD45RO and alpha 4 integrin (CD49d) were bound to mesenteric lymph node and small intestine than to peripheral lymph node. When expressed as a fraction of total ASC, the difference was statistically significant only for CD45RO binding to small intestine versus peripheral lymph node. No differences in expression of other homing markers on bound ASC were seen.

Antigen-specific B cells in tissues after oral typhoid vaccination.

Six human subjects who were to receive elective bowel surgery for a variety of diseases were vaccinated with the oral typhoid vaccine, Ty21a. Intestinal tissue (ileum in two, large intestine in four) removed 7-26 days after the first dose of vaccine was examined for the presence and distribution of antigen-specific B cells. This was compared with intestinal tissue derived from two unvaccinated controls. A number of B cell differentiation antigens were also assessed on these cells by immunofluorescence using dual-labelling. Antigen-specific cells were found randomly distributed in the lamina propria of all the vaccinated subjects in low frequency (6 +/- 0.5 to 37 +/- 31 [mean +/- s.e.m.] antigen specific cells/10 mm2 of tissue). The lymphocyte differentiation antigens CD45RA, CD45RO, L-selectin, CD-11a CD-38, CD-44 and VLA-4 were all found on antigen-specific cells, but no particular pattern was recognizable in this small series of six subjects with different disease processes affecting the intestine.

Detection and distribution of hepatitis C-specific antigens in naturally infected liver.

Hepatitis C virus antigen expression was examined using peptide antibodies in liver tissue taken at biopsy from four chronic carriers of hepatitis C virus. Hepatitis C virus antigens E2/NS1, NS3, NS4 and NS5 were widespread in unfixed frozen liver sections and were present as distinct granules or foci within the cytoplasm of hepatocytes and in infiltrating lymphocytes in portal tracts. Fixation of frozen sections with 1% formalin improved the histological appearance of the tissue section without reducing the sensitivity of antigen detection. However, in tissue sections fixed in acetone, chloroform, carbon tetrachloride or methyl carnoys, detection of all hepatocyte-specific hepatitis C virus antigens was significantly reduced. Dual immunostaining of liver sections for lymphocyte cluster of differentiation markers and hepatitis C virus antigens determined that a high proportion of cluster of differentiation 20-positive B cells and cluster of differentiation 4-and cluster of differentiation 8-positive T cells, predominant in lymphoid aggregates, were positive for hepatitis C virus antigens.

A comparison of hepatitis C virus (HCV)-RNA and--antibody as markers of infection and predictors of infectivity.

BACKGROUND: The diagnosis of hepatitis C virus (HCV) infection currently relies on the detection of antibody to HCV (anti-HCV). However, anti-HCV positivity may indicate past infection, current infection or possibly non-specific reactivity. For confirmation of current infection the virus needs to be assayed directly and this is possible by the polymerase chain reaction (PCR). AIMS: The aims were to compare HCV RNA and anti-HCV as markers of infection in two groups of individuals: (i) a heterogeneous group with suspected HCV infection and (ii) a small group of blood and bone marrow donors, and their respective recipients. METHODS: Serum samples were tested for alanine aminotransferase (ALT) as part of a liver function screen, for anti-HCV by ELISA II, and HCV RNA was detected by PCR. Single round and nested PCR was performed using primers designed from the sequence of the 5'-untranslated region of the HCV genome. RESULTS: Of the 36 subjects in the heterogeneous group, 19/22 anti-HCV-positive patients with chronic non-A non-B hepatitis (NANBH) were viraemic, and the majority (17/19) demonstrated elevated ALT. However, HCV RNA was undetected in seven anti-HCV-positive patients, four of whom suffered autoimmune hepatitis Type I and three were low risk blood donors. Of the remaining subjects (seven/36) who were anti-HCV-negative, three/seven were HCV-RNA-positive and included two with acute post-transfusion (PT) NANBH and a recent needlestick victim who contracted HCV. In the second group, four individuals (donors), including a mother with a history of drug use, were implicated in transmission to three recipients. ALT levels were normal in all donors but raised in two of the recipients. PCR determined which of two anti-HCV-negative blood donors was infectious, confirmed transmission between a bone marrow donor and recipient, and indicated that anti-HCV detected in a newborn child represented passive transfer of antibody. CONCLUSIONS: Anti-HCV detected by ELISA II is a useful marker of chronic HCV infection, particularly in association with raised ALT. However, HCV RNA is a superior marker of acute HCV infection, a more reliable predictor of infectivity and is more specific.

Dual staining of lymphocyte membrane antigens with colloidal gold and biotinylated horseradish peroxidase.

Double immunoenzymatic labelling procedures for the localization of antigens on cells in tissue sections using horseradish peroxidase (HRP) and alkaline phosphatase have been described previously, but mainly for detecting antigens on different cells. With this type of staining when two antigens are present on the same cell, an optimal colour combination that shows a high contrast between the basic colour of each enzyme substrate product is difficult to achieve and the interpretation of their mixed colour intermediate is subjective. We present a method for the simultaneous demonstration of two antigens on the same cell. The method can be used to label either single cells in suspension, or cells in paraffin fixed tissue, using a combination of a particulate label, colloidal immunogold-silver, and an enzymatic label HRP-DAB. The method is easy to perform and utilises commercially available staining kits.

Immunohistochemical detection of the NS4 antigen of hepatitis C virus and its relation to histopathology.

The immunohistochemical localization of the hepatitis C virus (HCV) nonstructural antigen 4 (NS4) was investigated in formalin-fixed human liver biopsy samples taken from 10 patients who were anti-HCV positive. NS4 was detected within the cytoplasm of hepatocytes in all HCV-positive patients studied, but not in the mononuclear cell infiltrates, bile duct epithelium, or endothelial cells. A high proportion of hepatocytes appeared positive, but the staining intensity was variable. After a coded histological evaluation of the liver tissue, the pattern of liver injury was shown to have no significant correlation with antigen-positive hepatocytes, and no direct relationship was observed between the distribution of antigen-positive hepatocytes and areas of hepatocyte necrosis. The staining pattern was considered to be specific because liver samples from patients chronically infected with hepatitis B virus or from uninfected individuals were negative. Furthermore, no staining was noted when either preimmune rabbit serum or anti-NS4 adsorbed against the specific synthetic peptide was substituted for the primary antibody.

Localisation of hepatitis C virus proteins in infected liver tissue by immunofluorescence.

Hepatitis C virus (HCV) antigen expression was examined by immunohistochemical staining in liver tissue taken at biopsy from 8 anti-HCV positive patients. Frozen liver sections were stained by indirect immunofluorescence for capsid, E2/NS1, NS3, NS4 and NS5 using polyclonal antibodies raised to synthetic peptides from these regions. The antigens E2 and NS3 were localised in scattered hepatocytes and also in cells within and around areas of inflammation. A weaker signal was observed for NS4 and NS5 and no signal was seen for capsid antigen. No staining was seen in liver tissue from 9 individuals, including 3 hepatitis B virus-positive and 2 hepatitis delta virus/positive patients, who were negative for serological markers of HCV. The specificity of the staining reaction was also confirmed by the lack of staining in HCV-positive liver samples, after the antisera was pre-adsorbed against the specific peptide. Collectively, the data suggests that HCV may not only be hepatotropic but also lymphotropic, and this may be an important factor in the pathogenesis of HCV infection.

Effect of parenteral immunization on the intestinal immune response to Salmonella typhi Ty21a as measured using peripheral blood lymphocytes.

The effects of parenteral administration of a killed typhoid vaccine on the intestinal immune response to live orally administered Salmonella typhi Ty21a in human subjects was assessed using an assay of in vitro specific antibody production by circulating peripheral blood lymphocytes (PBL). Previous priming with the parenterally administered vaccine had no effect on secondary immune responses to a live oral vaccine in humans with this response not differing in magnitude or duration from that following the primary oral vaccination course (p = 0.38). Following the primary PBL anti-LPS IgA response to an oral course of live vaccine in all subjects (6/6), boosting with a single oral dose of live vaccine resulted in 0/6 responders, while 2/11 subjects responded after a single dose of parenteral vaccine. No additional responses were evident after the second parenterally administered booster dose. PBL IgG and IgM responses were also demonstrated following oral vaccination, with 1/6 and 2/6 subjects parenterally vaccinated demonstrating IgM PBL responses after the first and second vaccination respectively. This study confirmed that parenteral vaccination did not impair the PBL IgA immune response to a secondary course of live orally administered organisms, and that multiple parenteral booster doses did not induce primary or secondary recirculation of antigen-specific PBL.

Effect of parenteral immunization on the intestinal immune response to Salmonella typhi Ty21a.

The effects of parenteral administration of a killed typhoid vaccine on the intestinal immune response to live orally administered Salmonella typhi Ty21a in human subjects was evaluated. Priming with parenteral vaccination neither enhanced nor suppressed the subsequent specific serum and intestinal immunoglobulin A (IgA) immune responses to a booster course of live oral vaccine. Neither a single oral dose of live vaccine nor a single dose of parenteral vaccine had any measurable booster effect on the observed primary intestinal IgA response to the live oral vaccine. Two booster doses of subcutaneously administered killed typhoid vaccine did result in a significant increase in the specific intestinal IgA antibody in those subjects primed with the oral live vaccine. This response was comparable in magnitude to the primary intestinal response. No evidence of this response could be found in serum IgA, although nonsignificant rises in serum IgG were evident. Previous parenteral priming had no effect on secondary immune responses to a live oral vaccine in humans. Serum immune responses were generally found to be of little value as indicators of local intestinal immunity. This study confirmed that parenteral vaccination was only able to induce an intestinal immune response following priming with live, orally administered organisms and that multiple parenteral booster doses were necessary to induce a measurable effect on intestinal immune responses.

Cellular and humoral responses in coeliac disease. 2. Protein extracts from different cereals.

The humoral and cellular immune responses to grain protein extracts from coeliac-toxic and non-toxic cereals were compared by use of a number of ELISA and immunoblotting methods and the indirect leucocyte migration inhibition factor (LMIF) assay. Both adult and child coeliacs had elevated levels of serum antibody to proteins from the coeliac-toxic cereals, namely bread wheat, durum wheat, rye and barley and low levels of proteins from other cereals. Using protein blotting techniques, antibody binding was greatest to gliadins/low mol mass glutenin subunits and homologous prolamins from rye and barley, consistent with the ELISA findings. Competition ELISA and preabsorption tests indicated that antibody reaction to maize storage proteins did not simply result from cross-reaction of antigliadin antibodies. In LMIF assays, only the wheat extracts had activity in coeliac patients. This is most likely partly due to loss of some of T-cell epitopes from the extraction technique required for these proteins, as well as the relatively small effects seen for even very active fractions in the LMIF assay.

Cellular and humoral responses in coeliac disease. 1. Wheat protein fractions.

The humoral and cellular immune response of coeliac individuals to various wheat protein fractions was studied using serum antibody ELISA assays and the indirect leucocyte migration inhibition factor (LMIF) assays. Greater migration inhibition factor activity was seen in coeliacs on a gluten-free-diet having low serum antibody titres, and using purified T-cells instead of total peripheral blood mononucleocytes. Gliadin was the most active fraction in both assays. Raised antibodies to low-molecular weight and high-molecular weight glutenin polypeptides was observed, though these proteins had little migration inhibition factor activity. No cellular or humoral response was seen to albumins or globulins. Proteins associated with the granules of well-washed wheat starch are distinct from gluten proteins and had little T-cell activity, correlating with clinical observations that properly prepared wheat starch is devoid of coeliac toxicity. The greater specificity of the humoral response for individual wheat protein fractions in this study, compared with the earlier reports, likely results from cross-contamination in the earlier work of each fraction with gliadin.