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A W Joss

Publications and source records attributed to A W Joss.

At least 19 recordsLinked to original sources

Lyme disease--what is the cost for Scotland?

This paper analyses likely Lyme disease costs incurred by patients tested in the authors' laboratory over an 18 month period, based on patient histories and test results relating to 2110 samples submitted from laboratories serving 59% of the Scottish population. Cost analysis takes account of the direct costs of consultation, laboratory tests, antibiotic treatment and management of any sequelae, as well as indirect costs of the loss of healthy time through illness. Standard costs for each element are derived from published information, and the proportions applied to each patient category are estimated from studies described elsewhere in the literature. Of the sample, 295 patients had evidence of early Lyme disease and 31 had late Lyme disease symptoms. Based on these figures, the total annual cost for Lyme disease, when projected to the whole of Scotland, is estimated to be significant at 331,000 Pounds (range 47,000-615,000 Pounds). The range is inevitably wide because it was not possible to document complete clinical and management histories on individual patients. In addition, some late Lyme disease sequelae will require management for more than 1 year, and costs are also identified that could justifiably be included for all the other patients who tested negative for Lyme disease. These data raise the question of whether there is sufficient focus on prevention and the best management of this disease.

Cost of Illness↗

Success in the toxoplasma dye test.

OBJECTIVES: To compare the success rate of the toxoplasma dye test using different accessory factors (human serum as a source of complement) and different batches of tachyzoites produced in vivo and in vitro. METHODS: Twenty-five accessory factors were used in the dye test to assess both types of tachyzoite. Batches of tachyzoites were produced in vivo (n = 49) and in vitro (n = 23) and their performance assessed against panels of accessory factors. Performance was recorded as success or failure (incorrect results, total killing or no killing). RESULTS: With in vivo tachyzoites 21/25 accessory factors were successful in P > or = 1 dye test runs, whereas with in vitro tachyzoites all 25 were successful. One or more failure was recorded for 19/25 and 12/25 accessory factors using in vivo and in vitro tachyzoites, respectively (P < 0.05). The number of successful dye test runs was less for in vivo (92/141, 65%) than in vitro (140/163, 86%) tachyzoites (P < 0.001). This was due to a higher success rate in citrated accessory factors used for in vitro tachyzoites compared to the corresponding uncitrated accessory factors used for in vivo tachyzoites (P < 0.001). Success in the dye test was recorded for 48/49 and 23/23 batches of in vivo and in vitro tachyzoites, respectively. The number of successful dye test runs was lower with in vivo (156/234, 67%) than in vitro (116/142, 82%) tachyzoites (P < 0.01). CONCLUSIONS: Success in the dye test may be due to the accessory factor, tachyzoites, or a combination of both. Problems due to the accessory factor can be minimized by careful quality control or use of modification procedures. Tachyzoites produced in vitro may also increase success in the dye test. Careful selection of accessory factor/tachyzoite combination makes it possible to use the dye test in a district general hospital.

Animals↗

Toxoplasma dye test using cell culture derived tachyzoites.

AIMS: To assess the diagnostic usefulness of Toxoplasma gondii tachyzoites produced by serial passage in HeLa cell culture. METHODS: Tachyzoites derived from serial passage in cell culture were used in the dye test. Human sera were also examined to determine their suitability for use as accessory factor. Using the optimum conditions, the dye test using cell culture derived tachyzoites was compared with the current method of production (animal culture) on 105 randomly selected sera. Start up and maintenance costs of each system were compared. RESULTS: Tachyzoites in most cell culture harvests (84%) from both early and later passes were useable. Tachyzoite yield and viability were maintained during serial passage in cell culture. Sodium citrate was used to modify accessory factor and improve its suitability. The performance of the accessory factor was improved by the addition of 1% and 3% sodium citrate for the current and cell culture systems, respectively. Under optimum conditions, dye test titres using cell culture and current systems were compared on 105 randomly selected sera. The results from 92 of 105 (87.6%) patients agreed or were within one dilution, but all discrepancies were resolved on re-testing. Start up costs for the current system would be 2.5 times and overall maintenance three times more expensive than cell culture. CONCLUSIONS: Tachyzoites derived from cell culture can be used routinely in the dye test. Production in cell culture is more cost effective than animal culture. It is possible for general hospitals to perform the dye test, thus obtaining faster and more specific results.

Animals↗

Identification of different Borrelia burgdorferi genomic groups from Scottish ticks.

AIM: To characterise 12 Borrelia burgdorferi sensu lato isolates cultured from ticks collected in the Highlands of Scotland. METHODS: Three molecular methods were used: an outer surface membrane protein A (OSP A) gene polymerase chain reaction (PCR) designed to give different molecular weight products with different genomic groups, randomly amplified polymorphic DNA (RAPD) analysis, and ribosomal RNA (rRNA) gene PCRs using genomic group specific primers. RESULTS: All of the molecular methods used were quick and easy to perform and capable of differentiating between the different genomic groups of B burgdorferi sensu lato. All 12 tick isolates were characterised successfully with each method: five were characterised as B afzelii and seven were characterised as B burgdorferi sensu stricto. RAPD also identified differences within these genomic groups. CONCLUSIONS: From this study, it is now known that at least two different B burgdorferi sensu lato genomic groups are present in the Highlands of Scotland: B afzelii and B burgdorferi sensu stricto. This information can now be used to develop appropriate serological tests, which should improve the diagnosis and management of patients with Lyme disease in Scotland. The molecular methods chosen were found to be useful typing tools and will allow rapid identification of any future isolates.

Animals↗

Evidence-based diagnosis of Lyme disease.

The aim of this study was to make an evidence-based comparison of four commercial enzyme immunoassays (EIAs) (Serion Classics, Sigma Diagnostics, Cambridge Biotech and ICN Diagnostics) and an in-house enzyme immunoassay (EIA) in order to select the most appropriate screening assay for diagnosis of Lyme disease. Borrelia burgdorferi sensu stricto cultured in BSK-H medium was used to develop the in-house assay. Escherichia coli antigen (0.9 mg/ml) was included in the serum diluent to reduce non-specific background. Comparison of the number of tests needed to diagnose (i.e. to indicate a positive result) and the cost per positive diagnosis for the five assays was made using a panel of 176 Western blot-characterised sera. The Cambridge Biotech and Sigma assays had the highest sensitivity but poorer specificity, whereas the Serion and ICN assays had highest specificity but poorer sensitivity. The in-house assay had average sensitivity and specificity, the number of tests needed to diagnose being 2.32 compared to 1.92 for Serion, 2.17 for ICN, 2.5 for Sigma and 2.7 for Cambridge Biotech. In a diagnostic protocol that uses an EIA as screening test, with confirmation by Western blot, a good balance of sensitivity and specificity is essential. The in-house assay was the most cost-effective (lowest cost per positive diagnosis), and is probably the best option for specialist laboratories in Europe.

Borrelia burgdorferi Group↗

Cell-culture system for continuous production of Toxoplasma gondii tachyzoites.

The aim of this study was to identify a sustainable cell line and culture method that could continuously provide a sufficient quantity of Toxoplasma gondii tachyzoites to serve the needs of a general hospital laboratory. Three continuous cell lines (HeLa, LLC and Vero) and three cell-culture methods (culture in conventional flasks, culture in membrane-based flasks and an automated culture system) were investigated. In multiplicity-of-infection and time-course experiments, HeLa was the cell line of choice. Harvests from HeLa cells had significantly higher tachyzoite yields than those from LLC cells (P<0.00005) or Vero cells (P<0.05). Membrane-based flasks gave higher yields (6.15x10(6) tachyzoites/ml) than conventional flasks (1-2x10(6) tachyzoites/ml) initially, but these were not sustained. The automated cell-culture system was unsuitable for parasite culture. Continuous passage in 25 cm2 flasks was successful, yielding 1x10(6) tachyzoites/ml; viability exceeded 90% after 96-120 h of infection throughout 38 passes, during which time the viability improved and the time to harvest became more consistent. Toxoplasma gondii grown in continuous culture in HeLa cells can provide a regular supply of viable tachyzoites. Demonstration that HeLa-derived tachyzoites could be used for the dye test confirms the potential of this in vitro system for use in general hospital laboratories.

Animals↗

Progression of Pneumocystis carinii infection in an animal model.

The development of Pneumocystis carinii infection in immunosuppressed rats is important not only in understanding the infection, but also as a source of P. carinii antigen for use in diagnostic serological tests. The aims of this study were to monitor the progression of P. carinii infection in the Sprague Dawley rat model and then determine parameters that indicate the maximum production of P. carinii antigen. Seventeen Sprague Dawley rats were killed at intervals up to 9 weeks after the start of immunosuppressive therapy. The progression of P. carinii lung infection was observed by Giemsa staining of lung imprints and by a hemi-nested polymerase chain reaction (PCR). Body weight, food and water intake and the appearance and activity of the rats were measured daily. Seven control rats were kept under the same conditions. P. carinii infection was detected in the lung 2 weeks after immunosuppression by hemi-nested PCR and after 3 weeks by Giemsa staining. No P. carinii DNA was detected in any of the blood samples. Rats with moderate or severe lung infection had been immunosuppressed for > or = 6 weeks. Body weight was significantly greater in control rats than in the immunosuppressed rats. Six weeks of immunosuppression was used as a cut-off to determine measurements to identify those rats with moderate or severe infections in their lungs. A combination of > 34% body weight loss at 6 weeks after immunosuppression and the condition of the animals with scores < or = 9 used in conjunction with duration of immunosuppression may be useful to maximise the yield of P. carinii infection from individual rats.

Animals↗

Do IgA, IgE, and IgG avidity tests have any value in the diagnosis of toxoplasma infection in pregnancy?

AIM: To determine the value of tests for specific IgA, IgE, and IgG avidity in diagnosing Toxoplasma gondii infection during pregnancy. METHODS: In a retrospective study, current serological tests (dye test and three IgM assays with different sensitivities) were compared with immunosorbent agglutination assays (ISAGA) for specific IgA and IgE and an IgG avidity enzyme linked immunosorbent assay (ELISA). Patient group 1 comprised six women with definite or probable infection during pregnancy determined by congenital toxoplasmosis or laboratory results. Group 2 comprised seven women infected during or before 11 pregnancies (two consecutive pregnancies in two patients and three in a third). RESULTS: One patient in group 1 seroconverted during pregnancy. IgA ISAGA and avidity confirmed acute infection when confirmatory IgM ELISA remained negative. In five of six patients from group 1, IgA and IgE ISAGA and avidity confirmed acute infection. In group 2, the dye test titre was raised in seven of 11 pregnancies (six of seven patients). Specific IgM and IgA were positive during all 11 pregnancies. IgE ISAGA was positive in only four of 11 pregnancies (three of seven patients), but negative results in the remainder may exclude acute infection. High avidity antibodies indicative of past infection were found in four of 11 pregnancies (two of seven patients). CONCLUSIONS: Each test improved diagnosis or timing of infection but no single test was ideal. The IgA ISAGA was sensitive and detected seroconversion. Positive IgE ISAGA and low avidity both confirmed infection, whereas negative IgE may exclude acute infection. High avidity diagnosed past infection but persistence of low avidity reduced its value to differentiate acute and past infection. Further studies with larger patient groups are needed to determine the optimum diagnostic strategy. These techniques are valuable in complementing existing tests.

Animals↗

Improved diagnosis of reactivated toxoplasmosis.

AIMS: To identify antigens detected by western blotting in primary Toxoplasma gondii infection and determine their role in diagnosis of reactivated toxoplasmosis. METHODS: Twenty three immunocompromised patients were tested by IgG western blotting. Patients were grouped retrospectively. Group 1 comprised 15 human immunodeficiency (HIV)/AIDS patients and included: group 1A (six patients with clinical and/or serological evidence of reactivation), group 1B (five patients with clinical evidence only), and group 1C (four asymptomatic patients). Group 2 comprised eight non-HIV/AIDS immunocompromised patients with clinical and/or serological evidence of reactivation. Immunocompetent patients (n = 23) with primary toxoplasmosis were a control group used to determine the progression of the antigens detected. RESULTS: In primary toxoplasmosis, antibodies against 6, 20, 22, 23, 25, 28, 29, and 36 kDa antigens predominated. Detection of four or more of the 6, 20, 22, 23, 25, and 36 kDa antigens was considered to be western blot positive. In two group 1A patients, western blotting indicated past infection. During reactivation, this reverted to being western blot positive. Three other group 1A patients were western blot positive. In three of five group 1B patients, western blot positive results improved serological diagnosis of reactivated toxoplasmosis (p < 0.05). In two of five group 1B patients and all four group 1C patients, western blot indicated past infection. In group 2, two of eight patients reverted from a pattern of past infection to western blot positive. Five other patients from group 2 were western blot positive. CONCLUSIONS: Detection of some low molecular weight antigens is diagnostic of reactivated toxoplasmosis. These antigens can be detected even with normal dye test titres and their detection improves the diagnosis of reactivated toxoplasmosis. They might be the result of the release of bradyzoites from ruptured tissue cysts.

Animals↗

The effect of sample storage on polymerase chain reaction-based detection of Toxoplasma gondii in amniotic fluids.

The effects of sample storage, target preparation and annealing temperature on a nested polymerase chain reaction (PCR) test for Toxoplasma gondii DNA were investigated with experimentally seeded amniotic fluids to simulate congenital infection. Aliquots of 17 amniotic fluid samples remaining after investigation for conditions other than toxoplasmosis and seeded to contain small numbers of T. gondii tachyzoites, were tested after storage for up to 2 weeks. There was no deterioration in test sensitivity on samples stored for 1-2 weeks at room temperature; thus, samples sent by post to reference laboratories are acceptable for examination. There was no significant difference in the results from two target preparation methods or two different annealing temperatures. One-to-ten parasites were detectable in 13 of 17 test samples. The significant feature in the remaining four samples was the use of washed stored parasites to seed the amniotic fluids rather than any feature of the amniotic fluids used or the test. False positive results were found in up to 15% of unseeded amniotic fluid samples, which contrasts with only 1.9% for the same PCR test in other routine or negative control samples tested as part of the laboratory's reference diagnostic work. The difference illustrates the increased risk of cross-contamination in PCR when the majority of specimens tested are positive. The results suggest that PCR techniques are likely to be sensitive and effective tools for the routine diagnosis of congenital toxoplasma infection.

Amniotic Fluid↗

Improved serodiagnosis of Lyme disease.

Aims-To determine whether enzyme linked immunosorbent assay (ELISA) results for Borrelia burgdorferi require confirmation by immunoblotting and how immunoblotting may best be used in the diagnosis of Lyme disease.Methods-Over one year, all referrals for Lyme disease to a district general hospital with a large tick population in its catchment area were tested by ELISA. Positive, low positive and negative serum samples were subjected to immunoblotting and the reactive bands analysed.Results-In total, 633 samples were received; 38 were ELISA positive and 97 low positive. More serum samples were from rural (n = 356) than from urban (n = 277) areas but a higher percentage of serum samples from urban areas were ELISA positive. The ELISA results were confirmed by immunoblotting in 15/38 positive samples but in only four of 37 with a low positive titre. An IgM positive blot required a 41 kDa band plus >/=1 specific band; for IgG a 41 kDa band plus >/=2 specific bands were necessary. Five serum samples were IgM positive with a 41 kDa plus one or more other specific bands. For IgG blots, the best discrimination was seen with the 21, 31, 46, and 92 kDa bands. Nonspecific, weakly reacting bands at 55, 60 and 67 kDa were frequently seen. Infection was confirmed in four of six patients with arthritis, but in only one of 10 patients with erythema chronicum migrans.Conclusions-ELISA alone is insufficient for diagnosis. All positive and low positive or negative serum samples with a good clinical history should be examined by immunoblotting. A higher percentage of modified ELISA positive than low positive results were confirmed. There are significant differences between European and American immunoblotting patterns. Local results show similarity to American results, highlighting the need for a local Borrelia isolate.

Journal Article↗

Differences exist in the immunoblotting profiles of cyst and trophozoite antigens of Pneumocystis carinii.

The antigenic profiles of Pneumocystis carinii trophozoites and cysts were compared by immunoblotting with hyperimmune rat sera against cyst and trophozoite antigens. Strong bands corresponding to proteins of 50-60 kDa and 104 kDa were demonstrated in cyst and trophozoite antigens by all antisera. Additional prominent proteins of 81 and 63 kDa and less prominent proteins of 88, 73, 69 and 37 kDa were found only in trophozoite antigen. The latter proteins were recognised by anti-trophozoite and anti-cyst antisera but the 81- and 63-kDa proteins were associated specifically with trophozoites. With cyst-rich antigen, antibodies to the 50-60-kDa protein were detected in only two of 14 sera from P. carinii pneumonia (PCP)-positive rats. With trophozoite-rich antigen, 11 of 24 rats with PCP and one of 18 PCP-negative animals had antibodies to both the 50-60 kDa and 104-kDa antigens. Antibodies to the 81- or 63-kDa antigens were demonstrated in 15 of 24 PCP-positive animals and none of the PCP-negative animals. The use of trophozoites rather than cysts increased the sensitivity of immunoblotting. As trophozoites predominate in PCP, antibody to trophozoite-specific antigens rather than common cyst and trophozoite antigens is likely to be a more useful marker of current infection.

Animals↗

The use of a nested polymerase chain reaction for detecting Pneumocystis carinii from lung and blood in rat and human infection.

A nested polymerase chain reaction (PCR) assay was developed to detect both rat- and human-derived Pneumocystis carinii DNA. The nested PCR product was 125 bp long and was representative of part of the gene coding for the large subunit of mitochondrial ribosomal RNA. Twenty serial blood samples and 24 tissues from six immunosuppressed Sprague-Dawley rats were examined by nested PCR. All lung samples were positive by PCR and Toluidine blue O staining. Buffy coat samples and all the other tissues were PCR-negative during up to 6 weeks of immunosuppression. Thirty-five clinical bronchoalveolar lavage, induced sputum or tracheal aspirate samples from human patients were tested. Twelve of 35 were positive by both PCR and indirect fluorescence assay (IFA) and 19 of 35 were both PCR- and IFA-negative. Four of 35 were IFA-negative but PCR-positive and there were good responses in these patients to specific therapy, indicating that PCR may be more useful than IFA in clinical samples. P. carinii DNA was not detected in three blood samples. The nested PCR is a sensitive and specific DNA amplification method suitable for the routine diagnosis of P. carinii in human respiratory samples.

Animals↗

Specificity and usefulness of an IgE immunosorbent agglutination assay for toxoplasmosis.

AIMS: To develop an immunosorbent agglutination assay for the detection of Toxoplasma gondii IgE antibodies (IgE-ISAGA); to assess its specificity; and to determine the role of specific IgE in the diagnosis of current toxoplasma infection. METHODS: Rabbit antihuman IgE capture antibody was adsorbed onto microtitre plates and formaldehyde fixed tachyzoites were used to identify specific antibody. Specificity was assessed in 513 serum samples (blood donor, potentially interfering and difficult, elevated and low total IgE and myeloma). Serum samples (n = 108) from 65 patients with documented toxoplasmosis were tested, as were 26 serum samples from nine pregnant women positive for specific IgM and 30 from 20 HIV positive patients. RESULTS: IgE-ISAGA was highly specific with only three of 513 (0.58%) positive reactions amongst the control groups, one of which (0.19%) was regarded as a false positive. Elevated total IgE did not influence specific IgE results nor did the presence of abnormal immunoglobulin concentrations. Sixty (92.3%) patients with toxoplasma associated lymphadenopathy had specific IgE in one or more samples. Positive or borderline results were obtained in 68 of 77 (88.3%) serum samples taken up to four months after onset and were also detected for up to 11 months in 21 of 31 (67.7%) sera. Of the nine pregnant women with detectable specific IgM, specific IgE was absent in five (12 specimens). Specific IgE was also detected in 10 of 30 (33.3%) serum samples from the 20 HIV positive patients, which was similar to the number with specific IgM. Neither the specific IgE nor IgM tests could distinguish symptomatic from asymptomatic HIV positive patients. CONCLUSIONS: IgE-ISAGA is sensitive, specific, and easy to perform. Although results suggest that specific IgE may be less helpful than previously claimed, specific IgE has a useful role in the diagnosis of current toxoplasma infection when used in conjunction with other tests.

AIDS-Related Opportunistic Infections↗

The role of a nested polymerase chain reaction in the diagnosis of Pneumocystis carinii pneumonia.

Aim-To compare the techniques and results of a nested PCR and an immunofluorescence assay (IFA) for the detection of Pneumocystis carinii infection; to consider the role of the nested PCR in the diagnosis of P carinii pneumonia (PCP).Methods-Serial dilutions of two known P carinii positive samples were tested by IFA and PCR to determine their relative sensitivities. Seventy eight respiratory samples (15 from 11 patients with HIV infection/acquired immunodeficiency syndrome (AIDS) and 63 from 42 patients with other forms of immunodeficiency) were tested using both assays, and the costs and technical requirements of each assay were assessed.Results-The PCR had a greater relative sensitivity over the IFA of 2 x 10(1) to 2 x 10(3) fold in a postmortem lung sample and 2 x 10(5) to 2 x 10(6) fold in a bronchoalveolar lavage sample from a patient with PCP. P carinii was detected in all 15 samples from the patients with HIV/AIDS by both IFA and PCR. Of the 63 samples from the patients with immunodeficiencies other than HIV/AIDS, the PCR was more sensitive than IFA.Conclusions-The nested PCR is a more sensitive assay than the IFA. It may be useful in the diagnosis of PCP in patients with immunodeficiencies other than HIV/AIDS. Similarly, PCR may be of benefit for this patient group as less invasive specimens are needed. PCR has an increasing role to play in the diagnosis of PCP in the routine laboratory.

Journal Article↗