D. O. Ho-Yen

Primary cutaneous B-cell lymphoma and Borrelia burgdorferi infection in patients from the Highlands of Scotland.

Although a link beteen primary cutaneous B-cell lymphoma (PCBCL) and Borrelia burgdorferi infection has long been suspected, previous studies have not demonstrated a significant association. The authors looked for evidence of B. burgdorferi in 20 cases of PCBCL from the Scottish Highlands, an area with endemic Lyme disease, and compared their findings with those in 40 control patients (20 undergoing wide reexcision at sites of malignant melanoma and 20 biopsies of inflammatory dermatoses). All studies were performed on formalin-fixed, paraffin-embedded tissues. The cases of PCBCL were classified according to criteria described by the European Organization for Research and Treatment of Cancer Cutaneous Lymphoma Project Group using a combination of morphology, immunohistochemistry, and seminested polymerase chain reaction (PCR) for immunoglobulin heavy chain gene rearrangement. A nested PCR was performed on deoxyribonucleic acid (DNA) extracts from the lymphoma and control cases using primers to a unique conserved region of the B. burgdorferi flagellin gene. B. burgdorferi-specific DNA was detected in seven of 20 lymphoma cases (five of 12 marginal zone lymphomas, one of five primary cutaneous follicle center cell lymphomas, one of three diffuse, large B-cell lymphomas of the leg) and in one melanoma reexcision patient of 40 control subjects. The relationship between B. burgdorferi and PCBCL was significant when compared with the control groups separately (p <0.05) or in combination (p <0.01). These results provide strong evidence to support the concept of B. burgdorferi-driven lymphomagenesis in the skin.

Borrelia burgdorferi-associated cutaneous marginal zone lymphoma: a clinicopathological study of two cases illustrating the temporal progression of B. burgdorferi-associated B-cell proliferation in the skin.

A relationship between Borrelia burgdorferi and primary cutaneous B‐cell lymphoma (PCBCL) has recently been confirmed following demonstration of the organism in lesional skin of patients with PCBCL. We report herein two cases of B. burgdorferi‐associated PCBCL which strengthen this association by demonstrating the organism in cutaneous B‐cell infiltrates present at sites in which PCBCL subsequently developed.

Simultaneous Diagnosis of Toxoplasmosis in Goats and Goatowner's Family

Four out of 6 goats from a small British goat herd gave birth to weak or stillborn kids. All were seropositive for Toxoplasma gondii. The parasite was isolated from the tissues of one kid and the milk of one goat. Concurrently, one of the goatowners sons developed a mononucleosis-like illness with serological evidence of current toxoplasma infection. Investigation of the family showed past infection in the other son, but both parents were seronegative. The source of infection for both boys appeared to be the consumption of unpasteurized goats milk.

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.

Cell-culture system for continuous production of Toxoplasma gondii tachyzoites

Abstract 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 (6.15×106 tachyzoites/ml) than conventional flasks (1–2×106 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 1×106 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.

Toxoplasma polymerase chain reaction on experimental blood samples

A two-stage polymerase chain reaction (PCR) assay employing oligonucleotide primers from the B1 gene of Toxoplasma gondii was developed and assessed for sensitivity and specificity. It was able to detect T. gondii DNA from as little as one parasite/sample in mock-infected rat or mouse leucocyte preparations. Parasitaemia was also identified in animals at five stages between 16 and 66 h after infection with the virulent RH strain, and at 12 stages between 2 and 38 days after infection with the cyst-forming Beverley strain. In the latter case, PCR was more sensitive than animal culture. No cross-reactions were observed in samples containing various opportunist pathogens which may also be found in the blood of immunocompromised patients.

Morphoea and Borrelia burgdorferi: results from the Scottish Highlands in the context of the world literature

Aims: Previous studies investigating the link between infection with Borrelia burgdorferi and morphoea have produced conflicting results. Often, these studies have been undertaken in patients from different regions or countries, and using methods of varying sensitivity for detecting Borrelia burgdorferi infection. This study aimed to establish whether a relation could be demonstrated in the Highlands of Scotland, an area with endemic Lyme disease, with the use of a sensitive method for detecting the organism. Methods: The study was performed on biopsies of lesional skin taken from 16 patients from the Highlands of Scotland with typical clinical features of morphoea. After histological confirmation of the diagnosis, a nested polymerase chain reaction (PCR) using primers to a unique conserved region of the Borrelia burgdorferi flagellin gene was performed on DNA extracts from each biopsy. A literature search was also performed for comparable studies. Results: None of the 16 patients had documented clinical evidence of previous infection with B burgdorferi. DNA was successfully extracted from 14 of the 16 cases but all of these were negative using PCR for B burgdorferi specific DNA, despite successful amplification of appropriate positive controls in every test. The results were compared with those of other documented studies. Conclusions: Examination of the literature suggests that there is a strong geographical relation between B burgdorferi and morphoea. These results, in which no such association was found, indicate that morphoea may not be associated with the subspecies of B burgdorferi found in the Highlands of Scotland.

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.

Toxoplasma gondii in vitro culture for experimentation

The aim of this study was to develop a culture method for Toxoplasma gondii that could provide fresh viable tachyzoites as and when required. When T. gondii was continuously maintained in HeLa cell cultures at 37 degrees C, the time to harvest varied from 48 to 144 h. Tachyzoite yields of > or = 1 x 10(6)/ml and > or = 90% viable were obtained from 519/882 (58.8%) cultures and 120/155 (77.4%) harvests were successfully used in the dye test. When cultures were transferred from 37 to 25 degrees C when maximally infected (48-54-h post-infection), they could be stabilised and tachyzoites could be harvested as required, up to 168 h later. When harvested from 25 degrees C, significantly more cultures 783/811 (96.5%) produced tachyzoite yields > or = 1 x 10(6)/ml > or = 90% viable (p < 0.001). Tachyzoite quality also significantly improved and 206/224 harvests (91.9%) (p < 0.001) were successfully used in the dye test. We have demonstrated that tachyzoites can be maintained at dye test quality for at least 7 days in HeLa cultures at 25 degrees C. The system is flexible and robust and provides a means whereby tachyzoites of standard quality can be stored for use in experimental models as and when required.

The use of an IgM immunosorbent agglutination assay to diagnose congenital toxoplasmosis

An IgM immunosorbent agglutination assay (ISAGA) was compared with a standard ELISA IgM test for the diagnosis of congenital toxoplasmosis. It was more sensitive, detecting all of five mothers of infected babies whereas the IgM ELISA was positive in two of three mothers tested at delivery and neither of two mothers referred 10-12 months after delivery. Five women infected in a previous pregnancy had IgM detectable by ISAGA in a subsequent pregnancy. The assays were comparable when sera from patients with past infection were tested or following toxoplasma-associated miscarriage or abortion. Four cord sera from congenitally-infected babies were positive by the ISAGA but only three of these were positive by ELISA for IgM. The ISAGA also detected IgM in another four sera from congenitally-infected babies referred late (10-18 months old); none were IgM positive by ELISA. The increased sensitivity of the ISAGA is an improvement in the diagnosis of congenital toxoplasmosis.