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1.  Do Toxoplasma gondii RH strain tachyzoites evolve during continuous passage? 
Journal of Clinical Pathology  2004;57(6):609-611.
Aim: To examine three lineages of Toxoplasma gondii RH strain in terms of performance in the dye test, culture, and gene expression.
Methods: Historical data (culture growth and performance in the dye test) from three lineages of RH strain tachyzoites (B, J, and Q) that had been continuously cultured in HeLa cells was assessed. Tachyzoite harvests obtained during continuous cell culture were retrieved from liquid nitrogen and cultured in HeLa cells, providing mRNA that was extracted and used to study gene expression using random amplified polymorphic DNA analysis at different stages of lineage adaptation to continuous culture.
Results: The B and Q lineages consistently produced tachyzoites that were successfully used in the dye test and their gene expression was stable after multiple passages. The J lineage had unpredictable growth, tachyzoites unsuitable for use in the dye test, and changing gene expression with multiple passage.
Conclusion: This study has explained some anomalies in the performance of different stocks of T gondii, and suggests that lineages that are still evolving in cell culture should be avoided.
doi:10.1136/jcp.2003.013763
PMCID: PMC1770330  PMID: 15166265
random amplified polymorphic DNA analysis; RH strain; Toxoplasma gondii; continuous cell culture; gene expression
2.  Identification of different Borrelia burgdorferi genomic groups from Scottish ticks 
Molecular Pathology  2000;53(2):94-98.
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.
PMCID: PMC1186912  PMID: 10889909
Borrelia burgdorferi; randomly amplified polymorphic DNA analysis; ribosomal RNA gene polymerase chain reaction
3.  Improved serodiagnosis of Lyme disease 
Clinical Molecular Pathology  1996;49(2):M80-M84.
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.
Images
PMCID: PMC408026  PMID: 16696055
Lyme disease; ELISA; immunoblotting; Borrelia burgdorferi
4.  The role of a nested polymerase chain reaction in the diagnosis of Pneumocystis carinii pneumonia 
Clinical Molecular Pathology  1995;48(6):M347-M350.
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 × 101 to 2 × 103 fold in a postmortem lung sample and 2 × 105 to 2 × 106 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.
PMCID: PMC408003  PMID: 16696036
Pneumocystis carinii; PCR; immunofluorescence assay; acquired immunodeficiency syndrome

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