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Our diagnostic criteria for T. whipplei encephalitis required at least two positive PCR assays targeting 2 different sequences on 2 different cerebrospinal fluid specimens, performed as previously reported, or positive PCR assays on brain biopsies [ 16, 17] and negative results PAS staining of gastric and small-bowel specimens, plus an absence of meningitis, myelitis, and other organ involvement.
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Notably, ELISA of single (acute-phase) serum specimens has performed as well or better than MAT or indirect hemagglutination of single serum specimens, so those strategies are not advised (10 ).
Culture and PCR diagnostics of ulcer specimens were performed as described elsewhere (13 ).
Freeze substitution and low-temperature embedding of the specimens was performed as described elsewhere [ 16– 16].
Hematoxylin eosin and Giemsa staining of paraffin-embedded specimens were performed, as were electron microscopic examinations following a conventional method.
Whenever a (pre)malignancy is found in the RRS specimen, RRO will be performed as soon as possible, as well as additional surgery or treatment if necessary, e.g. staging procedure.
Direct examination from specimens with 15% potassium hydroxide was performed, as well as cytological tests with methenamine silver (Grocott) and Periodic acid-Schiff (PAS) staining, in Sabouraud dextrose-agar (SDA) cultures, and SDA with antibiotics (chloramphenicol, 50 mg/L, and cycloheximide, 500 mg/L).
Compressive and flexural strength and mid-span beam deflection capacity testing of sound specimens were performed as part of the mechanical characterization.
Isolation of RNA and quantification of specific mRNAs by real-time PCR in brain specimens was performed as described previously [55].
After washing, immunofluorescent staining of the fixed specimens was performed as described above.
Analysis of the paraffin-embedded specimens was performed as previously described [ 3, 15].
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