Volume 19, Number 2—February 2013
Human Granulocytic Anaplasmosis, Japan
|Days after symptom onset†||Nested PCR result‡
|SFG rickettsia 16S rDNA||Orientia tsutsugamushi 16S rDNA||Anaplasma phagocytophilum p44/msp2||Ehrlichia sp. p28/omp-1|
*SFG, spotted fever group; NA, not available.
†After in-hospital treatment with minocycline (200 mg/d), both case-patients improved clinically and were discharged on days 20 and 12, respectively, after symptom onset.
‡Before being used in PCR, blood clots from the patients were homogenized by using BioMasher (Nippi Inc., Tokyo, Japan) and treated overnight with 100 U of streptokinase (WAKO Pure Chemical Industries Ltd, Osaka, Japan). DNA then was extracted by using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA). Multiplex nested first-step PCR for SFG rickettsiae and O. tsutsugamushi was performed by using the following primers: RO-1F (5'-CCGTAAACGATGAGTGCTAGA-3') and RO-R1 (5'-CCGAGAACGTATTCACCGC-3'). Multiplex nested second-step PCR for SFG rickettsiae 16S rDNA was performed by using the following primers: R-2F (5'-GAAGATTCTCTTTCGGTTTCGC-3') and R-2R (5'-GTCTTGCTTCCCTCTGTAAAC-3'). Multiplex nested second-step PCR for O. tsutsugamushi 16S rDNA was performed by using the following primers: O-2F (5'-GACATGGTAGTCGCGAAAAATG-3') and O-2R (5'-TGCAATCCGAACTGAGATACC-3'). A. phagocytophilum p44/msp2 was amplified by using primers p3726, p4257, p3761, and p4183, and Ehrlichia spp. p28/omp-1 was amplified by using primers conP28-F1, conP28-R1, conP28-F2, and conP28-R2, as described (3,9).
1These authors contributed equally to this article.
2Current affiliation: Ehime Prefectural Central Hospital, Matsuyama, Ehime, Japan.
3Current affiliation: Kochi Medical School, Nankoku, Kochi, Japan.