Human Infection with Candidatus Neoehrlichia mikurensis, China

To identify Candidatus Neoehrlichia mikurensis infection in northeastern China, we tested blood samples from 622 febrile patients. We identified in 7 infected patients and natural foci for this bacterium. Field surveys showed that 1.6% of ticks and 3.8% of rodents collected from residences of patients were also infected.

For broad-range assay, a nested PCR specific for the 16S rRNA (rrs) gene was used to detect all known species of the family Anaplasmataceae (Technical Appendix Table). PCR amplifications were performed in a 30-µL reaction volume in GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA).
For initial amplification, the reaction mixture contained 0.8 µmol/L each of primers Eh-out1 (1) and 3-17U, 200 mmol/L of each dNTP, 1 unit of Taq polymerase, 3 µL of 1 PCR buffer, and 3 µL of purified DNA. Cycling conditions were an initial 5-min denaturation at 94°C; 40 cycles at 94°C for 40 s, 55°C for 40 s, and 72°C for 1 min 45 s; and a final extension at 72°C for 7 min.
For nested amplification, the components were similar to those used in the initial amplification, except that 0.5 µmol/L of EHR16SD and 0.5 µmol/L of EHR16SR (2) were used as primers and 1 µL of the primary PCR product was used as template. Cycling conditions were 94°C for 5 min; 35 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s; and a final at 72°C extension for 7 min. Nested amplicons were directly sequenced by using primers EHR16SD and EHR16SR.
For positive samples, 2 heminested PCRs were performed to amplify the entire rrs gene.
Components and conditions in the 2 PCRs were similar to those in the nested PCR, except that Page 2 of 4 primers Eh-out1 and Eh-out2U were used to amplify 5 -end fragments, and primers Eh-out2fU and 3-17U were used to amplify 3 -end fragments. Amplified 5 -end fragments were sequenced by using primer Eh-out2U, and amplified 3 -end fragments were sequenced by using primers Eh-out2fU and CNM1050f.
For confirmation of identification of Candidatus Neoehrlichia mikurensis, a nested PCR specific for the 60-kDa heat shock protein (groEL) gene was performed. Components in the nested PCR were the same as those used in amplification of the rrs gene. Primers HS3-f and HSVR (3) was used for the initial amplification. Primers groEL-2f and groEL-2r were used for nested amplification. Cycling conditions were 94°C for 5 min; 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min 30s; and a final extension at 72°C for 7 min. Nested amplicons were sequenced by using primers groEL-Sf and groEL-Sr.
All positive amplicons were purified by using E.Z.N.A Gel Extraction Kit (Omega Bio-Tek, Norcross, GA, USA). These amplicons were then sequenced by using an automated DNA sequencer (3730 DNA Sequencer; Applied Biosystems).
To minimize risk for contamination, template isolation and PCR were performed by using specified pipettor sets in separate rooms. Certified DNA/RNase-free filter barrier tips were used to prevent aerosol contamination. All PCRs were performed with appropriate controls.

Fresh peripheral blood smears from patients with PCR-confirmed Candidatus
Neoehrlichia mikurensis infection were stained with Wright-Giemsa (BaSO Diagnostics, Inc., Zhuhai, China) and examined with a light microscope (BX43; Olympus, Center Valley, PA, USA) for intracellular morulae.