We report a case of HGE in a patient with newly diagnosed CML in the chronic phase. The diagnosis of HGE was confirmed by the recovery of A phagocytophilum
in culture from blood and bone marrow in the HL-60 cell line, positive PCR on blood, and seroconversion. This patient presented with multiorgan failure and many of the clinical manifestations of severe ehrlichial infection previously reported.14–17
Because of the underlying CML, the patient did not present with the thrombocytopenia and leucopenia characteristic of HGE. This patient shows that given the proper epidemiological setting, the presence of leucocytosis should not exclude HGE infection from the differential diagnosis of an acute febrile illness.18
HL-60 is the cell line most frequently used for the cultivation of A phagocytophilum
The observation that A phagocytophilum
could be propagated in vitro in the patient’s own leukaemic cells was unanticipated. Under normal conditions of A phagocytophilum
culture, patients’ leucocytes do not survive for more than a few days, and become overgrown by the HL-60 cells (unpublished observations, 1998). This patient’s cells survived until day 10 of culture, and probably beyond, in culture; furthermore, his cells supported infection earlier than did the HL-60 cells, both in the initial diagnostic culture and later in the in vitro experimental cultures. It is likely that the prolonged survival of this patient’s cells was a result of their malignant nature. The higher rate of infection in the patient’s leukaemic cells than in HL-60 cells in the original coculture might have been the result of the development of visible inclusions in cells originally infected in vivo, but this explanation could not account for the same observations made in the in vitro experiments. Our findings support the notion of the preferential infection of mature granulocytes by A phagocytophilum
In agreement with previous reports,20
control cells from a healthy donor did not survive in culture, whereas our patient’s cells were still viable at day 10. Although Yoshiie et al
were able to infect normal donor granulocytes in vitro, they used cell free organisms and they examined the cells every few hours for up to 96 hours.21
Our experiment, using infected HL-60 cells that contained mostly intracellular organisms, might have required a longer incubation period to initiate (or transmit) infection, and our healthy donor cells did not survive long enough.
Analogous to our findings, Klein et al
have shown that bone marrow progenitor cells (CD34+, HLA-DR+) were more susceptible in vitro to A phagocytophilum
infection after they were allowed to differentiate to the myelomonocytic pathway for four to five days in culture.22
Cells with increased granularity were more susceptible to infection, whereas undifferentiated cells were less susceptible. Whether A phagocytophilum
infected cells in peripheral blood are derived from the infection of progenitor cells in the bone marrow or from the infection of granulocytes in peripheral locations is currently unknown. Information regarding infection of bone marrow cells in vivo is scanty, because bone marrow is not normally sampled in patients with HGE. Our patient’s bone marrow was minimally infected, if at all, based on the negative PCR result and the delayed detection of culture positivity. The infected cells seen in the bone marrow smear could have been the result of contamination with peripheral blood. It could also be speculated that the apparent low infection rate of bone marrow results from the presence of only a small fraction of cells susceptible to infection out of the much larger heterogeneous cell population in bone marrow.
“The promyelocytic HL-60 cell line might not be the ideal cell line for the culture of Anaplasma phagocytophilum, but currently a more mature granulocytic cell line is not available”
Several factors could contribute to successful infection and propagation of A phagocytophilum
within the short lived peripheral blood granulocyte pool. In vitro and in vivo studies have shown that there is a reduction in apoptosis of infected neutrophils.21,23
It has also been reported that infected granulocytes lose PSGL-1 and L selectin, thus decreasing their binding to endothelial cells,24
and this may prolong the time that the granulocytes are present in blood. In addition, preliminary studies in our laboratory indicate that the replication time of A phagocytophilum
in vitro may be as short as three hours (unpublished data, 2003).
The reason for preferential infection of later stages of the myeloid series is unclear. It might be speculated that different myeloid stages differentially express ligands necessary for the binding or internalisation of the organism. Cells from a bone marrow sample showed a slower rate of expansion of A phagocytophilum infection than did the peripheral blood, suggesting that peripheral blood may be the principal site of infection and/or that the bone marrow milieu is less conducive for propagation of infection. Thus, the promyelocytic HL-60 cell line might not be the ideal cell line for the culture of A phagocytophilum, but currently a more mature granulocytic cell line is not available. Leukaemic cells induced to differentiate may be more suitable for culturing A phagocytophilum in vitro.
Take home messages
- The occurrence of human granulocytic ehrlichiosis (HGE) in a patient with chronic myelogenous leukaemia (CML) enabled us to study whether Anaplasma phagocytophilum infects mature or immature cells
- Anaplasma phagocytophilum seems to infect mature granulocytes preferentially: the organism grew better in CML cells than in HL-60 cells, there was a higher rate of infection in the patient’s mature cells than in immature granulocytes before treatment, and there was a minimal level of infection of the patient’s bone marrow
- The primary site of infection in HGE may be the peripheral mature granulocytic population