- Email: [email protected]
Direct detection of Anaplasma phagocytophilum by polymerase chain reaction followed by electrospray ionization mass spectrometry from human blood
International Journal of Infectious Diseases 60 (2017) 61–63
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Case Report
Direct detection of Anaplasma phagocytophilum by polymerase chain reaction followed by electrospray ionization mass spectrometry from human blood Heimo Laglera , Nicole Harrisona , Manuel Kussmanna , Markus Obermüllera , Heinz Burgmanna , Athanasios Makristathisb , Michael Ramhartera,c,* a
Department of Medicine 1, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna, Austria Department of Laboratory Medicine, Division of Clinical Microbiology, Medical University of Vienna, Vienna, Austria c Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Tübingen, Germany b
A R T I C L E I N F O
Article history: Received 28 February 2017 Received in revised form 5 May 2017 Accepted 10 May 2017 Corresponding Editor: Eskild Petersen, Aarhus, Denmark Keywords: Tick-borne disease Anaplasma phagocytophilum HGA IRIDICA PCR/ESI-MS
S U M M A R Y
Bacterial pathogens not detectable via commercial blood culture assays represent an important challenge for infectious disease physicians, in particular if clinical symptoms of the illness are non-specific. In this report, Anaplasma phagocytophilum was detected directly in a peripheral blood sample from a febrile patient reporting a tick bite. This was done using a commercial system based on PCR followed by electrospray ionization mass spectrometry (ESI-MS). The diagnosis of a human granulocytic anaplasmosis infection was established using this diagnostic methodology for the first time. Human granulocytic anaplasmosis is a neglected zoonotic disease in Europe. Its seroprevalence is similar in North America and Europe, but in contrast to the USA, it is rarely diagnosed in the old world. PCR followed by ESI-MS is a novel, complex, but highly promising diagnostic methodology for the rapid assessment of rare or exotic pathogens, including intracellular bacteria. © 2017 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
Case report In April 2016, a 38-year-old man without significant medical history presented to the outpatient clinic with symptoms of a flulike illness, including low-grade fever, malaise, headache, arthralgia, and myalgia. He had not travelled abroad recently, but mentioned frequent recreational outdoor activities. He recalled having been bitten by a tick 9 days before the onset of symptoms. He reported an uneventful removal of the tick and receiving a subsequent booster vaccination for tick-borne encephalitis virus by his general practitioner 1 week after the incident. No abnormal findings were observed on physical examination; in particular, there was no apparent rash on the skin. Clinical chemistry and haematology results showed a normal differential blood count and a moderately increased C-reactive protein (1.45 mg/dl; normal range <0.5 mg/dl) and creatine kinase
* Corresponding author at: Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstrasse 27, Tübingen 72074, Germany. Tel.: +49 7071 29 82191; fax: +49 7071/29 46 84. E-mail address: [email protected] (M. Ramharter).
(265 U/l; normal range <190 U/l). For further microbiological work-up, blood cultures and commercial nucleic acid amplification testing (NAAT) (SeptiFAST; Roche Diagnostics, Risch-Rotkreuz, Switzerland) were performed for bacterial pathogens; serology and NAAT were ordered for the detection of viral pathogens. In addition, a serological analysis was performed for rare bacterial pathogens including Borrelia burgdorferi, Brucella spp, Coxiella burnetii, and Anaplasma phagocytophilum. Finally, a novel commercial PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) (IRIDICA BAC BSI assay; Abbott Diagnostics, Lake Forest, IL, USA) of ethylenediaminetetraacetic acid (EDTA) peripheral blood was conducted (Metzgar et al., 2016). Awaiting the test results and due to the clinical picture and considerable distress, empiric antibiotic treatment for a suspected bacterial tick-borne disease was prescribed, consisting of doxycycline 300 mg once daily, and a follow-up visit was scheduled 3 days later. On follow-up the patient was afebrile and reported a rapid resolution of the clinical symptoms. Microbiological test results showed negative blood cultures, SeptiFAST, and NAAT testing for viral pathogens, and serology did not indicate an elevated antibody titre at initial presentation. However, the PCR/ESI-MS demonstrated the presence of A. phagocytophilum DNA in peripheral blood on
http://dx.doi.org/10.1016/j.ijid.2017.05.006 1201-9712/© 2017 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
62
H. Lagler et al. / International Journal of Infectious Diseases 60 (2017) 61–63
the same day. Based on this finding, the diagnosis of human granulocytic anaplasmosis (HGA) was established and treatment with doxycycline was continued for a total of 14 days. The PCR/ESI-MS results were then confirmed by Anaplasmataceae-specific nested PCR (Li et al., 2012) on DNA extracted from EDTA blood (QIAamp DNA Mini Kit; Qiagen, Hilden, Germany), followed by bidirectional sequencing (MWG Eurofins, Munich, Germany) and nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) with known sequences in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih. gov). The sequence of the 344-bp amplicon showed 100% homology with various A. phagocytophilum strains (for instance GenBank accession number AY776165.1). Interestingly, follow-up blood samples did not show an increase in antibody titres 1:64 or higher against A. phagocytophilum using an indirect immunofluorescence antibody assay (Anaplasma phagocytophilum IFA IgG; Focus Diagnostics, Cypress, CA, USA) (Chapman et al., 2006). Discussion The obligate intracellular pathogen A. phagocytophilum (previously known as Ehrlichia phagocytophila or Ehrlichia equi) has been known as a veterinary agent since 1932, but has only been recognized as a human pathogen since the 1990s (Dumler et al., 2005). The first human case was reported in Wisconsin, USA, where the patient died 2 weeks after a tick bite and subsequent severe febrile illness, indicating the life-threatening potential of the agent (Chen et al., 1994). A. phagocytophilum mostly infects neutrophils, and seroprevalence rates in humans are similar in the USA and Europe (Blanco and Oteo, 2002). However, in contrast to the USA, symptomatic HGA infections are rarely diagnosed in Europe (Dumler et al., 2005). For instance, only a few diagnosed cases have been reported in Austria in the last 15 years (Vogl et al., 2010; Walder et al., 2006; Haschke-Becher et al., 2010; Markowicz et al., 2016). It may be speculated that HGA is associated with fewer symptoms in patients infected in Europe compared to patients infected with strains circulating in the USA, leading to fewer confirmed cases in the clinical setting. The symptoms of this acute illness after a recent tick bite are non-specific. A meta-analysis of clinical manifestations in patients with HGA in North America and Europe showed malaise (94%), fever (92%), myalgia (77%), and headache (75%) to be the main symptoms in general. Interestingly, malaise was reported significantly less frequently by infected patients in Europe than by those in North America (47% versus 96%) (Dumler et al., 2005). This might be an indication as to why the illness has remained unrecognized in Europe. Importantly, A. phagocytophilum does not grow in standard blood cultures and is commonly not covered by commercial NAAT systems. Specific NAATs are not yet standardized for HGA, but are recommended for suspected A. phagocytophilum infection prior to the initiation of antimicrobial therapy (Wormser et al., 2006). It is important to determine which specific NAAT is used, as previous studies have estimated a sensitivity of 60–70% (Bakken et al., 2001), whereas more recent reports have indicated a sensitivity of up to 100% (Bell and Patel, 2005; Brouqui et al., 2004). Blood smears were not tested; however it is well known that only up to 60% of these are positive in patients with HGA (Bakken and Dumler, 2000). A. phagocytophilum-specific serology (IFA) can help in establishing the diagnosis retrospectively after 1 month antimicrobial therapy was started. Even though the IFA has a sensitivity estimated at 99.2%, its diagnostic accuracy may be limited if adequate treatment is initiated within a certain period of time after the tick bite. Thus, the fact that adequate antimicrobial therapy was
administered within 3 days of symptom onset in the case presented here may explain why no seroconversion was observed in this case (Bakken et al., 2002). PCR/ESI-MS is a novel molecular assay that is able to detect more than 750 bacterial pathogens in a reasonable turnaround time, thereby making results available directly from whole blood specimens on the same day (Wolk et al., 2012). An observational study on critically ill patients showed that in bloodstream infections with cultivatable bacteria, the PCR/ESI-MS assay yielded a sensitivity of 81%, specificity of 69%, and a negative predictive value of 97% when compared to blood culture, at 6 h from sample acquisition (Vincent et al., 2015). By introducing a new specimen preparation technique and replicate testing, the sensitivity was increased to 91% and the specificity to 99% (Bacconi et al., 2014). Assessment of the diagnostic accuracy for pathogens of the Anaplasmataceae family, which are typically not detectable in commercially available blood culture systems, is not yet available. However, PCR/ESI-MS seems to be particularly helpful for the diagnostic workup of patients with potentially rare or exotic pathogens, as it allows direct pathogen detection without the need for multiple specific NAAT protocols (Metzgar et al., 2016). Unfortunately, the availability of PCR/ESI-MS has remained limited due to the considerable costs associated with this diagnostic methodology (Wolk et al., 2012). A. phagocytophilum was detected in this patient by PCR/ESI-MS, representing the first such detection. Furthermore this was the only diagnostic assay that detected the underlying pathogen, which it did in a reasonable detection time. This method was therefore paramount for establishing the correct diagnosis. The technique holds the potential to greatly improve the turnaround time and diagnostic capacity for patients with rare and exotic bacterial infections. Funding This work was supported financially by the Karl Landsteiner Gesellschaft (Austria). We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing fund of University Tuebingen (Germany). Conflict of interest The authors have no conflicts of interest to declare. References Bacconi A, Richmond GS, Baroldi MA, Laffler TG, Blyn LB, Carolan HE, et al. Improved sensitivity for molecular detection of bacterial and Candida infections in blood. J Clin Microbiol 2014;52:3164–74. Bakken JS, Dumler JS. Human granulocytic ehrlichiosis. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2000;31:554–60. Bakken JS, Aguero-Rosenfeld ME, Tilden RL, Wormser GP, Horowitz HW, Raffalli JT, et al. Serial measurements of hematologic counts during the active phase of human granulocytic ehrlichiosis. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2001;32:862–70. Bakken JS, Haller I, Riddell D, Walls JJ, Dumler JS. The serological response of patients infected with the agent of human granulocytic ehrlichiosis. Clinical infectious diseases an official publication of the Infectious Diseases Society of America 2002;34:22–7. Bell CA, Patel R. A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Diagn Microbiol Infect Dis 2005;53:301–6. Blanco JR, Oteo JA. Human granulocytic ehrlichiosis in Europe. Clin Microbiol Infect 2002;8:763–72. Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoersdorff A, Blanco JR, et al. Guidelines for the diagnosis of tick-borne bacterial diseases in Europe. Clin Microbiol Infect 2004;10:1108–32. Chapman AS, Bakken JS, Folk SM, Paddock CD, Bloch KC, Krusell A, et al. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever, ehrlichioses, and anaplasmosis–United States: a practical guide for
H. Lagler et al. / International Journal of Infectious Diseases 60 (2017) 61–63 physicians and other health-care and public health professionals. MMWR Recomm Rep 2006;55:1–27. Chen SM, Dumler JS, Bakken JS, Walker DH. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol 1994;32:589–95. Dumler JS, Choi KS, Garcia-Garcia JC, Barat NS, Scorpio DG, Garyu JW, et al. Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg Infect Dis 2005;11:1828–34. Haschke-Becher E, Bernauer R, Walleczek AM, Apfalter P, Afazel-Saeedi S, Kraus J, et al. First detection of the Anaplasma phagocytophilum groEL-A genotype in man. J Infect 2010;60:300–5. Li H, Jiang JF, Liu W, Zheng YC, Huo QB, Tang K, et al. Human infection with Candidatus Neoehrlichia mikurensis, China. Emerg Infect Dis 2012;18: 1636–9. Markowicz M, Schotta AM, Wijnveld M, Stanek G. Human granulocytic anaplasmosis acquired in Connecticut, USA, diagnosed in Vienna, Austria, 2015. Diagn Microbiol Infect Dis 2016;84:347–9. Metzgar D, Frinder MW, Rothman RE, Peterson S, Carroll KC, Zhang SX, et al. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood. PLoS One 2016;11:e0158186.
63
Vincent JL, Brealey D, Libert N, Abidi NE, O’Dwyer M, Zacharowski K, et al. Rapid Diagnosis of Infections in the Critically Ill T. Rapid Diagnosis of Infection in the Critically Ill, a Multicenter Study of Molecular Detection in Bloodstream Infections, Pneumonia, and Sterile Site Infections. Crit Care Med 2015;43:2283– 91. Vogl UM, Presterl E, Stanek G, Ramharter M, Gattringer KB, Graninger W. First described case of human granulocytic anaplasmosis in a patient in Eastern Austria. Wien Med Wochenschr 2010;160:91–3. Walder G, Fuchs D, Sarcletti M, Berek K, Falkensammer B, Huber K, et al. Human granulocytic anaplasmosis in Austria: epidemiological, clinical, and laboratory findings in five consecutive patients from Tyrol, Austria. Int J Med Microbiol 2006;296(Suppl 40):297–301. Wolk DM, Kaleta EJ, Wysocki VH. PCR-electrospray ionization mass spectrometry: the potential to change infectious disease diagnostics in clinical and public health laboratories. J Mol Diagn 2012;14:295–304. Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, et al. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2006;43:1089–134.
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Case Report
Direct detection of Anaplasma phagocytophilum by polymerase chain reaction followed by electrospray ionization mass spectrometry from human blood Heimo Laglera , Nicole Harrisona , Manuel Kussmanna , Markus Obermüllera , Heinz Burgmanna , Athanasios Makristathisb , Michael Ramhartera,c,* a
Department of Medicine 1, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna, Austria Department of Laboratory Medicine, Division of Clinical Microbiology, Medical University of Vienna, Vienna, Austria c Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Tübingen, Germany b
A R T I C L E I N F O
Article history: Received 28 February 2017 Received in revised form 5 May 2017 Accepted 10 May 2017 Corresponding Editor: Eskild Petersen, Aarhus, Denmark Keywords: Tick-borne disease Anaplasma phagocytophilum HGA IRIDICA PCR/ESI-MS
S U M M A R Y
Bacterial pathogens not detectable via commercial blood culture assays represent an important challenge for infectious disease physicians, in particular if clinical symptoms of the illness are non-specific. In this report, Anaplasma phagocytophilum was detected directly in a peripheral blood sample from a febrile patient reporting a tick bite. This was done using a commercial system based on PCR followed by electrospray ionization mass spectrometry (ESI-MS). The diagnosis of a human granulocytic anaplasmosis infection was established using this diagnostic methodology for the first time. Human granulocytic anaplasmosis is a neglected zoonotic disease in Europe. Its seroprevalence is similar in North America and Europe, but in contrast to the USA, it is rarely diagnosed in the old world. PCR followed by ESI-MS is a novel, complex, but highly promising diagnostic methodology for the rapid assessment of rare or exotic pathogens, including intracellular bacteria. © 2017 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
Case report In April 2016, a 38-year-old man without significant medical history presented to the outpatient clinic with symptoms of a flulike illness, including low-grade fever, malaise, headache, arthralgia, and myalgia. He had not travelled abroad recently, but mentioned frequent recreational outdoor activities. He recalled having been bitten by a tick 9 days before the onset of symptoms. He reported an uneventful removal of the tick and receiving a subsequent booster vaccination for tick-borne encephalitis virus by his general practitioner 1 week after the incident. No abnormal findings were observed on physical examination; in particular, there was no apparent rash on the skin. Clinical chemistry and haematology results showed a normal differential blood count and a moderately increased C-reactive protein (1.45 mg/dl; normal range <0.5 mg/dl) and creatine kinase
* Corresponding author at: Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstrasse 27, Tübingen 72074, Germany. Tel.: +49 7071 29 82191; fax: +49 7071/29 46 84. E-mail address: [email protected] (M. Ramharter).
(265 U/l; normal range <190 U/l). For further microbiological work-up, blood cultures and commercial nucleic acid amplification testing (NAAT) (SeptiFAST; Roche Diagnostics, Risch-Rotkreuz, Switzerland) were performed for bacterial pathogens; serology and NAAT were ordered for the detection of viral pathogens. In addition, a serological analysis was performed for rare bacterial pathogens including Borrelia burgdorferi, Brucella spp, Coxiella burnetii, and Anaplasma phagocytophilum. Finally, a novel commercial PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) (IRIDICA BAC BSI assay; Abbott Diagnostics, Lake Forest, IL, USA) of ethylenediaminetetraacetic acid (EDTA) peripheral blood was conducted (Metzgar et al., 2016). Awaiting the test results and due to the clinical picture and considerable distress, empiric antibiotic treatment for a suspected bacterial tick-borne disease was prescribed, consisting of doxycycline 300 mg once daily, and a follow-up visit was scheduled 3 days later. On follow-up the patient was afebrile and reported a rapid resolution of the clinical symptoms. Microbiological test results showed negative blood cultures, SeptiFAST, and NAAT testing for viral pathogens, and serology did not indicate an elevated antibody titre at initial presentation. However, the PCR/ESI-MS demonstrated the presence of A. phagocytophilum DNA in peripheral blood on
http://dx.doi.org/10.1016/j.ijid.2017.05.006 1201-9712/© 2017 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
62
H. Lagler et al. / International Journal of Infectious Diseases 60 (2017) 61–63
the same day. Based on this finding, the diagnosis of human granulocytic anaplasmosis (HGA) was established and treatment with doxycycline was continued for a total of 14 days. The PCR/ESI-MS results were then confirmed by Anaplasmataceae-specific nested PCR (Li et al., 2012) on DNA extracted from EDTA blood (QIAamp DNA Mini Kit; Qiagen, Hilden, Germany), followed by bidirectional sequencing (MWG Eurofins, Munich, Germany) and nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) with known sequences in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih. gov). The sequence of the 344-bp amplicon showed 100% homology with various A. phagocytophilum strains (for instance GenBank accession number AY776165.1). Interestingly, follow-up blood samples did not show an increase in antibody titres 1:64 or higher against A. phagocytophilum using an indirect immunofluorescence antibody assay (Anaplasma phagocytophilum IFA IgG; Focus Diagnostics, Cypress, CA, USA) (Chapman et al., 2006). Discussion The obligate intracellular pathogen A. phagocytophilum (previously known as Ehrlichia phagocytophila or Ehrlichia equi) has been known as a veterinary agent since 1932, but has only been recognized as a human pathogen since the 1990s (Dumler et al., 2005). The first human case was reported in Wisconsin, USA, where the patient died 2 weeks after a tick bite and subsequent severe febrile illness, indicating the life-threatening potential of the agent (Chen et al., 1994). A. phagocytophilum mostly infects neutrophils, and seroprevalence rates in humans are similar in the USA and Europe (Blanco and Oteo, 2002). However, in contrast to the USA, symptomatic HGA infections are rarely diagnosed in Europe (Dumler et al., 2005). For instance, only a few diagnosed cases have been reported in Austria in the last 15 years (Vogl et al., 2010; Walder et al., 2006; Haschke-Becher et al., 2010; Markowicz et al., 2016). It may be speculated that HGA is associated with fewer symptoms in patients infected in Europe compared to patients infected with strains circulating in the USA, leading to fewer confirmed cases in the clinical setting. The symptoms of this acute illness after a recent tick bite are non-specific. A meta-analysis of clinical manifestations in patients with HGA in North America and Europe showed malaise (94%), fever (92%), myalgia (77%), and headache (75%) to be the main symptoms in general. Interestingly, malaise was reported significantly less frequently by infected patients in Europe than by those in North America (47% versus 96%) (Dumler et al., 2005). This might be an indication as to why the illness has remained unrecognized in Europe. Importantly, A. phagocytophilum does not grow in standard blood cultures and is commonly not covered by commercial NAAT systems. Specific NAATs are not yet standardized for HGA, but are recommended for suspected A. phagocytophilum infection prior to the initiation of antimicrobial therapy (Wormser et al., 2006). It is important to determine which specific NAAT is used, as previous studies have estimated a sensitivity of 60–70% (Bakken et al., 2001), whereas more recent reports have indicated a sensitivity of up to 100% (Bell and Patel, 2005; Brouqui et al., 2004). Blood smears were not tested; however it is well known that only up to 60% of these are positive in patients with HGA (Bakken and Dumler, 2000). A. phagocytophilum-specific serology (IFA) can help in establishing the diagnosis retrospectively after 1 month antimicrobial therapy was started. Even though the IFA has a sensitivity estimated at 99.2%, its diagnostic accuracy may be limited if adequate treatment is initiated within a certain period of time after the tick bite. Thus, the fact that adequate antimicrobial therapy was
administered within 3 days of symptom onset in the case presented here may explain why no seroconversion was observed in this case (Bakken et al., 2002). PCR/ESI-MS is a novel molecular assay that is able to detect more than 750 bacterial pathogens in a reasonable turnaround time, thereby making results available directly from whole blood specimens on the same day (Wolk et al., 2012). An observational study on critically ill patients showed that in bloodstream infections with cultivatable bacteria, the PCR/ESI-MS assay yielded a sensitivity of 81%, specificity of 69%, and a negative predictive value of 97% when compared to blood culture, at 6 h from sample acquisition (Vincent et al., 2015). By introducing a new specimen preparation technique and replicate testing, the sensitivity was increased to 91% and the specificity to 99% (Bacconi et al., 2014). Assessment of the diagnostic accuracy for pathogens of the Anaplasmataceae family, which are typically not detectable in commercially available blood culture systems, is not yet available. However, PCR/ESI-MS seems to be particularly helpful for the diagnostic workup of patients with potentially rare or exotic pathogens, as it allows direct pathogen detection without the need for multiple specific NAAT protocols (Metzgar et al., 2016). Unfortunately, the availability of PCR/ESI-MS has remained limited due to the considerable costs associated with this diagnostic methodology (Wolk et al., 2012). A. phagocytophilum was detected in this patient by PCR/ESI-MS, representing the first such detection. Furthermore this was the only diagnostic assay that detected the underlying pathogen, which it did in a reasonable detection time. This method was therefore paramount for establishing the correct diagnosis. The technique holds the potential to greatly improve the turnaround time and diagnostic capacity for patients with rare and exotic bacterial infections. Funding This work was supported financially by the Karl Landsteiner Gesellschaft (Austria). We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing fund of University Tuebingen (Germany). Conflict of interest The authors have no conflicts of interest to declare. References Bacconi A, Richmond GS, Baroldi MA, Laffler TG, Blyn LB, Carolan HE, et al. Improved sensitivity for molecular detection of bacterial and Candida infections in blood. J Clin Microbiol 2014;52:3164–74. Bakken JS, Dumler JS. Human granulocytic ehrlichiosis. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2000;31:554–60. Bakken JS, Aguero-Rosenfeld ME, Tilden RL, Wormser GP, Horowitz HW, Raffalli JT, et al. Serial measurements of hematologic counts during the active phase of human granulocytic ehrlichiosis. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2001;32:862–70. Bakken JS, Haller I, Riddell D, Walls JJ, Dumler JS. The serological response of patients infected with the agent of human granulocytic ehrlichiosis. Clinical infectious diseases an official publication of the Infectious Diseases Society of America 2002;34:22–7. Bell CA, Patel R. A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Diagn Microbiol Infect Dis 2005;53:301–6. Blanco JR, Oteo JA. Human granulocytic ehrlichiosis in Europe. Clin Microbiol Infect 2002;8:763–72. Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoersdorff A, Blanco JR, et al. Guidelines for the diagnosis of tick-borne bacterial diseases in Europe. Clin Microbiol Infect 2004;10:1108–32. Chapman AS, Bakken JS, Folk SM, Paddock CD, Bloch KC, Krusell A, et al. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever, ehrlichioses, and anaplasmosis–United States: a practical guide for
H. Lagler et al. / International Journal of Infectious Diseases 60 (2017) 61–63 physicians and other health-care and public health professionals. MMWR Recomm Rep 2006;55:1–27. Chen SM, Dumler JS, Bakken JS, Walker DH. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol 1994;32:589–95. Dumler JS, Choi KS, Garcia-Garcia JC, Barat NS, Scorpio DG, Garyu JW, et al. Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg Infect Dis 2005;11:1828–34. Haschke-Becher E, Bernauer R, Walleczek AM, Apfalter P, Afazel-Saeedi S, Kraus J, et al. First detection of the Anaplasma phagocytophilum groEL-A genotype in man. J Infect 2010;60:300–5. Li H, Jiang JF, Liu W, Zheng YC, Huo QB, Tang K, et al. Human infection with Candidatus Neoehrlichia mikurensis, China. Emerg Infect Dis 2012;18: 1636–9. Markowicz M, Schotta AM, Wijnveld M, Stanek G. Human granulocytic anaplasmosis acquired in Connecticut, USA, diagnosed in Vienna, Austria, 2015. Diagn Microbiol Infect Dis 2016;84:347–9. Metzgar D, Frinder MW, Rothman RE, Peterson S, Carroll KC, Zhang SX, et al. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood. PLoS One 2016;11:e0158186.
63
Vincent JL, Brealey D, Libert N, Abidi NE, O’Dwyer M, Zacharowski K, et al. Rapid Diagnosis of Infections in the Critically Ill T. Rapid Diagnosis of Infection in the Critically Ill, a Multicenter Study of Molecular Detection in Bloodstream Infections, Pneumonia, and Sterile Site Infections. Crit Care Med 2015;43:2283– 91. Vogl UM, Presterl E, Stanek G, Ramharter M, Gattringer KB, Graninger W. First described case of human granulocytic anaplasmosis in a patient in Eastern Austria. Wien Med Wochenschr 2010;160:91–3. Walder G, Fuchs D, Sarcletti M, Berek K, Falkensammer B, Huber K, et al. Human granulocytic anaplasmosis in Austria: epidemiological, clinical, and laboratory findings in five consecutive patients from Tyrol, Austria. Int J Med Microbiol 2006;296(Suppl 40):297–301. Wolk DM, Kaleta EJ, Wysocki VH. PCR-electrospray ionization mass spectrometry: the potential to change infectious disease diagnostics in clinical and public health laboratories. J Mol Diagn 2012;14:295–304. Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, et al. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2006;43:1089–134.