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Rickettsia Typhi As Cause of Fatal Encephalitic Typhus in Hospitalized Patients, Hamburg, Germany, 1940–1944 Jessica Rauch, Birgit Muntau, Petra Eggert, Dennis Tappe

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Rickettsia Typhi As Cause of Fatal Encephalitic Typhus in Hospitalized Patients, Hamburg, Germany, 1940–1944 Jessica Rauch, Birgit Muntau, Petra Eggert, Dennis Tappe SYNOPSIS Rickettsia typhi as Cause of Fatal Encephalitic Typhus in Hospitalized Patients, Hamburg, Germany, 1940–1944 Jessica Rauch, Birgit Muntau, Petra Eggert, Dennis Tappe In support of improving patient care, this activity has been planned and implemented by Medscape, LLC and Emerging Infectious Diseases. Medscape, LLC is jointly accredited by the Accreditation Council for Continuing Medical Education (ACCME), the Accreditation Council for Pharmacy Education (ACPE), and the American Nurses Credentialing Center (ANCC), to provide continuing education for the healthcare team. Medscape, LLC designates this Journal-based CME activity for a maximum of 1.00 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity. All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME activity: (1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test with a 75% minimum passing score and complete the evaluation at http://www.medscape.org/journal/eid; and (4) view/print certificate. For CME questions, see page 2139. Release date: October 15, 2018; Expiration date: October 15, 2019 Learning Objectives Upon completion of this activity, participants will be able to: • Describe molecular and other findings related to pathogens identified in archived 73-year-old tissue blocks from patients diagnosed with typhus during World War II • Explain immunohistochemical and histopathological findings in archived 73-year-old tissue blocks from patients diagnosed with typhus during World War II • Determine clinical and public health implications of findings from archived 73-year-old tissue blocks from patients diagnosed with typhus clinically and histopathologically during World War II CME Editor Kristina B. Clark, PhD, Copyeditor, Emerging Infectious Diseases. Disclosure: Kristina B. Clark, PhD, has disclosed no relevant financial relationships. CME Author Laurie Barclay, MD, freelance writer and reviewer, Medscape, LLC. Disclosure: Laurie Barclay, MD, has disclosed the following relevant financial relationships: owns stock, stock options, or bonds from Pfizer. Authors Disclosures: Jessica Rauch, PhD; Birgit Muntau, MSc; Petra Eggert; and Dennis Tappe, MD, have disclosed no relevant financial relationships. We evaluated formalin-fixed paraffin-embedded tissue in Hamburg, Germany, during World War II. The archived specimens from 7 patients who died with encephalitic typhus specimens included only central nervous system tissues >70 years old that had been stored at room temperature. Author affiliation: Bernhard Nocht Institute for Tropical Medicine, We demonstrated successful detection of Rickettsia typhi Hamburg, Germany DNA by a nested qPCR specific toprsA in 2 patients. These results indicate that R. typhi infections contributed to ty- DOI: https://doi.org/10.3201/eid2411.171373 phus outbreaks during World War II. Immunohistochemical 1982 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 24, No. 11, November 2018 Page 1 of 1 R. typhi as Cause of Fatal Encephalitic Typhus analyses of brain tissue specimens of R. typhi DNA–posi- pathology diagnosis and received typhus specimens from tive and –negative specimens showed perivascular B-cell multiple hospitals in Hamburg. We retrieved from the accumulation. Around blood vessels, nodular cell accumu- archives formalin-fixed, paraffin-embedded (FFPE) tis- lations consisted of CD4-positive and CD8-positive T cells sue blocks, which had been stored at room temperature. and CD68-positive microglia and macrophages; neutrophils Clearance by the local ethics committee was obtained (no. were found rarely. These findings are similar to those of pre- WF-034/17) for our analyses. viously reported R. prowazekii tissue specimen testing. Be- cause R. typhi and R. prowazekii infections can be clinically and histopathologically similar, molecular analyses should Histology and Immunohistochemical Analyses be performed to distinguish the 2 pathogens. For each FFPE tissue block, we analyzed a standard he- matoxylin and eosin stained section microscopically for ickettsia typhi infection, also known as murine or en- typhus nodules and documented the presence and numbers Rdemic typhus, is, except for its often milder course, of lesions semiquantitatively. We screened sections for in- clinically indistinguishable from epidemic typhus caused tracellular rickettsiae using Giemsa stains. by R. prowazekii. Infection with R. typhi or R. prowaze- We performed immunohistochemical studies with kii causes a clinical syndrome of high fever, headache, antibodies against CD3 (1:400 dilution; EpitMics, Burl- and rash. The central nervous system (CNS), cardiac, and ingame, CA, USA), CD20 (1:150 dilution; Agilent, Santa pulmonary complications that occur are responsible for fa- Clara, CA, USA), CD4 (1:30 dilution; Cell Marque, Rock- tality rates of 4% for untreated endemic typhus and 30% lin, CA, USA), CD8 (1:20 dilution; Cell Marque), CD68 for epidemic typhus (1–3). Whereas R. typhi is transmitted (1:100 dilution; Agilent), CD177 (1:33 dilution; Zytomed, by fleas (oriental rat flea Xenopsylla cheopis and cat flea Berlin, Germany), and inducible nitric oxide synthase Ctenocephalides felis), R. prowazekii is transmitted by the (iNOS, 1:100 dilution; Zytomed). After pretreatment of human body louse Pediculus humanus corporis. Both of FFPE tissue sections with buffers containing Trilogy (me- these pathogens are obligate intracellular zoonotic bacteria dac diagnostika, Tornesch, Germany; at 95°C for CD177), and Biosafety Level 3 pathogens (1–3); R. prowazekii is EDTA (pH 8 for CD4), or citrate (pH 6 for CD3, CD20, classified as a Centers for Disease Control and Prevention CD8, CD68, and iNOS) and endogenous peroxidase block- category B bioweapon agent. ing, we incubated the sections with the respective antibod- Human infection with these bacteria occurs after in- ies in Antibody Diluent Solution (Zytomed) at 4°C over- oculation of flea or louse feces in the skin lesion caused night. Then, we incubated with either AEC 2-Component by the arthropod bite or by inhalation of dust containing Detection Kit and 3-amino-9-ethylcarbazole substrate dried vector feces. The appearance of epidemic louseborne (DCS, Hamburg, Germany) for immunoperoxidase stain- typhus is often attributed to overcrowding and unhygienic ing or AP Detection Kit and Fast Blue substrate (DCS) for conditions, such as those seen in prisons and refugee, labor, immunophosphatase staining. Brain tissue from 5 patients and concentration camps, and is associated with poverty without encephalitis served as negative controls, and lym- and war worldwide. In contrast, the occurrence of murine phatic tissue served as a positive control for immunologic fleaborne typhus is sporadic and linked to the presence of staining of immune cells. rats, often in coastal subtropical regions. Large epidemics of louseborne typhus occurred during World War I and II, Molecular Assays leading to high fatalities in civilian populations, forced la- We ran samples through 3 rounds of processing: FFPE borers, imprisoned persons, and military personnel. tissue block sectioning, DNA extraction, and quantitative We examined brain tissue samples from persons who PCR (qPCR). For each round, FFPE tissue blocks from ty- had died from typhus in an infectious disease hospital in phus patients and negative control patients (patients with Hamburg, Germany, during World War II. We character- unrelated conditions, e.g., liver amebiasis) were placed in ized R. typhi and R. prowazekii infections by using histo- alternating order (i.e., 2 typhus patient samples, 1 negative logic, immunohistochemical, and molecular techniques. control, 2 typhus patient samples, 1 negative control, and so on), cut into 5-µm slices, and deparaffinized. Before Materials and Methods and after each round of sectioning and between sectioning different samples, we cleaned the microtome and micro- Typhus Cases tome blades with DNA-ExitusPlus (AppliChem, Darm- We identified typhus cases by screening the books of ar- stadt, Germany). We performed DNA extractions with rivals from the Bernhard Nocht Institute Department of tissue sections in the same order as previously mentioned Pathology (Hamburg) for clinical and histopathologic de- using the QIAamp DNA FFPE Tissue Kit (QIAGEN, scriptions of typhus. The Bernhard Nocht Institute Depart- Hilden, Germany). We included 2 additional negative con- ment of Pathology served as a center for infectious disease trols (water) per round of extraction. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 24, No. 11, November 2018 1983 SYNOPSIS We performed panrickettsial real-time qPCRs target- nodules (Figure 2, panel D). No iNOS+ cells were found ing the ompB and gltA genes (4,5). In addition, we used (D. Tappe, unpub. data). a typhus group rickettsiae–specific qPCR targeting the The panrickettsial qPCR, typhus group rickettsiae– rpr331 gene (6) and nested species-specific qPCRs de- specific rpr331 qPCR, and R. prowazekii–specific nested tecting the prsA genes (7,8) of the R. typhi and R. prowa- qPCR results indicated all samples were negative for rick- zekii genomes (online Technical Appendix Table, https:// ettsial genomic material. However, the nested prsA qPCR wwwnc.cdc.gov/EID/article/24/11/17-1373-Techapp1. indicated tissue sections of the pons of 2 patients (patient pdf). We ran a β-actin gene qPCR (9) in
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