Serologic Diagnosis of Acute Rickettsiosis in Children in Southern Israel.

We assessed serology results of clinically suspected rickettsiosis episodes in the hospital setting. Overall, 322 of 963 (33%) cases were serology positive. Among those, rash rates were low (30%), murine typhus (MT) predominated over spotted fever and IgM positivity rate was higher in MT. These findings suggest that during acute rickettsiosis, serology may reliably identify MT infection but may underdiagnose spotted fever.

GroEL is an immunodominant surface-exposed antigen of Rickettsia typhi.

Rickettsioses are neglected and emerging potentially fatal febrile diseases that are caused by obligate intracellular bacteria, rickettsiae. Rickettsia (R.) typhi and R. prowazekii constitute the typhus group (TG) of rickettsiae and are the causative agents of endemic and epidemic typhus, respectively. We recently generated a monoclonal antibody (BNI52) against R. typhi. Characterization of BNI52 revealed that it specifically recognizes TG rickettsiae but not the members of the spotted fever group (SFG) rickettsiae. We further show that BNI52 binds to protein fragments of ±30 kDa that are exposed on the bacterial surface and also present in the periplasmic space. These protein fragments apparently derive from the cytosolic GroEL protein of R. typhi and are also recognized by antibodies in the sera from patients and infected mice. Furthermore, BNI52 opsonizes the bacteria for the uptake by antigen presenting cells (APC), indicating a contribution of GroEL-specific antibodies to protective immunity. Finally, it is interesting that the GroEL protein belongs to 32 proteins that are differentially downregulated by R. typhi after passage through immunodeficient BALB/c CB17 SCID mice. This could be a hint that the rickettsia GroEL protein may have immunomodulatory properties as shown for the homologous protein from several other bacteria, too. Overall, the results of this study provide evidence that GroEL represents an immunodominant antigen of TG rickettsiae that is recognized by the humoral immune response against these pathogens and that may be interesting as a vaccine candidate. Apart from that, the BNI52 antibody represents a new tool for specific detection of TG rickettsiae in various diagnostic and experimental setups.

Assessing human exposure to spotted fever and typhus group rickettsiae in Ontario, Canada (2013-2018): a retrospective, cross-sectional study.

BACKGROUND: Assessing the burden of rickettsial infections in Ontario, Canada, is challenging since rickettsial infections are not reportable to public health. In the absence of reportable disease data, we assessed the burden of rickettsial infections by examining patient serological data and clinical information. METHODS: Our retrospective, cross-sectional study included patients who had Rickettsia serological testing ordered by their physician, in Ontario, from 2013 to 2018. We tested sera from 2755 non-travel patients for antibodies against spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) using an indirect immunofluorescence assay (IFA) (positive IgG titers ≥1:64). We classified cases using a sensitive surveillance case definition: confirmed (4-fold increase in IgG titers between acute and convalescent sera with clinical evidence of infection), possible (single positive sera with clinical evidence) and previous rickettsial infection (single positive sera without clinical evidence). We classified cases seropositive for both SFGR and TGR as unspecified Rickettsia infections (URIs). RESULTS: Less than 5% of all patients had paired acute and convalescent sera tested, and of these, we found a single, laboratory-confirmed SFGR case, with a 4-fold increase in IgG titers and evidence of fever, maculopapular rash and headache. There were 45 possible (19 SFGR, 7 TGR, 19 URI) and 580 previous rickettsial infection (183 SFGR, 89 TGR, 308 URI) cases. The rate of positive tests for SFGR, TGR and URI combined (all case classifications) were 4.4 per 100,000 population. For confirmed and possible cases, the most common signs and symptoms were fever, headache, gastrointestinal complaints and maculopapular rash. The odds of having seropositive patients increased annually by 30% (odds ratio = 1.3, 95% confidence interval: 1.23-1.39). CONCLUSIONS: The rates of rickettsial infections in Ontario are difficult to determine. Based on confirmed and possible cases, rates are low, but inclusion of previous rickettsial infection cases would indicate higher rates. We highlight the need for education regarding the importance of testing acute and convalescent sera and consistent completion of the laboratory requisition in confirming rickettsial disease. We suggest further research in Ontario to investigate rickettsial agents in potential vectors and clinical studies employing PCR testing of clinical samples.

Serum C-reactive protein and procalcitonin values in acute Q fever, scrub typhus, and murine typhus.

BACKGROUND: Although C-reactive protein (CRP) and procalcitonin (PCT) are widely used inflammatory markers for infectious diseases, their role and potential application for rickettsioses were rarely studied. METHODS: A retrospective chart review and serological study were conducted in patients with rickettsioses. The clinical presentations, characteristics, laboratory data, and treatment responses were recorded and their associations with CRP and PCT values were analyzed. RESULTS: A total of 189 cases of rickettsioses, including 115 cases of acute Q fever (60.8%), 55 cases of scrub typhus (29.1%), and 19 cases of murine typhus (10.1%) were investigated. Both CRP and PCT values increased in the acute phase and declined in the convalescent phase. In the acute phase, mean CRP and PCT values were 78.2 ± 63.7 mg/L and 1.05 ± 1.40 ng/mL, respectively. Percentages of patients falling under different cut-off values of CRP and PCT were calculated systematically. Only 10.8% of CRP was > 150 mg/L and 14.2% of PCT was > 2.0 ng/mL. Patients with delayed responses to doxycycline treatment (> 3 days from treatment to defervescence) had significantly higher CRP values (102.7 ± 77.1 vs. 72.2 ± 58.2 mg/L, p = 0.041) and more PCT > 1.0 ng/ml (48.4% vs. 26.0%, p = 0.019) in the acute phase; higher CRP values (19.1 ± 37.4 vs. 3.6 ± 13.1 mg/L, p = 0.049) and more PCT > 0.5 ng/ml (19.2% vs. 1.4%, p = 0.005) in the convalescent phase. Correlation analysis was conducted for patients with acute Q fever. CRP and PCT values were positively correlated to each other, and both markers also had a positive correlation with serum aspartate transaminase values. Both CRP and PCT values and white blood cell counts were positively correlated to the days needed from doxycycline treatment to defervescence. CONCLUSION: CRP and PCT values might be useful in clinical investigations for patients with suspected rickettsioses and in predicting the response to doxycycline treatment for rickettsioses.

Underdiagnoses of Rickettsia in patients hospitalized with acute fever in Indonesia: observational study results.

BACKGROUND: Reports of human rickettsial infection in Indonesia are limited. This study sought to characterize the epidemiology of human rickettsioses amongst patients hospitalized with fever at 8 tertiary hospitals in Indonesia. METHODS: Acute and convalescent blood from 975 hospitalized non-dengue patients was tested for Rickettsia IgM and IgG by ELISA. Specimens from cases with seroconversion or increasing IgM and/or IgG titers were tested for Rickettsia IgM and IgG by IFA and Rickettsia genomes using primers for Rickettsia (R.) sp, R. typhi, and Orientia tsutsugamushi. Testing was performed retrospectively on stored specimens; results did not inform patient management. RESULTS: R. typhi, R. rickettsii, and O. tsutsugamushi IgG antibodies were identified in 269/872 (30.8%), 36/634 (5.7%), and 19/504 (3.8%) of samples, respectively. For the 103/975 (10.6%) non-dengue patients diagnosed with acute rickettsial infection, presenting symptoms included nausea (72%), headache (69%), vomiting (43%), lethargy (33%), anorexia (32%), arthralgia (30%), myalgia (28%), chills (28%), epigastric pain (28%), and rash (17%). No acute rickettsioses cases were suspected during hospitalization. Discharge diagnoses included typhoid fever (44), dengue fever (20), respiratory infections (7), leptospirosis (6), unknown fever (6), sepsis (5), hepatobiliary infections (3), UTI (3), and others (9). Fatalities occurred in 7 (6.8%) patients, mostly with co-morbidities. CONCLUSIONS: Rickettsial infections are consistently misdiagnosed, often as leptospirosis, dengue, or Salmonella typhi infection. Clinicians should include rickettsioses in their differential diagnosis of fever to guide empiric management; laboratories should support evaluation for rickettsial etiologies; and public policy should be implemented to reduce burden of disease.

Selection of Diagnostic Cutoffs for Murine Typhus IgM and IgG Immunofluorescence Assay: A Systematic Review.

Murine typhus is a neglected but widespread infectious disease that results in acute fever. The immunofluorescence assay (IFA) is the "gold standard" to identify IgM or IgG antibodies, although there is a lack of standardization in methodologies. The objective of this review is to summarize 1) the differences in published methodologies, 2) the diagnostic cutoff titers, and 3) the justification of diagnostic cutoffs. Searches were performed by combining the following search terms: "murine typhus," "rickettsia typhi," "immunofluorescence," "IFA," and "serologic" with restrictions (i.e., "rickettsia typhi" or "murine typhus," and "IFA" or "immunofluorescence," or "serologic*"). The search identified 78 studies that used IFA or immunoperoxidase assay (IIP) antibody cutoffs to diagnose murine typhus, 39 of which were case series. Overall, 45 studies (57.7%) provided little to no rationale as to how the cutoff was derived. Variation was seen locally in the cutoff titers used, but a 4-fold or greater increase was often applied. The cutoffs varied depending on the antibody target. No consensus was observed in establishing a cutoff, or for a single-value diagnostic cutoff. In conclusion, there is a lack of consensus in the establishment of a single-value cutoff. Further studies will need to be executed at each distinct geographic location to identify region-specific cutoffs, while also considering background antibody levels to distinguish between healthy and infected patients.

Comparison of Commercial Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay for Diagnosis of Acute Rickettsia typhi Infections.

Murine typhus is a tropical disease caused by Rickettsia typhi and is endemic in resource-limited settings such as Southeast Asian countries. Early diagnosis of R. typhi infection facilitates appropriate management and reduces the risk of severe disease. However, molecular detection of R. typhi in blood is insensitive due to low rickettsemia. Furthermore, the gold standard of sero-diagnosis by immunofluorescence assay (IFA) is cumbersome, subjective, impractical, and unavailable in many endemic areas. In an attempt to identify a practical diagnostic approach that can be applied in Indonesia, we evaluated the performance of commercial R. typhi IgM and IgG enzyme-linked immunosorbent assay (ELISA) and IFA using paired plasma from previously studied R. typhi PCR-positive cases and controls with other known infections. Sensitivity and specificity of combined ELISA IgM and IgG anti-R. typhi using paired specimens were excellent (95.0% and 98.3%, respectively), comparable to combined IFA IgM and IgG (97.5% and 100%, respectively); sensitivity of ELISA IgM from acute specimens only was poor (45.0%), but specificity was excellent (98.3%). IFA IgM was more sensitive (77.5%), but less specific (89.7%) for single specimens.

The temporal dynamics of humoral immunity to Rickettsia typhi infection in murine typhus patients.

OBJECTIVES: This study examined individuals with Rickettsia typhi infection in the Lao People's Democratic Republic (Lao PDR) to (a) investigate humoral immune dynamics; (b) determine the differences in reference diagnostic results and recommend appropriate cut-offs; (c) determine differences in immune response after different antibiotic treatments; and (d) determine appropriate diagnostic cut-off parameters for indirect immunofluorescence assay (IFA). METHODS: Sequential serum samples from 90 non-pregnant, adults were collected at seven time-points (days 0, 7, 14, 28, 90, 180 and 365) as part of a clinical antibiotic treatment trial. Samples were tested using IFA to determine IgM and IgG antibody reciprocal end-point titres against R. typhi and PCR. RESULTS: For all 90 individuals, reciprocal R. typhi IgM and IgG antibody titres ranged from <400 to ≥3200. The median half-life of R. typhi IgM was 126 days (interquartile range 36-204 days) and IgG was 177 days (interquartile range 134-355 days). Overall median patient titres for R. typhi IgM and IgG were significantly different (p < 0.0001) and at each temporal sample collection point (range p < 0.0001 to p 0.0411). Using Bayesian latent class model analysis, the optimal diagnostic cut-off reciprocal IFA titer on patient admission for IgM was 800 (78.6%, 95% CI 71.6%-85.2% sensitivity; 89.9%, 95% CI 62.5%-100% specificity), and for IFA IgG 1600 (77.3%; 95% CI 68.2%-87.6% sensitivity; 99%, 95% CI 95%-100% specificity). CONCLUSIONS: This study suggests suitable diagnostic cut-offs for local diagnostic laboratories and other endemic settings and highlights antibody persistence following acute infection. Further studies are required to validate and define cut-offs in other geographically diverse locations.

Case Report: Early Doxycycline Therapy for Potential Rickettsiosis in Critically Ill Patients in Flea-Borne Typhus-Endemic Areas.

Flea-borne typhus (FBT), although usually perceived as a self-resolving febrile illness, actually encompasses a wide spectrum of disease severity, including fulminant sepsis with multi-organ failure. In endemic Texas and California, the incidence of FBT has more than doubled over the last decade. Clinicians remain unfamiliar with severe septic presentations of FBT when considering the etiologies of acute undifferentiated febrile syndromes. The diagnostic challenges of FBT include the nonspecific and variable nature of both history and physical examination and the lack of diagnostic testing that can provide clinically relevant information early in the course of infection. These barriers perpetuate misdiagnoses in critically ill patients and lead to delay in initiating appropriate antibiotics, which may contribute to preventable morbidity and mortality. This case series describes the clinical and diagnostic trajectories of three patients who developed FBT-associated multi-organ dysfunction. These patients achieved resolution of infection after receiving doxycycline in the context of a high clinical suspicion. Patients residing in FBT-endemic areas presenting with a febrile illness of unknown etiology with a suggestive constellation of hyponatremia, elevated transaminase levels, and thrombocytopenia should be suspected of having FBT. Clinicians should proceed to serologic testing with early doxycycline therapy for potential rickettsiosis. Familiarizing clinicians with the presentation of rickettsiosis-associated septic syndromes and its early and appropriate antibiotic treatment can provide lifesaving care and reduce health-care costs through prevention of the morbidity associated with FBT.

Rickettsia typhi as Cause of Fatal Encephalitic Typhus in Hospitalized Patients, Hamburg, Germany, 1940-1944.

We evaluated formalin-fixed paraffin-embedded tissue specimens from 7 patients who died with encephalitic typhus in Hamburg, Germany, during World War II. The archived specimens included only central nervous system tissues >70 years old that had been stored at room temperature. We demonstrated successful detection of Rickettsia typhi DNA by a nested qPCR specific to prsA in 2 patients. These results indicate that R. typhi infections contributed to typhus outbreaks during World War II. Immunohistochemical analyses of brain tissue specimens of R. typhi DNA-positive and -negative specimens showed perivascular B-cell accumulation. Around blood vessels, nodular cell accumulations consisted of CD4-positive and CD8-positive T cells and CD68-positive microglia and macrophages; neutrophils were found rarely. These findings are similar to those of previously reported R. prowazekii tissue specimen testing. Because R. typhi and R. prowazekii infections can be clinically and histopathologically similar, molecular analyses should be performed to distinguish the 2 pathogens.

Typhus Group Rickettsiosis, Germany, 2010-20171.

Typhus group rickettsiosis is caused by the vectorborne bacteria Rickettsia typhi and R. prowazekii. R. typhi, which causes murine typhus, the less severe endemic form of typhus, is transmitted by fleas; R. prowazekii, which causes the severe epidemic form of typhus, is transmitted by body lice. To examine the immunology of human infection with typhus group rickettsiae, we retrospectively reviewed clinical signs and symptoms, laboratory changes, and travel destinations of 28 patients who had typhus group rickettsiosis diagnosed by the German Reference Center for Tropical Pathogens, Hamburg, Germany, during 2010-2017. Immunofluorescence assays of follow-up serum samples indicated simultaneous seroconversion of IgM, IgA, and IgG or concurrence in the first serum sample. Cytokine levels peaked during the second week of infection, coinciding with organ dysfunction and seroconversion. For 3 patients, R. typhi was detected by species-specific nested quantitative PCR. For all 28 patients, R. typhi was the most likely causative pathogen.

The Cat Flea (Ctenocephalides felis) Immune Deficiency Signaling Pathway Regulates Rickettsia typhi Infection.

Rickettsia species are obligate intracellular bacteria with both conserved and lineage-specific strategies for invading and surviving within eukaryotic cells. One variable component of Rickettsia biology involves arthropod vectors: for instance, typhus group rickettsiae are principally vectored by insects (i.e., lice and fleas), whereas spotted fever group rickettsiae are exclusively vectored by ticks. For flea-borne Rickettsia typhi, the etiological agent of murine typhus, research on vertebrate host biology is facilitated using cell lines and animal models. However, due to the lack of any stable flea cell line or a published flea genome sequence, little is known regarding R. typhi biology in flea vectors that, importantly, do not suffer lethality due to R. typhi infection. To address if fleas combat rickettsial infection, we characterized the cat flea (Ctenocephalides felis) innate immune response to R. typhi Initially, we determined that R. typhi infects Drosophila cells and increases antimicrobial peptide (AMP) gene expression, indicating immune pathway activation. While bioinformatics analysis of the C. felis transcriptome identified homologs to all of the Drosophila immune deficiency (IMD) and Toll pathway components, an AMP gene expression profile in Drosophila cells indicated IMD pathway activation upon rickettsial infection. Accordingly, we assessed R. typhi-mediated flea IMD pathway activation in vivo using small interfering RNA (siRNA)-mediated knockdown. Knockdown of Relish and Imd increased R. typhi infection levels, implicating the IMD pathway as a critical regulator of R. typhi burden in C. felis These data suggest that targeting the IMD pathway could minimize the spread of R. typhi, and potentially other human pathogens, vectored by fleas.

Newly Recognized Pediatric Cases of Typhus Group Rickettsiosis, Houston, Texas, USA.

An increase in typhus group rickettsiosis and an expanding geographic range occurred in Texas, USA, over a decade. Because this illness commonly affects children, we retrospectively examined medical records from 2008-2016 at a large Houston-area pediatric hospital and identified 36 cases. The earliest known cases were diagnosed in 2011.

Acute Febrile Illness and Complications Due to Murine Typhus, Texas, USA1,2.

Murine typhus occurs relatively commonly in southern Texas, as well as in California. We reviewed records of 90 adults and children in whom murine typhus was diagnosed during a 3-year period in 2 hospitals in southern Texas, USA. Most patients lacked notable comorbidities; all were immunocompetent. Initial signs and symptoms included fever (99%), malaise (82%), headache (77%), fatigue (70%), myalgias (68%), and rash (39%). Complications, often severe, in 28% of patients included bronchiolitis, pneumonia, meningitis, septic shock, cholecystitis, pancreatitis, myositis, and rhabdomyolysis; the last 3 are previously unreported in murine typhus. Low serum albumin and elevated procalcitonin, consistent with bacterial sepsis, were observed in >70% of cases. Rash was more common in children; thrombocytopenia, hyponatremia, elevated hepatic transaminases, and complications were more frequent in adults. Murine typhus should be considered as a diagnostic possibility in cases of acute febrile illness in southern and even in more northern US states.

Serological evidence of exposure to Rickettsia felis and Rickettsia typhi in Australian veterinarians.

BACKGROUND: Rickettsia felis and Rickettsia typhi are emerging arthropod-borne zoonoses causing fever and flu-like symptoms. Seroprevalence and risk factors associated with exposure to these organisms was explored in Australian veterinarians. METHODS: One hundred and thirty-one veterinarians from across Australia were recruited to participate in a cross-sectional survey. Veterinarians provided a single blood sample and answered a questionnaire on potential risk factors influencing their exposure to R. felis and R. typhi. Indirect microimmunofluorescence antibody testing (IFAT) was used to identify evidence of serological exposure of the participants to R. felis and R. typhi. Results were analyzed and a logistical regression model performed to predict risk factors associated with seropositivity. RESULTS: In total, 16.0% of participants were seropositive to R. felis, 4.6% to R. typhi and 35.1% seropositive to both, where cross-reactivity of the IFAT between R. felis and R. typhi precluded a definitive diagnosis. Veterinarians residing within the south-eastern states of Victoria and Tasmania were at a higher risk of exposure to R. felis or generalised R. felis or R. typhi exposure. Older veterinarians and those that recommended flea treatment to their clients were found to be significantly protected from exposure. CONCLUSIONS: The high exposure to R. felis amongst veterinary professionals suggests that flea-borne spotted fever is an important cause of undifferentiated fever conditions that may not be adequately recognized in Australia.

Cytotoxic effector functions of T cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model of infection: Role of TH1 and TH17 cytokines in protection and pathology.

Endemic typhus caused by Rickettsia (R.) typhi is an emerging febrile disease that can be fatal due to multiple organ pathology. Here we analyzed the requirements for protection against R. typhi by T cells in the CB17 SCID model of infection. BALB/c wild-type mice generate CD4+ TH1 and cytotoxic CD8+ T cells both of which are sporadically reactivated in persistent infection. Either adoptively transferred CD8+ or CD4+ T cells protected R. typhi-infected CB17 SCID mice from death and provided long-term control. CD8+ T cells lacking either IFNγ or Perforin were still protective, demonstrating that the cytotoxic function of CD8+ T cells is not essential for protection. Immune wild-type CD4+ T cells produced high amounts of IFNγ, induced the release of nitric oxide in R. typhi-infected macrophages and inhibited bacterial growth in vitro via IFNγ and TNFα. However, adoptive transfer of CD4+IFNγ-/- T cells still protected 30-90% of R. typhi-infected CB17 SCID mice. These cells acquired a TH17 phenotype, producing high amounts of IL-17A and IL-22 in addition to TNFα, and inhibited bacterial growth in vitro. Surprisingly, the neutralization of either TNFα or IL-17A in CD4+IFNγ-/- T cell recipient mice did not alter bacterial elimination by these cells in vivo, led to faster recovery and enhanced survival compared to isotype-treated animals. Thus, collectively these data show that although CD4+ TH1 cells are clearly efficient in protection against R. typhi, CD4+ TH17 cells are similarly protective if the harmful effects of combined production of TNFα and IL-17A can be inhibited.

CD4+ T Cells Are as Protective as CD8+ T Cells against Rickettsia typhi Infection by Activating Macrophage Bactericidal Activity.

Rickettsia typhi is an intracellular bacterium that causes endemic typhus, a febrile disease that can be fatal due to complications including pneumonia, hepatitis and meningoencephalitis, the latter being a regular outcome in T and B cell-deficient C57BL/6 RAG1-/- mice upon Rickettsia typhi infection. Here, we show that CD4+ TH1 cells that are generated in C57BL/6 mice upon R. typhi infection are as protective as cytotoxic CD8+ T cells. CD4+- as well as CD8+-deficient C57BL/6 survived the infection without showing symptoms of disease at any point in time. Moreover, adoptively transferred CD8+ and CD4+ immune T cells entered the CNS of C57BL/6 RAG1-/- mice with advanced infection and both eradicated the bacteria. However, immune CD4+ T cells protected only approximately 60% of the animals from death. They induced the expression of iNOS in infiltrating macrophages as well as in resident microglia in the CNS which can contribute to bacterial killing but also accelerate pathology. In vitro immune CD4+ T cells inhibited bacterial growth in infected macrophages which was in part mediated by the release of IFNγ. Collectively, our data demonstrate that CD4+ T cells are as protective as CD8+ T cells against R. typhi, provided that CD4+ TH1 effector cells are present in time to support bactericidal activity of phagocytes via the release of IFNγ and other factors. With regard to vaccination against TG Rickettsiae, our findings suggest that the induction of CD4+ TH1 effector cells is sufficient for protection.

Liver Necrosis and Lethal Systemic Inflammation in a Murine Model of Rickettsia typhi Infection: Role of Neutrophils, Macrophages and NK Cells.

Rickettsia (R.) typhi is the causative agent of endemic typhus, an emerging febrile disease that is associated with complications such as pneumonia, encephalitis and liver dysfunction. To elucidate how innate immune mechanisms contribute to defense and pathology we here analyzed R. typhi infection of CB17 SCID mice that are congenic to BALB/c mice but lack adaptive immunity. CB17 SCID mice succumbed to R. typhi infection within 21 days and showed high bacterial load in spleen, brain, lung, and liver. Most evident pathological changes in R. typhi-infected CB17 SCID mice were massive liver necrosis and splenomegaly due to the disproportionate accumulation of neutrophils and macrophages (MΦ). Both neutrophils and MΦ infiltrated the liver and harbored R. typhi. Both cell populations expressed iNOS and produced reactive oxygen species (ROS) and, thus, exhibited an inflammatory and bactericidal phenotype. Surprisingly, depletion of neutrophils completely prevented liver necrosis but neither altered bacterial load nor protected CB17 SCID mice from death. Furthermore, the absence of neutrophils had no impact on the overwhelming systemic inflammatory response in these mice. This response was predominantly driven by activated MΦ and NK cells both of which expressed IFNγ and is considered as the reason of death. Finally, we observed that iNOS expression by MΦ and neutrophils did not correlate with R. typhi uptake in vivo. Moreover, we demonstrate that MΦ hardly respond to R. typhi in vitro. These findings indicate that R. typhi enters MΦ and also neutrophils unrecognized and that activation of these cells is mediated by other mechanisms in the context of tissue damage in vivo.

Evaluation of a new serological test for the detection of anti-Coxiella and anti-Rickettsia antibodies.

Coxiella burnetii and members of the genus Rickettsia are obligate intracellular bacteria. Since cultivation of these organisms requires dedicated techniques, their diagnosis usually relies on serological or molecular biology methods. Immunofluorescence is considered the gold standard to detect antibody-reactivity towards these organisms. Here, we assessed the performance of a new automated epifluorescence immunoassay (InoDiag) to detect IgM and IgG against C. burnetii, Rickettsia typhi and Rickettsia conorii. Samples were tested with the InoDiag assay. A total of 213 sera were tested, of which 63 samples from Q fever, 20 from spotted fever rickettsiosis, 6 from murine typhus and 124 controls. InoDiag results were compared to micro-immunofluorescence. For acute Q fever, the sensitivity of phase 2 IgG was only of 30% with a cutoff of 1 arbitrary unit (AU). In patients with acute Q fever with positive IF IgM, sensitivity reached 83% with the same cutoff. Sensitivity for chronic Q fever was 100% whereas sensitivity for past Q fever was 65%. Sensitivity for spotted Mediterranean fever and murine typhus were 91% and 100%, respectively. Both assays exhibited a good specificity in control groups, ranging from 79% in sera from patients with unrelated diseases or EBV positivity to 100% in sera from healthy patients. In conclusion, the InoDiag assay exhibits an excellent performance for the diagnosis of chronic Q fever but a very low IgG sensitivity for acute Q fever likely due to low reactivity of phase 2 antigens present on the glass slide. This defect is partially compensated by the detection of IgM. Because it exhibits a good negative predictive value, the InoDiag assay is valuable to rule out a chronic Q fever. For the diagnosis of rickettsial diseases, the sensitivity of the InoDiag method is similar to conventional immunofluorescence.