Identification of Bacteria and Viruses Associated with Patients with Acute Febrile Illness in Khon Kaen Province, Thailand.

The majority of cases of undifferentiated acute febrile illness (AFI) in the tropics have an undefined etiology. In Thailand, AFI accounts for two-thirds of illnesses reported to the Ministry of Public Health. To characterize the bacterial and viral causes of these AFIs, we conducted molecular pathogen screening and serological analyses in patients who sought treatment in Chum Phae Hospital, Khon Kaen province, during the period from 2015 to 2016. Through integrated approaches, we successfully identified the etiology in 25.5% of cases, with dengue virus infection being the most common cause, noted in 17% of the study population, followed by scrub typhus in 3.8% and rickettsioses in 6.8%. Further investigations targeting viruses in patients revealed the presence of Guadeloupe mosquito virus (GMV) in four patients without other pathogen co-infections. The characterization of four complete genome sequences of GMV amplified from AFI patients showed a 93-97% nucleotide sequence identity with GMV previously reported in mosquitoes. Nucleotide substitutions resulted in amino acid differences between GMV amplified from AFI patients and mosquitoes, observed in 37 positions. However, these changes had undergone purifying selection pressure and potentially had a minimal impact on protein function. Our study suggests that the GMV strains identified in the AFI patients are relatively similar to those previously reported in mosquitoes, highlighting their potential role associated with febrile illness.

Retrospective survey of severe fever with thrombocytopenia syndrome in patients with suspected rickettsiosis in Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by the SFTS virus (SFTSV). The aim of this study was to clarify whether SFTS is potentially mis-diagnosed as rickettsioses, including spotted fever, typhus fever, and scrub typhus, which are also tick-borne disease. A total of 464 serum samples collected from 222 patients with clinically suspected rickettsiosis between 1999 and 2012 were tested for antibodies against the SFTSV. Of the 464 serum samples, one was positive for antibodies against the virus in an enzyme-linked immunosorbent assay and indirect immunofluorescence assay. The patient of SFTSV antibody-positive sample (15 days after disease onset) was positive for SFTSV genome in the acute phase sample (3 days after disease onset) as determined via reverse transcription-quantitative polymerase chain reaction. This patient, who was a resident of the Yamaguchi prefecture in Western Japan, was in his 40s when he showed symptoms in 2011. As the result, 1 of 222 patients, who was clinically suspected of rickettsiosis, was retrospectively diagnosed with SFTS. In this case, both the C-reactive protein and white blood cell count levels were lower than the ranges of these parameters for patients diagnosed with rickettsiosis. Therefore, SFTS should be considered in the differential diagnosis for rickettsiosis in Japan.

Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Rickettsia spp.

We developed a novel loop-mediated isothermal amplification (LAMP) method to detect Rickettsia spp., including Rickettsia prowazekii and R. typhi. Species-specific LAMP primers were developed for orthologous genes conserved among Rickettsia spp. The selected modified primers could detect all the Rickettsia spp. tested. The LAMP method was successfully used to detect 100 DNA copies of Rickettsia spp. within approximately 60 min at 63℃. Therefore, this method may be an excellent tool for the early diagnosis of rickettsiosis in a laboratory or in the field.

Prevalence of Rickettsia spp. in Ticks and Serological and Clinical Outcomes in Tick-Bitten Individuals in Sweden and on the Åland Islands.

Tick-transmitted diseases are an emerging health problem, and the hard tick Ixodes ricinus is the main vector for Borrelia spp., tick-borne encephalitis virus and most of the spotted fever Rickettsiae in Europe. The aim of the present study was to examine the incidence of rickettsial infection in the southernmost and south central parts of Sweden and the Åland Islands in Finland, the risk of infection in humans and its correlation with a bite of a Rickettsia-infected tick, the self-reported symptoms of rickettsial disease, and the prevalence of co-infection between Rickettsia spp. and Borrelia spp. Persons with a recent tick bite were enrolled through public media and asked to answer a questionnaire, provide a blood sample and bring detached ticks at enlistment and at follow-up three months later. Blood samples were previously analysed for Borrelia spp. antibodies and, for this report, analysed for antibodies to Rickettsia spp. by immunofluorescence and in 16 cases also using Western Blot. Ninety-six (44.0%) of the 218 participants were seropositive for IgG antibodies to Rickettsia spp. Forty (18.3%) of the seropositive participants had increased titres at the follow-up, indicating recent/current infection, while four (1.8%) had titres indicating probable recent/current infection (≥1:256). Of 472 ticks, 39 (8.3%) were Rickettsia sp. positive. Five (31.3%) of 16 participants bitten by a Rickettsia-infected tick seroconverted. Experience of the self-reported symptoms nausea (p = 0.006) and radiating pain (p = 0.041) was more common among those with recent, current or probable infection compared to those who did not seroconvert. Participants who showed seroreactivity or seroconversion to Rickettsia spp. had more symptoms than those who were seronegative. Seven (3.2%) participants showed seroconversion to Borrelia spp., and three (1.4%) of these showed seroconversion to both Rickettsia spp. and Borrelia spp., in accordance with previous studies in Sweden. Symptoms of rickettsial disease were in most of the cases vague and general that were difficult to differentiate from other tick-borne diseases.

Survey for hantaviruses, tick-borne encephalitis virus, and Rickettsia spp. in small rodents in Croatia.

In Croatia, several rodent- and vector-borne agents are endemic and of medical importance. In this study, we investigated hantaviruses and, for the first time, tick-borne encephalitis virus (TBEV) and Rickettsia spp. in small wild rodents from two different sites (mountainous and lowland region) in Croatia. In total, 194 transudate and tissue samples from 170 rodents (A. flavicollis, n=115; A. agrarius, n=2; Myodes glareolus, n=53) were tested for antibodies by indirect immunofluorescence assays (IIFT) and for nucleic acids by conventional (hantaviruses) and real-time RT-/PCRs (TBEV and Rickettsia spp.). A total of 25.5% (24/94) of the rodents from the mountainous area revealed specific antibodies against hantaviruses. In all, 21.3% (20/94) of the samples from the mountainous area and 29.0% (9/31) from the lowland area yielded positive results for either Puumala virus (PUUV) or Dobrava-Belgrade virus (DOBV) using a conventional RT-PCR. All processed samples (n=194) were negative for TBEV by IIFT or real-time RT-PCR. Serological evidence of rickettsial infection was detected in 4.3% (4/94) rodents from the mountainous region. Another 3.2% (3/94) rodents were positive for Rickettsia spp. by real-time PCR. None of the rodents (n=76) from the lowland area were positive for Rickettsia spp. by real-time PCR. Dual infection of PUUV and Rickettsia spp. was found in one M. glareolus from the mountainous area by RT-PCR and real-time PCR, respectively. To our knowledge, this is the first detection of Rickettsia spp. in small rodents from Croatia. Phylogenetic analyses of S- and M-segment sequences obtained from the two study sites revealed well-supported subgroups in Croatian PUUV and DOBV. Although somewhat limited, our data showed occurrence and prevalence of PUUV, DOBV, and rickettsiae in Croatia. Further studies are warranted to confirm these data and to determine the Rickettsia species present in rodents in these areas.

Update on rickettsioses in Slovakia.

The reported incidence of vector-borne diseases including various cases of Rickettsioses in humans is increasing due to a combination of climatic and social factors, escalating the opportunities for contact between people and ticks, fleas or lice. Many of the emerging infectious diseases currently challenging human health in Europe are transmitted by ticks which normally feed on domestic or wild animals. Each Rickettsia spp. has one or several tick vectors, and their geographical distribution varies according to geographical conditions; e.g.; altitude or temperature, which is gradually changing due to a global warming. Evidence of Rickettsia spp. particularly of a newly discovered species is a strong indication that a great number of diseases may be caused by so far undetected or unrecognized organisms. Their diagnosis relies mostly on rare "spot like" cooperation of clinicians with scientists, the members of the working groups that are devoted to the scientific studies of the corresponding research areas. The clinical picture of the disease caused by rickettsiae varies significantly from flu like symptoms to severe fatal outcomes, reflecting the various factors, e.g. a variability of virulence of rickettsial species due to cell invasion, dissemination of rickettsiae, genomics, immune response of an infected organism, or a tricky impact of a treatment. Several major reviews on rickettsioses have been previously published, e.g. in 1997 (Raoult and Roux, 1997a), in 2005 (Parola et al., 2005), and in 2011 (Botelho-Nevers and Raoult, 2011). In this work we intend to present a short historical overview and to describe new trends in research studies of rickettsiology. The main focus will be on rickettsioses affecting Europe΄s population.

[Molecular genetic variety of rickettsiae].

In this review modern data on the study of the genome of the representatives of the family Rickettsiaceae and, as follows from the accumulated information, on changes in the nomination and hierarchical place of its pathogenic and avirulent representatives are presented.

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo.

Brazilian spotted fever (BSF) cases have been increasing in the state of São Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of São Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.

Specific amplification of Rickettsia japonica DNA from clinical specimens by PCR.

The gene encoding the 17,000-molecular-weight genus-common antigen (17K genus-common antigen) has been cloned and sequenced from Rickettsia japonica. The primer pair used for PCR was designed from this sequence. A 357-bp fragment was observed by amplifying the genomic DNA from R. japonica and also the DNA from blood clots of patients with spotted fever group rickettsiosis. The results indicated that this method is suitable for the diagnosis of spotted fever group rickettsiosis in Japan.