MINIREVIEW

crossm

A Concise Review of the Epidemiology and Diagnostics of

Rickettsioses: Rickettsia and Orientia spp.
Mohammad Yazid Abdad,a,b Rita Abou Abdallah,c Pierre-Edouard Fournier,c John Stenos,d Shawn Vasooa,b,e

a National Centre for Infectious Diseases, Singapore

b Department of Infectious Diseases, Institute of Infectious Diseases and Epidemiology, Tan Tock Seng Hospital,
Singapore
c
Centre National de Référence des Rickettsia, Coxiella et Bartonella, Faculté de Médecine, Université de la
Méditerranée, Aix-en-Provence, France
d Australian Rickettsial Reference Laboratory, University Hospital, Geelong, Victoria, Australia
e Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore

ABSTRACT Rickettsioses are globally distributed and caused by the family Rickettsi-
aceae, which comprise a diverse and expanding list of organisms. These include two
genera, Rickettsia and Orientia. Serology has been traditionally the mainstay of diag-
nosis, although this has been limited by cross-reactions among closely related mem-
bers and diminished sensitivity/utility in the acute phase of illness. Other techniques,
such as nucleic acid amplification tests using blood specimens or tissue swabs/bi-
opsy specimens, sequencing, and mass spectrometry, have emerged in recent years
for both pathogen and vector identification. This paper provides a concise review of
the rickettsioses and the traditional and newer technologies available for their diag-
nosis.
KEYWORDS Orientia, Rickettsia, diagnostics, rickettsioses, scrub typhus, spotted
fever, vector-borne diseases

R ickettsioses are caused by members of the family Rickettsiaceae, which comprise

Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.

the two genera Rickettsia and Orientia. Members of Rickettsia spp. are obligate
intracellular bacteria first described by Ricketts in 1909 (1). Since then, the genus
Rickettsia has expanded to comprise 31 species (https://www.bacterio.net), with more
species being added to the genus every year (2). This increase can be attributed to the
advent of molecular techniques in the last 3 decades and the large reductions in cost
associated with utilizing the new molecular tools. The members of the genus Rickettsia
are traditionally characterized into two main groups, the spotted fever group (SFG) and
the typhus group (TG), with most of known species belonging to the SFG. Two species,
Rickettsia typhi and Rickettsia prowazekii, make up the TG. Classification into the two
groups was historically based on their physiological characteristics of intracellular
localization, optimal growth temperature, and cross-reaction of serum from an infected
Accepted manuscript posted online 16 May
patient with somatic antigens of three strains of Proteus (3). The classification of new 2018
members of Rickettsia today is done through genomic sequence comparison with Citation Abdad MY, Abou Abdallah R, Fournier
information for well-published strains. The advent of modern molecular methods in the P-E, Stenos J, Vasoo S. 2018. A concise review of
the epidemiology and diagnostics of
last couple of decades has not only eased the characterization of new rickettsial species
rickettsioses: Rickettsia and Orientia spp. J Clin
but also allowed rickettsiologists to review some members of the genus. Rickettsia Microbiol 56:e01728-17. https://doi.org/10
tsutsugamushi was renamed Orientia tsutsugamushi; this genus (Orientia) was geneti- .1128/JCM.01728-17.
cally distinct enough but is still closely related to Rickettsia and remains in the same ␣-1 Editor Colleen Suzanne Kraft, Emory University
subgroup (4). Recently, the discovery of another member of the genus Orientia (O. Copyright © 2018 American Society for
Microbiology. All Rights Reserved.
chuto) has increased its membership to two species (5). Another member of Rickettsia
Address correspondence to Mohammad Yazid
that was reclassified was Rickettsia burnetii, which was moved to the genus Coxiella and Abdad, yazid_abdad@ttsh.com.sg.
renamed Coxiella burnetii when it was discovered that its 16S rRNA sequence was more

August 2018 Volume 56 Issue 8 e01728-17 Journal of Clinical Microbiology jcm.asm.org 1

Minireview Journal of Clinical Microbiology

FIG 1 Major rickettsioses described by causative agent, clinical syndrome, and vector by region. From references 2, 15–17, 30, and 66 and the CDC Yellow Book
(https://wwwnc.cdc.gov/travel/yellowbook/2018/infectious-diseases-related-to-travel/rickettsial-spotted-and-typhus-fevers-and-related-infections-including-
anaplasmosis-and-ehrlichiosis). The map was created using mapchart.net.

similar to the ␥-subgroup (6). In this minireview, we briefly review the epidemiology,
transmission, pathogenesis, and clinical features of the Rickettsiaceae (which includes
Rickettsia and Orientia spp.) and discuss updates in clinical diagnostics.

EPIDEMIOLOGY AND TRANSMISSION

Rickettsial organisms have been found on all continents except Antarctica (Fig. 1).

Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.

Most rickettsial species are region-locked due to climatic conditions and vector and
natural host constraints. However, there are rickettsiae that are globally distributed,
such as Rickettsia felis and Rickettsia typhi (7, 8). These two species of Rickettsia are
transmitted by fleas, deviating significantly from most rickettsiae that require a tick
vector, which tend to be limited to the geographical distribution of the ticks. Other
vectors that are known to harbor and transmit rickettsiae are mites (Rickettsia akari) and
lice (Rickettsia prowazekii) (8). Rickettsia felis has been found in nonhematophagous
arthropods, such as booklice (9); however, recent reports of R. felis in mosquitoes (10,
11) have potentially changed our understanding of rickettsial vectors and transmission
to human hosts. Mosquito-to-human transmission has been hypothesized and is cur-
rently being investigated (12).
Rickettsiae require a vector for host transmission; however, infection is acquired by
different routes depending on the vector type and rickettsial species. Most SFG
rickettsiae are harbored by ixodid ticks and are transmitted by their bites during
feeding (saliva). This is also true for most other rickettsiae that are harbored by mites.
Flea- and louse-borne rickettsiae are known to cause infection via entry of fecal material
in bite sites and cuts on the host’s skin (13). TG rickettsiae can cause infection via
inhalation through the aerosolization or contamination of dust particles floating in the
air (14). An uncommon route of infection is via the conjunctivae, through exposure of
contaminated tick hemolymph on fingers from crushed ticks (13).
Scrub typhus has geographically been identified in the tropical Pacific triangle with
points in Australia, Japan, and central Asia. However, recent publications suggest that

August 2018 Volume 56 Issue 8 e01728-17 jcm.asm.org 2

Minireview Journal of Clinical Microbiology

Orientia agents may have a wider global presence, with O. chuto isolated from patient
blood in the United Arab Emirates and scrub typhus-seropositive results recognized in
patients from Chile (15). Evidence is emerging that scrub typhus may be widely
distributed in Africa and is causing disease with reports from Djibouti and Cameroon
(16, 17). The trombiculid mite is the sole vector for Orientia spp. for transmission to
humans.

ARTHROPOD VECTORS AND IDENTIFICATION

The identification of arthropods (ticks, fleas, and mites) traditionally requires a
parasitologist trained in acarology and/or entomology for species-level identification,
which is performed by comparing morphological characteristics against taxonomic
keys. The challenge to this is that taxonomic keys are specific to species of arthropods
according the region in which they are found. The advent of molecular methods
provides an alternative to identifying arthropods. This method allows for the identifi-
cation of tick species by analysis of submitted sequences of Ixodida 16S rRNA, 12S,
internal transcribed spacer 2 (ITS2), and cytochrome oxidase I (COI) genes. An analysis
of available analytical methods demonstrated that COI allows for accurate identification
of tick species when either BLASTn or nearest-neighbor methods are used (18). This
method is, however, limited by the sequences submitted to online databases, such as
GenBank and the Barcode of Life Database. A species can only be identified if its gene
sequences have been uploaded and available for analysis; thus, its widespread uptake
has been slow. The use of matrix-assisted laser desorption ionization–time of flight
mass spectrometry (MALDI-TOF MS) to identify arthropods to the species level has been
met with much better success than molecular methods and has recently been used in
the identification of ticks, fleas, mosquitoes, and mites (19–23).

VIRULENCE, PATHOGENESIS, AND CLINICAL PRESENTATION

Rickettsial disease has historically been associated with higher rates in men and
those of older age; however, recent studies on risk of rickettsial exposure demonstrated
that there are no significant differences between gender or age groups (24, 25). Other
factors that have been found to influence rickettsial disease severity include underlying
patient disease and enhanced oxidative stress (26). Rickettsial virulence has also been
linked to the degradation of the rickettsial genome, where genomes with higher
degradation rates were observed in more pathogenic species (27).

Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.

One of the main pathologies of rickettsial infection is increased vascular permea-
bility linked to bacterial load and tumor necrosis factor-␣ disrupting endothelial cell
junctions of small-to-medium-sized blood vessels (28, 29). Localized rickettsial infection
may present as an eschar (“tache noir”) at the site of arthropod inoculation, and a more
robust reaction is thought to be related to the local control of infection (30). However,
disseminated infection may result in severe vasculitis and endothelial damage, clinically
manifesting cutaneous necrosis and digital gangrene, pneumonitis, meningoencepha-
litis, and multiorgan failure; a case of antineutrophil cytoplasmic autoantibody (ANCA)-
positive vasculitis associated with Rocky Mountain spotted fever (RMSF) has also been
described (31). Misdiagnosis due to the nonspecific symptoms shared with other febrile
diseases is not uncommon. The traditional identifying factor for rickettsial infection is
the presence of an eschar and rash around the bite site. Escharless and spotless
rickettsioses do occur, and as a result, highly specific and sensitive rapid diagnostic
testing for rickettsial infections is recommended for febrile illness occurring in regions
known to be endemic for the disease (32).

DIAGNOSTICS FOR RICKETTSIOSES

Diagnosis of rickettsial infections can be tricky without the right tools available to
the attending physician. A physician is limited by the tests that can be conducted
according to the tissue type collected; thus, a general knowledge of the tests available
is recommended (Fig. 2). The diagnosis of a rickettsial infection is usually achieved
through the gathering of a complete clinical history indicating exposure to a potential

August 2018 Volume 56 Issue 8 e01728-17 jcm.asm.org 3

Minireview Journal of Clinical Microbiology

FIG 2 Illustration summarizing samples that can be obtained from patients and invertebrates and the testing that can be conducted with respect to sample
type.

source of rickettsial disease alongside laboratory testing. The submission of arthropods

collected at the bite site or eschar for rickettsial detection is also recommended if
available. General laboratory findings might include thrombocytopenia, transaminitis,
and hyponatremia. To further compound the matter, rickettsial infection may only be
considered by physicians in many locations of endemicity worldwide when the re-
quested initial diagnostics are unyielding.
Most national public health laboratories and larger medical reference laboratories in
developed countries are able to provide initial diagnostic testing for rickettsial diseases,
usually via serology. However, for more in-depth work into rickettsial diagnosis requir-
ing isolation of the pathogen in cell culture, specialized serologic and molecular assays,

Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.

and pathogen characterization. There are several reference laboratories located in the
United States, France, and Australia (Table 1) that can provide further diagnostic
services to clinicians worldwide.

SEROLOGY
For Rickettsia spp., early detection methods relied heavily on the Weil-Felix test (33)
and, subsequently, Gimenez staining (34). The adaptation of modern serological tech-
niques for rickettsial diagnosis (immunofluorescence) (35) increased diagnostic accu-
racy significantly and was deemed the gold standard before the wide acceptance of
molecular testing. Serological testing is still being conducted in many laboratories
worldwide due to quick turnaround time and need for minimal sample preparation.
They come in many forms, such as dipsticks, enzyme-linked immunosorbent assay
(ELISA) kits, and immunofluorescence assays (IFA). Western blotting is a technique
which may allow more specific identification of the causative agent, but a robust
rickettsial antigen collection must be available, and thus, this is only available in some
reference laboratories. Many kits on the market are plagued by poor specificity and
sensitivity due to their reliance on few established rickettsial species, such as Rickettsia
rickettsii and Rickettsia conorii. It is thus recommended that users of such kits be aware
of the rickettsial antigens used to validate such kits. For rickettsiae that are suspected
to be distinct from those used, rickettsial reference laboratories (Table 1) have available
in-house IFA and microimmunofluorescence (MIF) methods and a larger range of
rickettsial species to provide more accurate results and diagnosis. MIF is similar to IFA,

August 2018 Volume 56 Issue 8 e01728-17 jcm.asm.org 4

 TABLE 1 Reference laboratories for rickettsial diagnostics
Reference laboratory
Centers for Disease Control and Prevention SMB/STAT
Attn: Reference Diagnostic Laboratory Rickettsial
Zoonoses Branch (unit 78) 1600 Clifton Road NE
Atlanta, GA 30329-4027 Phone: (404) 639-1075
Email: rzbepidiag@cdc.gov Website: https://www
.cdc.gov/ncezid/dvbd/specimensub/rickettsial-
shipping.html
August 2018 Volume 56 Issue 8 e01728-17
Centre National de Référence des Rickettsia, Coxiella
et Bartonella, Faculte de Medicine Université de la
Mediteranee Contact: Prof Pierre-Edouard Fournier
264 rue Saint-Pierre 13385 Marseille Cedex 5,
France Phone: ⫹33 (0)4 91 385517 Fax:
⫹33 (0)4 91 387772
Email: pierre-edouard.fournier@univ-amu.fr
Website: http://www.mediterranee-infection.com/
article.php?laref⫽349&titre⫽centre-national-
de-reference-
jcm.asm.org 5
Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.
Method(s) (target gene[s], for NAAT),
thermocycler utilized (from published
Genus/species references)a
Spotted fever group IFA with IgG
Typhus group Single-stage or nested PCR (17-kDa
Orientia protein-encoding gene and gltA
[Rickettsia-genus] or ompA [SFG],
sequencing
Real-time PCR panrickettsia- and R.
rickettsii-specific assays (23S rRNA
and gene encoding hypothetical
protein A1G_04230) (Applied
Biosystems 7500 FastDX)
Culture
Immunohistochemistry
Spotted fever group IFA
Typhus group Other methods used were MIF, specific
serology, WB, WB ⫾ cross-adsorption
test
Real-time PCR, gltA citrate synthase A
gene (Rickettsia genus specific),
RC0338 gene encoding hypothetical
protein (Rickettsia genus specific),
glycosyltransferase gene (TG) and
periplasmic serine protease gene (O.
tsutsugamushi), with ␤-actin gene as
control (LightCycler 3.5 instrument;
Roche Diagnostics, Mannheim,
Germany)
Nested PCR technique with single-use
primers targeting single-use DNA
fragments (“suicide PCR”) (PTC200
DNA thermal cycler; MJ Research);
conventional PCR ompA and gltA
(PTC200 DNA thermal cycler, MJ
Research)
Specimen(s)b
Serum
Blood (serum, EDTA-whole blood) and
tissue specimens (fresh and FFPE),
skin ulcer swab and eschar
Whole blood, tissue (fresh)
Tissue (fresh or FFPE)
Serum
Skin biopsy specimens, cutaneous
swab specimens, whole-blood
serum
Selected
Minireview
Notesc reference(s)
Paired samples should be 2–6 wk apart; cutoff of 49, 50
ⱖ1:64 is used
TAT 1–2 days, may not be highly sensitive in blood 49–51
samples during acute disease (except for
advanced/fatal cases); historically, the standard
method was used for testing blood/fresh tissue
specimens
TAT ⬍1 h; analytical sensitivity range of 10 to 104
fg, and limit of 8 to 9 genome copies with 95%
reproducibility; the real-time assay identified
more positives than did nested PCR in 223
banked DNA specimens; most sensitive in first
week of acute illness and within 24 h of
antibiotic therapy
e.g., in Vero E6 cells 49, 50
Immunoalkaline phosphatase technique with
mono- or polyclonal rabbit antibodies;
immunologic reagents are generally group
rather than species specific
Specific serologies for R. conorii, R. typhi, R. slovaca, 52–54
other Rickettsia spp., O. tsutsugamushi
For IFA/MIF, titers of 1:128 for IgG and 1:32 for IgM
in acute-phase serum specimens and/or
evidence of seroconversion with 4-fold increases
in IgG titers are considered evidence of recent
rickettsial infection
If SFG gltA PCR positive, this was reflexed to 55, 56
specific real-time PCR for, e.g., R. conorii, R.
africae, R. slovaca, R. raoultii, and R. australis,
depending on epidemiologic exposures; TG
tested for depending on epidemiologic
exposures; in a 2-yr study of 643 clinical samples
screened for Rickettsia DNA, 45 positive samples
were detected; positive samples were detected
mainly from cutaneous biopsy specimens and
swabs (31/45)
In one study of skin biopsy specimens from 103 43, 57
patients with a definite rickettsiosis, sensitivity of
suicide PCR was 68%, compared to 31% for
culture and 45.6% for regular PCR
(Continued on next page)
Journal of Clinical Microbiology
 TABLE 1 (Continued)
Method(s) (target gene[s], for NAAT),
thermocycler utilized (from published Selected
Minireview

Reference laboratory Genus/species references)a Specimen(s)b Notesc reference(s)

Culture, including shell vial cultures
Skin biopsy specimens, blood
Specimen in sterile container or blood in 58
heparinized tube; with samples intended for
culture (except the blood), freezing at ⫺80°C
and transport in ⫺20°C (dry ice) is preferable;
TAT 3 days to 3 mo for Rickettsia spp. and 7
days to 3 mo for O. tsutsugamushi; culture cell
lines include HEL and L929, detected with IFA/
Gimenez stain/PCR; shell via protocol detected
Rickettsia spp. in 52/949 human samples;
definitive identification to the species level was
systematically done by specific PCR and
sequencing

August 2018 Volume 56 Issue 8 e01728-17

WHO Collaborating Centre for Reference and Spotted fever group MIF, ELISA, immunoblot Serum Titer threshold of 1:128 is used to determine 59, 60
Research of Rickettsioses Australian Rickettsial (Rickettsia reactivity in the MIF assay to detect IgG
Reference Laboratory University Hospital Geelong australis, R. honei, antibodies
Entrance 3, Bellarine Street Victoria 3220, Australia R. conorii, R.
Contact: Dr. John Stenos Phone: ⫹61 (3) 4215 africae, R.
1357 Fax: ⫹61 (3) 4215 1370 rickettsii, R. felis)
Email: johns@barwonhealth.org.au Website: Typhus group (R. Real-time PCR targeting Rickettsia- EDTA-blood, biopsy specimens, Rickettsial assay had a sensitivity of one target 61–63
https://www.rickettsialab.org.au/contact prowazekii, R. specific gltA (citrate synthase gene) cerebrospinal fluid copy no. per reaction by serial plasmid dilution
typhi) O. tsutsugamushi rrs (16S rRNA
gene); (RotorGene 3000 instrument;
Corbett Lifesciences, Qiagen)
Scrub typhus group Conventional gel-based PCR rickettsial Isolates, blood/buffy coat May be used for confirmation 64, 65
(Orientia 17-kDa antigen and sequencing
tsutsugamushi
[serotypes
Gilliam, Karp,
Kato, Cowley
Beach], Orientia
chuto)
Culture EDTA-blood, tissue e.g., in Vero E6, XTC-2 cell lines 65
aNAAT, nucleic acid amplification test; IFA, indirect immunofluorescence assay; MIF, indirect microimmunofluorescence assay; WB, Western blotting; ELISA, enzyme-linked immunosorbent assay.
bFFPE, formalin-fixed paraffin-embedded;
cTAT, turnaround time.

jcm.asm.org 6
Journal of Clinical Microbiology

Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.

Minireview Journal of Clinical Microbiology

except that wells are spotted with multiple rickettsial antigens for simultaneous de-
tection. It has been suggested that a rickettsial antigen eliciting an antibody titer with
a 4-fold higher dilution than those for antigens from other species in the MIF format
suggests a causative agent (36). However, this may not definitively be so due to
cross-reactivity, and paired sera (acute and convalescent) for IgG are recommended
(37). Other strategies to mitigate this, such as cross-adsorption assays, are technically
challenging, costly, and lie in the realm of research and/or reference laboratories.
It should be noted that IgM detection may not be diagnostic for acute disease, as
there could be cross-reactivity with other species and persistence of IgM beyond acute
illness. Serologic tests for Rickettsia spp. generally turn positive only after 7 to 10 days
of illness, and this may be delayed up to 25 days or later for certain species, like R.
africae (38). Serology for scrub typhus also traditionally utilizes IFA as the gold standard;
however, in contrast to the SFG and TG Rickettsia spp., the ELISA format and rapid
diagnostic tests (e.g., for IgM) have shown promise, with acceptable sensitivity and
specificity (37, 39).

MOLECULAR TESTING
Nucleic acid amplification tests (NAATs), such as PCR, may be useful in the diagnosis
of rickettsioses. The genomes of many Rickettsia spp. are well described, allowing for
the adaptation of PCR assays to detect different genes, such as citrate synthase (gltA),
outer membrane protein A (ompA), outer membrane protein B (ompB), 16S rRNA, and
gene D (sca4). The genes for 47-kDa periplasmic serine protease (htrA), 16S rRNA,
56-kDa antigen, and heat shock protein 60 (HSP60) (groEL) have been used as PCR
targets for Orientia tsutsugamushi (37). The reader is referred to recent reviews on
potential PCR targets and protocols for further details (40, 41). The quick turnaround
time allows for prompt diagnosis without the need to wait for seroconversion (serol-
ogy) or lengthy growth time (culture of blood [EDTA] or eschar biopsy, e.g., in Vero or
L929 cells), which can take anywhere between 10 days and 4 weeks. PCR (either
real-time or conventional) can be performed on whole-blood, buffy coat, or eschar
material (crust, swabs, or biopsy samples) (42, 43). A nested conventional PCR format
can improve diagnostic sensitivity and allow more data to be obtained from sequenc-
ing because of the longer amplicon but be more prone to amplicon contamination. To
overcome this, nested conventional PCR with single-use primers (“suicide PCR”) has
been proposed (43). The identification of rickettsial species by sequence analysis is now

Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.

commonplace, although for clinical reasons, species identification may not be as vital,
since rickettsioses are generally similarly treated with doxycycline. Other limitations of
NAATs include the fact that they are more sensitive in acute illness (e.g., febrile phase,
ideally days 1 to 5 of illness, possibly up to days 7 to 10; or when an eschar is still
present) (37). A combinatorial approach using PCR in acute illness in addition to
serology may improve diagnostic yield.

TISSUE BIOPSIES: CULTURE, IMMUNOHISTOCHEMISTRY, AND PCR

Tissue biopsy specimens (e.g., punch biopsy specimens) may be fresh, frozen, or
formalin-fixed and paraffin-embedded (FFPE) and be submitted for testing at special-
ized laboratories, for example, the Reference Diagnostic Laboratory, Rickettsial Zoono-
ses Branch at the Centers of Disease Control and Protection in Atlanta, GA. The
advantage of fresh tissue is that it can be subject to immunohistochemistry (IHC) in
addition to culture and PCR. In cases of a negative PCR result, culture may be
considered for use as a backup test. The yields of culture and IHC are lower for frozen
tissue, although PCR can be performed. For FFPE specimens (tissue blocks), PCR and IHC
can be performed. For optimal sensitivity, specimens should be obtained prior to or
within 24 h of antibiotic administration (https://www.cdc.gov/ncezid/dvbd/pdf/collection
-submission-skin-biopsy-specimens-rickettsial-disease.pdf). IHC is limited to reference lab-
oratories due to the need for special reagents comprising group-reactive polyclonal or
species-specific monoclonal antibodies. Older data (up to 1974) indicate that 15% of
3,921 laboratory-associated infections were caused by rickettsioses; however, these

August 2018 Volume 56 Issue 8 e01728-17 jcm.asm.org 7

Minireview Journal of Clinical Microbiology

data are largely from an era when rickettsial cultures were more widely performed and
biosafety practices (e.g., use of biosafety cabinets) were not as stringent (44). Non-
propagative laboratory procedures for rickettsioses can be performed under biosafety
level 2 (BSL2) conditions; however, cell culture and manipulation of infectious material
should be performed in a BSL3 laboratory.
It is not uncommon for rickettsiae to be isolated from a patient sample where a BSL2
viral pathogen is the suspected cause of disease. This may pose a biosecurity risk if the
laboratory in question and operators are unaware that they have isolated rickettsiae in
their monolayer culture. It is thus advisable that any laboratory attempting to isolate
viral pathogens have an assay to determine that their isolate is a free rickettsial
organism, as this may require them to transfer any isolated rickettsiae to a BSL3
laboratory before further work can be done.

NEXT-GENERATION SEQUENCING STRATEGIES

Whole-genome sequences and next-generation sequencing technologies have been
useful in clarifying phylogeny (2), studying virulence (27), and defining new and known
species of Rickettsiaceae carried by vectors/hosts (45). Whole-genome next-generation
sequencing performed directly from human clinical samples has been used to provide
clinically actionable results for organisms not detected by routine cultures, and it is
anticipated to prove useful for the diagnosis of clinical infection caused by Rickettsi-
aceae, although to our knowledge, this has not been reported yet.

MATRIX-ASSISTED LASER DESORPTION IONIZATION–TIME OF FLIGHT MASS

SPECTROMETRY
Few studies have examined the use of MALDI-TOF MS for the diagnosis of rickettsial
infection. When applied to tick leg protein extracts (hemolymph), MALDI-TOF MS has
been shown promise in identifying ticks and determining if they are infected with
Rickettsiaceae (e.g., Rhipicephalus sanguineus with Rickettsia conorii subsp. conorii and
Dermacentor marginatus with Rickettsia slovaca) and other tick-borne diseases (19, 46,
47), although spectra need to be added, and as these are not available in the
commercially available libraries. This may assist clinical decision-making with respect to
more targeted antimicrobial prophylaxis and surveillance for the development of
disease. One study with the Bruker Autoflex II instrument using whole-cell MALDI-TOF
MS found that O. tsutsugamushi elicited specific macrophage responses which differed

Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.

from those of Coxiella burnetii and other extracellular bacterial pathogens (48), but such
work is preliminary and has not yet been applied to routine clinical diagnostics.

CONCLUSIONS
While serology remains the most widely used clinical diagnostic for the rickettsioses
worldwide, molecular methods complement serology and broaden the diagnostic
window in the acute phase of illness and may allow a definitive diagnosis. Currently,
however, molecular assays are mostly offered in reference laboratories as laboratory-
developed tests, as with the case with culture. Further work in improving serologic
diagnostics includes developing more specific panels to rickettsioses found in different
geographic locales and improving the performance of nonreference (but more acces-
sible) serologic methods, such as ELISA and rapid antigen tests for Rickettsia species.
Apart from identification via traditional taxonomic keys, MALDI-TOF MS and molecular
methods represent newer and more objective methods for vector identification. Sur-
veillance work remains critical for our understanding of disease patterns and so affects
diagnostic testing. For this, and the description of new members of the Rickettsiaceae,
traditional culture-based methods, in conjunction with sequencing, remain important.
The known members of the family Rickettsiaceae causing human disease are likely to
continue to expand. Given that the rickettsioses may clinically resemble many other
febrile illness, or sometime manifest atypically, clinicians will do well to remember that
appropriate diagnostic testing may only be considered if the rickettsioses are consid-
ered part of the differential diagnosis.

August 2018 Volume 56 Issue 8 e01728-17 jcm.asm.org 8

Minireview
REFERENCES
1. Ricketts HT. 1909. A micro-organism which apparently has a specific
relationship to Rocky Mountain spotted fever: a preliminary report.
JAMA 52:379 –380. https://doi.org/10.1001/jama.1909.25420310039002.
2. Abdad MY, Abdallah RA, El Karkouri K, Beye M, Stenos J, Owen H,
Unsworth N, Robertson I, Blacksell SD, Nguyen T-T, Nappez C, Raoult D,
Fenwick S, Fournier P-E. 2017. Rickettsia gravesii sp. nov.: a novel spotted
fever group rickettsia in Western Australian Amblyomma triguttatum
triguttatum ticks. Int J Syst Evol Microbiol 67:3156 –3161. https://doi.org/
10.1099/ijsem.0.001865.
3. Hechemy KE, Stevens RW, Sasowski S, Michaelson EE, Casper EA, Philip
RN. 1979. Discrepancies in Weil-Felix and microimmunofluorescence test
results for Rocky Mountain spotted fever. J Clin Microbiol 9:292–293.
4. Tamura A, Ohashi N, Urakami H, Miyamura S. 1995. Classification of
Rickettsia tsutsugamushi in a new genus, Orientia gen. nov., as Orientia
tsutsugamushi comb. nov. Int J Syst Evol Microbiol 45:589 –591.
5. Izzard L, Fuller A, Blacksell SD, Paris DH, Richards AL, Aukkanit N, Nguyen
C, Jiang J, Fenwick S, Day NP, Graves S, Stenos J. 2010. Isolation of a
novel Orientia species (O. chuto sp. nov.) from a patient infected in
Dubai. J Clin Microbiol 48:4404 – 4409. https://doi.org/10.1128/JCM.0152
6-10.
6. McDade JE. 1990. Historical aspects of Q fever. In Marrie TJ (ed), Q fever,
vol I: the disease. CRC Press, Boca Raton, FL.
7. Abdad MY, Stenos J, Graves S. 2011. Rickettsia felis, an emerging flea-
transmitted human pathogen. Emerg Health Threats J 4:7168. https://
doi.org/10.3402/ehtj.v4i0.7168.
8. Telford SR, III, Parola P. 2007. Chapter 3: arthropods and rickettsiae. In
Raoult D, Parola P (ed), Rickettsial diseases. Informa Healthcare, New
York, NY.
9. Behar A, McCormick LJ, Perlman SJ. 2010. Rickettsia felis infection in a
common household insect pest, Liposcelis bostrychophila (Psocoptera:
Liposcelidae). Appl Environ Microbiol 76:2280 –2285. https://doi.org/10
.1128/AEM.00026-10.
10. Socolovschi C, Pages F, Raoult D. 2012. Rickettsia felis in Aedes albopictus
mosquitoes, Libreville, Gabon. Emerg Infect Dis 18:1687–1689. https://
doi.org/10.3201/eid1810.120178.
11. Socolovschi C, Pages F, Ndiath MO, Ratmanov P, Raoult D. 2012. Rick-
ettsia species in African Anopheles mosquitoes. PLoS One 7:e48254.
https://doi.org/10.1371/journal.pone.0048254.
12. Dieme C, Bechah Y, Socolovshi C, Audoly G, Berenger J-M, Faye O, Raoult
D, Parola P. 2015. Transmission potential of Rickettsia felis infection by
Anopheles gambiae mosquitoes. Proc Natl Acad Sci U S A 112:
8088 – 8093. https://doi.org/10.1073/pnas.1413835112.
13. Walker DH, Ismail N, Olano JP, Valbuena GA, McBride J. 2007. Chapter 2:
pathogenesis, immunity, pathology, and pathophysiology in rickettsial
diseases. In Raoult D, Parola P (ed), Rickettsial diseases. Informa Health-
care, New York, NY.
14. Raoult D, Woodward T, Dumler JS. 2004. The history of epidemic typhus.
Infect Dis Clin North Am 18:127–140. https://doi.org/10.1016/S0891
-5520(03)00093-X.
15. Balcells ME, Rabagliati R, Garcia P, Poggi H, Oddó D, Concha M, Abarca
K, Jiang J, Kelly DJ, Richards AL, Fuerst PA. 2011. Endemic scrub typhus-
like illness, Chile. Emerg Infect Dis 17:1659 –1663. https://doi.org/10
.3201/eid1709.100960.
16. Horton KC, Jiang J, Maina A, Dueger E, Zayed A, Ahmed AA, Pimentel G,
Richards AL. 2016. Evidence of Rickettsia and Orientia infections among
abattoir workers in Djibouti. Am J Trop Med Hyg 95:462– 465. https://
doi.org/10.4269/ajtmh.15-0775.
17. Ghorbani RP, Ghorbani AJ, Jain MK, Walker DH. 1997. A case of scrub
typhus probably acquired in Africa. Clin Infect Dis 25:1473–1474. https://
doi.org/10.1086/516990.
18. Lv J, Wu S, Zhang Y, Chen Y, Feng C, Yuan X, Jia G, Deng J, Wang C, Wang
Q, Mei L, Lin X. 2014. Assessment of four DNA fragments (COI, 16S rDNA,
ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida).
Parasit Vectors 7:93. https://doi.org/10.1186/1756-3305-7-93.
19. Yssouf A, Flaudrops C, Drali R, Kernif T, Socolovschi C, Berenger J-M,
Raoult D, Parola P. 2013. Matrix-assisted laser desorption ionization–time
of flight mass spectrometry for the rapid identification of tick vectors. J
Clin Microbiol 51:522–528. https://doi.org/10.1128/JCM.02665-12.
20. Yssouf A, Socolovschi C, Leulmi H, Kernif T, Bitam I, Audoly G, Almeras L,
Raoult D, Parola P. 2014. Identification of flea species using MALDI-TOF/
August 2018 Volume 56 Issue 8 e01728-17
Journal of Clinical Microbiology
MS. Comp Immunol Microbiol Infect Dis 37:153–157. https://doi.org/10
.1016/j.cimid.2014.05.002.
21. Rothen J, Githaka N, Kanduma EG, Olds C, Pfluger V, Mwaura S, Bishop
RP, Daubenberger C. 2016. Matrix-assisted laser desorption/ionization
time of flight mass spectrometry for comprehensive indexing of East
African ixodid tick species. Parasit Vectors 9:151. https://doi.org/10.1186/
s13071-016-1424-6.
22. Kumsa B, Laroche M, Almeras L, Mediannikov O, Raoult D, Parola P. 2016.
Morphological, molecular and MALDI-TOF mass spectrometry identifica-
tion of ixodid tick species collected in Oromia, Ethiopia. Parasitol Res
115:4199 – 4210. https://doi.org/10.1007/s00436-016-5197-9.
23. Tandina F, Almeras L, Kone AK, Doumbo OK, Raoult D, Parola P. 2016.
Use of MALDI-TOF MS and culturomics to identify mosquitoes and their
midgut microbiota. Parasit Vectors 9:495. https://doi.org/10.1186/s1307
1-016-1776-y.
24. Walker DH, Fishbein DB. 1991. Epidemiology of rickettsial diseases. Eur J
Epidemiol 7:237–245. https://doi.org/10.1007/BF00145672.
25. Abdad MY, Cook A, Dyer J, Stenos J, Fenwick SG. 2014. Seroepidemio-
logical study of outdoor recreationists’ exposure to spotted fever group
rickettsia in Western Australia. Am J Trop Med Hyg 91:584 –588. https://
doi.org/10.4269/ajtmh.14-0102.
26. Eremeeva ME, Silverman DJ. 1998. Rickettsia rickettsii infection of the
EA.hy 926 endothelial cell line: morphological response to infection and
evidence for oxidative injury. Microbiology 144:2037–2048. https://doi
.org/10.1099/00221287-144-8-2037.
27. Fournier PE, El Karkouri K, Leroy Q, Robert C, Guiumelli B, Renesto P,
Socolovschi C, Parola P, Audic S, Raoult D. 2009. Analysis of Rickettsia
africae genome reveals that virulence acquisition in Rickettsia species
may be explained by genome reduction. BMC Genomics 10:166. https://
doi.org/10.1186/1471-2164-10-166.
28. Valbuena G, Walker DH. 2008. Changes in the adherens junctions of
human endothelial cells infected with spotted fever group rickettsiae.
Virchows Arch 446:379–382. https://doi.org/10.1007/s00428-004-1165-3.
29. Woods ME, Olano JP. 2008. Host defenses to Rickettsia rickettsii infection
contribute to increased microvascular permeability in human cerebral
endothelial cells. J Clin Immunol 28:174 –185. https://doi.org/10.1007/
s10875-007-9140-9.
30. Parola P, Paddock C, Socolovschi C, Labruna M, Mediannikov O, Kernif T,
Abdad MY, Stenos J, Bitam I, Fournier PE, Raoult D. 2013. Update on
tick-borne rickettsioses around the world: a geographic approach. Clin
Microbiol Rev 26:657–702. https://doi.org/10.1128/CMR.00032-13.
31. Nickerson A, Marik PE. 2012. Life-threatening ANCA-positive vasculitis
Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.
associated with rickettsial infection. BMJ Case Rep 2012:bcr0320125993.
10.1136/bcr.03.2012.5993.
32. Raoult D, Lakos A, Fenollar F, Beytout J, Brouqui P, Fournier PE. 2002.
Spotless rickettsiosis caused by Rickettsia slovaca and associated with
Dermacentor ticks. Clin Infect Dis 34:1331–1336. https://doi.org/10.1086/
340100.
33. Cruickshank R. 1927. The Weil-Felix reaction in typhus fever. Epidemiol
Infect 27:64 – 69. https://doi.org/10.1017/S0022172400031818.
34. Giménez DF. 1964. Staining rickettsiae in yolk-sac cultures. Stain Technol
39:135–140. https://doi.org/10.3109/10520296409061219.
35. Philip RN, Casper EA, Ormsbee RA, Peacock MG, Burgdorfer W. 1976.
Microimmunofluorescence test for the serological study of Rocky Moun-
tain spotted fever and typhus. J Clin Microbiol 3:51– 61.
36. Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoersdorff A, Blanco JR,
Caruso G, Cinco M, Fournier PE, Francavilla E, Jensenius M, Kazar J, Laferl
H, Lakos A, Lotric Furlan S, Maurin M, Oteo JA, Parola P, Perez-Eid C, Peter
O, Postic D, Raoult D, Tellez A, Tselentis Y, Wilske B, ESCMID Study Group
on Coxiella, Anaplasma, Rickettsia, and Bartonella; European Network for
Surveillance of Tick-Borne Diseases. 2004. Guidelines for the diagnosis of
tick-borne bacterial diseases in Europe. Clin Microbiol Infect 10:
1108 –1132. https://doi.org/10.1111/j.1469-0691.2004.01019.x.
37. Paris DH, Dumler JS. 2016. State of the art of diagnosis of rickettsial
diseases: the use of blood specimens for diagnosis of scrub typhus,
spotted fever group rickettsiosis, and murine typhus. Curr Opin Infect
Dis 29:433– 439. https://doi.org/10.1097/QCO.0000000000000298.
38. Fournier P-E, Jensenius M, Laferl H, Vene S, Raoult D. 2002. Kinetics of
antibody responses in Rickettsia africae and Rickettsia conorii infections.
Clin Diagn Lab Immunol 9:324 –328. https://doi.org/10.1128/CDLI.9.2
.324-328.2002.
jcm.asm.org 9
Minireview
39. Kim YJ, Yeo SJ, Park SJ, Woo YJ, Kim MW, Kim SH, Chang IA, Jeon SH, Park
BJ, Song GJ, Lee MG, Kim IS, Kim YW. 2013. Improvement of the
diagnostic sensitivity of scrub typhus using a mixture of recombinant
antigens derived from Orientia tsutsugamushi serotypes. J Korean Med
Sci 28:672– 679. https://doi.org/10.3346/jkms.2013.28.5.672.
40. Portillo A, de Sousa R, Santibanez S, Duarte A, Edouard S, Fonseca IP,
Marques C, Novakova M, Palomar AM, Santos M, Silaghi C, Tomassone L,
Zuquete S, Oteo JA. 2017. Guidelines for the detection of Rickettsia spp.
Vector Borne Zoonotic Dis 17:23–32. https://doi.org/10.1089/vbz.2016
.1966.
41. Luce-Fedrow A, Mullins K, Kostik AP, St. John HK, Jiang J, Richards AL.
2015. Strategies for detecting rickettsiae and diagnosing rickettsial dis-
eases. Future Microbiol 10:537–564. https://doi.org/10.2217/fmb.14.141.
42. Mouffok N, Socolovschi C, Benabdellah A, Renvoise A, Parola P, Raoult D.
2011. Diagnosis of rickettsioses from eschar swab samples, Algeria. Emerg
Infect Dis 17:1968–1969. https://doi.org/10.3201/eid1710.110332.
43. Fournier PE, Raoult D. 2004. Suicide PCR on skin biopsy specimens for
diagnosis of rickettsioses. J Clin Microbiol 42:3428 –3434. https://doi.org/
10.1128/JCM.42.8.3428-3434.2004.
44. Pike RM. 1976. Laboratory-associated infections: summary and analysis
of 3921 cases. Health Lab Sci 13:105–114.
45. Vayssier-Taussat M, Moutailler S, Michelet L, Devillers E, Bonnet S, Cheval
J, Hebert C, Eloit M. 2013. Next generation sequencing uncovers unex-
pected bacterial pathogens in ticks in Western Europe. PLoS One
8:e81439. https://doi.org/10.1371/journal.pone.0081439.
46. Yssouf A, Almeras L, Terras J, Socolovschi C, Raoult D, Parola P. 2015.
Detection of Rickettsia spp. in ticks by MALDI-TOF MS. PLoS Negl Trop
Dis 9:e0003473. https://doi.org/10.1371/journal.pntd.0003473.
47. Yssouf A, Almeras L, Berenger J-M, Laroche M, Raoult D, Parola P. 2015.
Identification of tick species and disseminate pathogen using hemo-
lymph by MALDI-TOF MS. Ticks Tick Borne Dis 6:579 –586. https://doi
.org/10.1016/j.ttbdis.2015.04.013.
48. Ouedraogo R, Daumas A, Ghigo E, Capo C, Mege J-L, Textoris J. 2012.
Whole-cell MALDI-TOF MS: a new tool to assess the multifaceted acti-
vation of macrophages. J Proteomics 75:5523–5532. https://doi.org/10
.1016/j.jprot.2012.07.046.
49. Paddock CD, Sumner JW, Comer JA, Zaki SR, Goldsmith CS, Goddard J,
McLellan SL, Tamminga CL, Ohl CA. 2004. Rickettsia parkeri: a newly
recognized cause of spotted fever rickettsiosis in the United States. Clin
Infect Dis 38:805– 811. https://doi.org/10.1086/381894.
50. Paddock CD, Greer PW, Ferebee TL, Singleton J, Jr, McKechnie DB,
Treadwell TA, Krebs JW, Clarke MJ, Holman RC, Olson JG, Childs JE, Zaki
SR. 1999. Hidden mortality attributable to Rocky Mountain spotted fever:
immunohistochemical detection of fatal, serologically unconfirmed dis-
ease. J Infect Dis 179:1469 –1476. https://doi.org/10.1086/314776.
51. Kato CY, Chung IH, Robinson LK, Austin AL, Dasch GA, Massung RF. 2013.
Assessment of real-time PCR assay for detection of Rickettsia spp. and
Rickettsia rickettsii in banked clinical samples. J Clin Microbiol 51:
314 –317. https://doi.org/10.1128/JCM.01723-12.
52. Rolain JM, Bourry O, Davoust B, Raoult D. 2005. Bartonella quintana and
Rickettsia felis in Gabon. Emerg Infect Dis 11:1742–1744. https://doi.org/
10.3201/eid1111.050861.
August 2018 Volume 56 Issue 8 e01728-17
Journal of Clinical Microbiology
53. Teysseire N, Chiche-Portiche C, Raoult D. 1992. Intracellular movements
of Rickettsia conorii and R. typhi based on actin polymerization. Res
Microbiol 143:821– 829. https://doi.org/10.1016/0923-2508(92)90069-Z.
54. La Scola B, Raoult D. 1997. Laboratory diagnosis of rickettsioses: current
approaches to diagnosis of old and new rickettsial diseases. J Clin
Microbiol 35:2715–2727.
55. Bechah Y, Socolovschi C, Raoult D. 2011. Identification of rickettsial
infections by using cutaneous swab specimens and PCR. Emerg Infect
Dis 17:83– 86. https://doi.org/10.3201/eid1701.100854.
56. Renvoisé A, Rolain JM, Socolovschi C, Raoult D. 2012. Widespread use of
real-time PCR for rickettsial diagnosis. FEMS Immunol Med Microbiol
64:126 –129. https://doi.org/10.1111/j.1574-695X.2011.00899.x.
57. Fournier P-E, Roux V, Raoult D. 1998. Phylogenetic analysis of spotted
fever group rickettsiae by study of the outer surface protein rOmpA. Int
J Syst Bacteriol 48:839 – 849. https://doi.org/10.1099/00207713-48-3-839.
58. Gouriet F, Fenollar F, Patrice JY, Drancourt M, Raoult D. 2005. Use of
shell-vial cell culture assay for isolation of bacteria from clinical
specimens: 13 years of experience. J Clin Microbiol 43:4993–5002.
https://doi.org/10.1128/JCM.43.10.4993-5002.2005.
59. Graves SR, Dwyer BW, McColl D, McDade JE. 1991. Flinders Island
spotted fever: a newly recognised endemic focus of tick typhus in Bass
Strait. Part 2. Serological investigations. Med J Aust 154:99 –104.
60. Teoh YT, Hii SF, Stevenson MA, Graves S, Rees R, Stenos J, Traub RJ. 2017.
Serological evidence of exposure to Rickettsia felis and Rickettsia typhi
in Australian veterinarians. Parasit Vectors 10:129. https://doi.org/10
.1186/s13071-017-2075-y.
61. Stenos J, Graves S, Izzard L. 2010. Rickettsia, p 197–200. In Schuller M,
Sloots TP, James G, Halliday CL, Carter IWJ (ed), PCR for clinical
microbiology: an Australian and international perspective. Springer,
Heidelberg, Germany.
62. Stenos J, Graves SR, Unsworth NB. 2005. A highly sensitive and specific
PCR assay for the detection of spotted fever and typhus group rickett-
siae. Am J Trop Med Hyg 73:1083–1085.
63. Harris PNA, Oltvolgyi C, Islam A, Hussain-Yusuf H, Loewenthal MR,
Vincent G, Stenos J, Graves S. 2016. An outbreak of scrub typhus in
military personnel despite protocols for antibiotic prophylaxis: doxycy-
cline resistance excluded by a quantitative PCR-based susceptibility
assay. Microbes Infect 18:406 – 411. https://doi.org/10.1016/j.micinf.2016
.03.006.
64. Webb L, Carl M, Malloy DC, Dasch GA, Azad AF. 1990. Detection of
murine typhus infection in fleas by using the polymerase chain reaction.
J Clin Microbiol 28:530 –534.
65. Unsworth NB, Stenos J, Graves SR, Faa AG, Cox GE, Dyer JR, Boutlis CS,
Downloaded from https://journals.asm.org/journal/jcm on 12 June 2022 by 118.70.17.135.
Lane AM, Shaw MD, Robson J, Nissen MD. 2007. Flinders Island spotted
fever rickettsioses caused by “marmionii” strain of Rickettsia honei,
eastern Australia. Emerg Infect Dis 13:566 –573. https://doi.org/10.3201/
eid1304.060087.
66. Derne B, Weinstein P, Musso D, Lau C. 2015. Distribution of rickettsioses
in Oceania: past patterns and implications for the future. Acta Trop
143:121–133. https://doi.org/10.1016/j.actatropica.2014.10.012.
jcm.asm.org 10