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Author affiliation: US Centers for Disease Control and Prevention, Central Asia Office, Almaty, Kazakhstan (Y.V. Bumburidi, D.V. Berezovskiy, B.T. Zhakipbayeva, R.Z. Horth, A.J. Millman); Centers for Disease Control and Prevention, Atlanta, Georgia, USA (W.L. Nicholson, G.E. Zemtsova, V. Seffren); Almaty Branch of the National Center for Biotechnology, Almaty (E.R. Maltseva, Z.A. Berdygulova, Y.O. Ostapchuk, Y.V. Perfilyeva, A.M. Dmitrovskiy); Pavlodar Regional Hospital named after Sultanov, Pavlodar, Kazakhstan (Z.K. Dukayeva); Scientific-Practical Center for Sanitary-Epidemiological Expertise and Monitoring, National Center for Public Healthcare, Ministry of Health, Almaty (Z.Z. Shapiyeva)
Spotted fever group rickettsioses (SFGR) are tickborne diseases that are typically characterized by nonspecific symptoms such as fever, headache, and muscle pain caused by closely related bacteria. A rash or eschar (a black scab) is present in some but not all infections, although manifestations vary depending on the rickettsia species. That variability in clinical manifestations creates challenges for diagnosing SFGR. SFGR are treatable; however, untreated SFGR cases can result in severe complications and death in otherwise healthy persons. The severity of rickettsiosis depends on the Rickettsia species with which the patient is infected (1–3).
Tickborne rickettsial bacteria cannot survive on their own in the environment; they depend on a complex lifecycle involving small animal hosts, such as rodents, and can be transmitted transovarially, with ticks serving as both the primary vector and reservoirs. Infected ticks transmit spotted fever group Rickettsia to humans through bites. Although SFGR are globally distributed, the actual burden of illness remains unknown because of underrecognition, limited testing, and underreporting. The geographic expansion and increase in cases of SFGR worldwide can be attributed to increased human–animal contact resulting from land-use changes, climate variations, improved clinical awareness of tickborne diseases, and the increased availability of novel molecular diagnostic techniques (4–7).
Rickettsial diseases are notifiable in Kazakhstan. By the end of 2019, SFGR were recognized as endemic in 4 regions (East Kazakhstan, Kyzylorda, Pavlodar, and North Kazakhstan). Several different Rickettsia species have been identified in ticks in Kazakhstan, including R. sibirica, R. raoultii, R. slovaca, R. aeschlimannii–like organism, and R. conorii (8–12). In addition, R. sibirica, R. rauoltii, and R. heilongjiangensis were identified in Russia in a region bordering the Pavlodar oblast in Kazakhstan (13,14).
The Pavlodar region has the second highest reported rate of rickettsioses in Kazakhstan. Unlike Kyzylorda, the region with the highest reported burden, no studies have documented circulating rickettsial diseases in Pavlodar (15). On the basis of data available in the national surveillance system (Infectious Diseases in the Republic of Kazakhstan), during 2016–2018 the Pavlodar region reported 146 clinically identified cases of SFGR. Because SFGR signs and symptoms can be nonspecific and routine laboratory confirmation is not available in Kazakhstan, cases are often missed or misdiagnosed. Therefore, the true burden of SFGR is likely higher than reported. We assessed the clinical and epidemiologic characteristics of the disease in humans (including disease burden, clinical features, and etiologic agent) and the molecular identification of Rickettsia species in ticks collected from the study region.
Study Design
We conducted a cross-sectional study during April–October 2019 among persons hospitalized with signs and symptoms consistent with SFGR. We selected 2 regional infectious disease hospitals in the city of Pavlodar (including a pediatric hospital), which serve as referral hospitals, and 4 district hospitals that reported rickettsial disease cases in 2016–2018. Participants >6 years of age were recruited during April–October, the period when the risk for seasonal tickborne transmission was highest on the basis of active surveillance for ticks in the region.
Enrollment and Inclusion Criteria
Participants were persons hospitalized during the study period with symptoms consistent with Rickettsia-like illnesses (suspected cases of rickettsiosis), such as fever (>38°C) and >1 of the following clinical signs or symptoms: rash, eschar, headache, myalgia, anemia, thrombocytopenia, hepatic transaminase elevation, or clinician suspicions of rickettsiosis (16). Participants provided written informed consent; for persons <18 years of age, the primary caregiver consented. We excluded persons who were not residents of the Pavlodar region (residence was defined as having lived <2 weeks outside the Pavlodar region during the study period before disease onset) and children <6 years of age.
Data Collection
Clinicians at the sentinel sites were trained to collect data at the time of patient’s inclusion to apply the standard case definition of Rickettsia-like illness and to notify the health department of new suspected cases of rickettsiosis. Upon receiving notifications, trained surveillance officers visited the hospitals and obtained written informed consent from hospitalized patients or their primary caregivers to participate in the study. Surveillance officers completed paper case investigation forms containing demographic, clinical, laboratory, and behavioral data. They also abstracted clinical and laboratory data from the medical and laboratory records and entered data into EpiInfo 7.2 (https://www.cdc.gov/epiinfo).
Specimen Collection
We collected acute-phase samples, including rash or eschar biopsies or swab specimens and serum samples, at admission (17,18). We collected second serum samples (convalescent) 2–3 weeks after admission from patients with no rash or eschar or whose rash or eschar samples were PCR negative. We collected second serum samples were collected at the hospital before discharge; however, if duration of hospitalization was <2 weeks, we invited patients by phone to the territorial polyclinic for specimen collection. We collected rash and eschar biopsies and swab samples under sterile conditions.
We stored specimens in refrigerators (4°C) at the hospital for up to 24 hours before being transported to the Pavlodar regional laboratory for PCR testing (rash and eschar samples) and freezing (at −20°C). Samples were subsequently sent to the Central Reference Laboratory for sequencing.
Laboratory Testing of Human Specimens
PCR Testing and Sequencing
We extracted DNA from rash and eschar specimens using the QIAGEN DNA Mini Kit (QIAGEN, https://www.qiagen.com) according to the manufacturer’s instructions. We used a real-time genus-specific Rickettsia (pan-Rickettsia) assay targeting the 23S rRNA as a screening assay (19). We further tested specimens positive for rickettsial DNA in the pan-Rickettsia assay by using a seminested PCR to amplify and subsequently sequence a fragment of the gene encoding the protein rOmpA, appropriate for differentiation of 21 Rickettsia species, including the most commonly reported species circulating in the investigated region (20). We used the human housekeeping glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, yielding a 157-bp fragment as an internal control (21). We reported PCR test results to the hospital within 7 days.
We performed elution of DNA fragments from the agarose gel using a QIAquick gel extraction kit (QIAGEN). We then sequenced the purified DNA fragments in both directions using a BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI 3500XL genetic analyzer (Thermo Fisher Scientific, https://www.thermofisher.com).
Individual gene sequences underwent multiple sequence alignment using the MUSCLE algorithm in the MEGA X program (22). We constructed a maximum-likelihood phylogenetic tree using Rickettsia species from various phylogenetic groups as references. We used nonparametric bootstrapping with 500 replicates to calculate confidence estimates.
Indirect Immunofluorescence Antibody Assay for Serum Samples
We tested serum samples using an indirect immunofluorescence antibody assay (IFA). We prepared multiantigen slides with strains of R. raoultii, R. sibirica, and R. slovaca. We used serum containing human IgG to spotted fever group Rickettsia as positive controls. Slides and positive controls were provided by the Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA). For determination of acute case status, positive samples were titrated to determine endpoint titer and to examine 4-fold titer increases. Comparison of titers provided presumptive etiology for this study (23). We defined in IFA the species of Rickettsia implicated in the disease for patients with paired or single serum samples as the species with the highest difference in titers or the highest titer. In case of equivalent titers for >1 species, we considered the results unidentified species of SFGR. We considered a single serum IgG titer of 1/64 a minimum positive titer, which supports Rickettsiosis for patients for surveillance purposes at CDC with a clinically compatible acute illness and with an epidemiologic history of the disease (5). However, to increase the specificity for identification of clinical cases, we included only patients with 1 serum sample with titers >1:128 (probable cases in our study) in the data analysis (24).
Sampling and Data Collection of Ticks
Two groups collected ticks for 4 days (May 13–16) in 2 districts of Pavlodar region. Using a map and a hand-held global positioning system tracker, we selected 4 ordinal points within each designated village for tick collection. Groups conducted sampling for >3 person-hours at each location within a suitable habitat (i.e., woods, grassland, and trail margins) (25).
We collected ticks using the dragging method, in which light-colored flannel material is dragged behind the collector by a rope handle attached to a wooden base. We used a flagging method in areas with high vegetation density in which the drag cloth could not be pulled between plants. For the flagging method, we used the material attached to 1 end of a wooden rod like a flag to sweep it through vegetation (26). We collected ticks in the second week of May 2019. This window coincided with peak activity for Dermacentor ticks, which are known to carry Rickettsia species (27–29).
We identified tick species, development stage, and sex morphologically using a stereomicroscope according to published tick specification guidelines (30,31). We then placed adult ticks individually into tubes containing 70% ethanol for storage before sending them to the Central Reference Laboratory for testing.
Laboratory Testing of Ticks
We homogenized each tick specimen individually using the TissueLyser II (QIAGEN). We extracted total nucleic acid using the PrepMan Ultra Sample Preparation Reagent (ThermoFisher Scientific) according to the manufacturer’s instructions. We used Rickettsia genus-specific (pan-Rickettsia) real-time PCRfor testing each tick individually. We tested positive samples using a seminested PCR for amplification and sequencing targeting rOmpA gene to determine species, as described previously for human samples.
Key Definitions
We defined a suspected case of rickettsiosis as illness in a person hospitalized with fever (temperature >38°C) and >1 of the following clinical signs or symptoms: rash, eschar, headache, myalgia, anemia, thrombocytopenia, hepatic transaminase elevation (16), or suspicion of rickettsiosis by the clinician. We defined a probable case of rickettsiosis as illness in a person meeting the definition of a suspected case of rickettsiosis with only 1 serum sample available, with IFA Rickettsia IgG titers >1:128 (24). We defined a laboratory-confirmed case of rickettsiosis as illness in a person meeting the definition of aa suspected case of rickettsiosis with a positive PCR result for blood, eschar, or skin, or a 4-fold increase in IFA Rickettsia IgG titers (R. raoultii, R. sibirica, or R. slovaca) from a sample taken upon admission and a convalescent sample (16). Severe disease was defined by clinicians as having any of the following: acute respiratory distress syndrome (shortness of breath [respiration rate >25 breaths/min], cyanosis, bilateral infiltrates on the lung radiograph, progressive hypoxemia [SpO2 ≤90%]); disseminated intravascular coagulation (blood clotting time >12 s, plasma D-dimer >250 µg/L, platelet count <120 × 109/L, plasma fibrinogen <1.5 g/L, prothrombin time (PT) >16 s); or renal failure (oliguria up to anuria and accompanied by proteinuria of >0.5 g/L, serum creatinine >117 mkmol/L, blood serum urea >8 mmol/L).
Data Analysis
Our data analysis only included laboratory-confirmed and probable cases. Electronic questionnaire development, data entry, data cleaning, and analysis were done in EpiInfo 7.2. We used the χ2 or Fisher exact tests to assess the differences in clinical symptoms by Rickettsia species. We considered a p value of <0.05 significant.
Ethics Statement
This study was approved by the Ethical Committee of the Kazakh Medical University (“The Highest School of Public Health”) (protocol no. IRB-A096, April 2019) and extended (protocol no. IRB-A12, September 2020). This activity was reviewed by CDC, deemed not research, and was conducted consistent with applicable federal law and CDC policy (e.g., 45 C.F.R. part 46, 21 C.F.R. part 56; 42 U.S.C. §241(d); 5 U.S.C. §552a; 44 U.S.C. §3501 et seq.).
Characteristics of Participants
We obtained consent from and enrolled 105 persons hospitalized with suspected rickettsiosis; among those, 62 (59%) were determined to be laboratory-confirmed or probable rickettsiosis. Among participants with rickettsiosis, 20 (32%) were confirmed by IFA in paired samples (a 4-fold increase of IgG titers to 1 of 3 investigated Rickettsia species), 35 (56%) by PCR, and 7 (11%) as probable cases in a single available acute sample with a titer of >1:128 by IFA. We did not include 10 patients with a titer of 1:64 with a single available IFA in the analysis. We identified 3 Rickettsia species among the participants: R. sibirica (47%), R. raoultii (31%), and R. slovaca (6%). No Rickettsia-specific species were identified in 16% of participants. (Appendix Table 1).
Of the 62 participants with acute rickettsiosis, 85% were adults (>18 years of age), 48% were male, 66% lived in rural areas or suburbs, 55% owned a cat or dog, and 23% owned livestock (Table 1). In the 2 weeks before disease onset, 53% had worked in a garden or orchard, 48% had noted a tick bite, and 31% had been in an open countryside area. Fifty-eight percent of participants reported usually removing ticks with their bare hands. To protect themselves against tick bites, 32% reported taking some protective measures, such as wearing long-sleeved clothing or gloves or using repellents; 68% reported taking no measures (Table 1).
We detected rickettsiosis cases in all sentinel sites. Eleven percent of cases were primarily referrals from nonsentinel sites to the regional hospitals in Pavlodar City. Based on the place of residence, we identified rickettsioses cases in 85% (11/13) of the administrative territories of Pavlodar region.
Rickettsiosis cases peaked in May (53% of cases). Although R. sibirica cases occurred throughout the endemic season, R. raoultii cases occurred predominantly in the summer months (Appendix Figure).
Clinical Features
The most common signs and symptoms among those with rickettsiosis were fever (100%), headache (90%), rash (79%), myalgia (61%), and eschar (48%). More than half (53%) had thrombocytopenia, and 39% had elevated aspartate aminotransferase. Fifteen percent had severe disease; no deaths occurred (Table 2).
Rash, eschar, or swollen lymph nodes were present in 87% and absent in 13% of patients. Eschar was the most common among participants with R. sibirica and least common among those with R. raoultii with a difference in prevalence of 7.5 times (crude prevalence ratio 7.5 [95% CI –2.0 to 28.3]; p<0.001). Severe disease was also most common among participants with R. sibirica (21%) and least common among those with R. raoultii (5%) or R. slovaca (0%). (Table 2).
Rickettsia Species in D. marginatus and D. reticulatus Ticks
We collected 959 ticks; 64% were identified as D. reticulatus ticks and 36% were D. marginatus ticks. R. raoultii was detected in 46 (5%), SFG rickettsiae in 37 (4%), and R. sibirica in only 2 ticks. In both tick species, R. raoultii was in almost equivalent proportions of 4% (13) in D. marginatus ticks to 5% (33) in D. reticulatus ticks (Appendix Table 2).
Rickettsioses were detected in ticks or humans in 85% of districts in the Pavlodar region. By establishing sentinel sites and enhanced laboratory testing for surveillance of rickettsioses, we observed a 27% increase in the number of detected cases compared with the mean of the previous 3 years (62 vs. 49). R. sibirica and R. raoultii were the most frequent etiologic agents identified among cases. Those findings highlight missed opportunities for diagnosis and treatment of rickettsioses.
Our observation of a 27% increase in the number of detected cases compared with the mean of the previous 3 years after we established sentinel sites for surveillance is concerning because of potential gaps in medical interventions for persons with rickettsioses. Although most persons had mild illness, 15% had severe disease. Empirical treatment is most effective at preventing death and severe illness when started within the first 5 days after symptom onset. Although our findings do not enable us to evaluate missed opportunities for early treatment, the substantial underdetection of cases in an endemic region suggests there could be infected persons who develop severe disease or are at risk for developing severe disease who do not receive the appropriate diagnosis and early treatment by healthcare providers (32,33).
Consistent with known clinical manifestations of SFGR, fever, headache, and myalgia were the most common symptoms among case-patients (5). Cutaneous findings occurred frequently, but 13% of case-patients had none. Cutaneous signs can differ widely by species. The presence of eschar has been reported to be 7 times less common among persons with R. raoultii than R. sibirica (5,34,35), and we found that 26% of participants with R. raoultii did not have cutaneous signs. Because of the lack of a standard case definition and clinical protocol for rickettsioses in Kazakhstan, patients without cutaneous signs might not receive appropriate diagnostics or treatment for rickettsioses. The inclusion of headache, fever, or myalgia in the clinical case definition for suspected rickettsioses could contribute to improved recognition of the disease and faster initiation of appropriate treatment.
D. marginatus and D. reticulatus ticks were the only species identified in our study. The detection of R. raoultii and R. sibirica in those ticks suggests that they might serve as vectors of SFGR in the Pavlodar region. However, given that only 3%–5% of the tested ticks were positive for any Rickettsia species, this association should be interpreted with caution. The vector capacity of D. marginatus and D. reticulatus ticks in transmitting R. raoultii has been demonstrated in previous studies (11). Transmission of R. sibirica is generally associated with Haemaphysalis concinna, Dermacentor spp., and Hyalomma spp. ticks (8,13,36). H. concinna ticks have been found in Pavlodar region by a previous study (37). Therefore, tick species other than those identified in this study might also contribute to the transmission of Rickettsia in the region. This information can assist in prompting clinicians to suspect rickettsioses among persons who are bitten by ticks of those species. In addition, persons bitten by those ticks can be given information about the need to self-monitor for fever, headache, myalgia, eschar, or rash and to seek medical care if those signs develop.
We also identified gaps in the use of protective clothing and tick removal practices. Regular use of repellents and protective clothing that covers the skin are recommended to decrease the risk for tick bites when spending time outdoors in tick environments in rickettsioses-endemic regions (38,39). Public health risk communication about preventing tick exposure can help increase awareness of tickborne diseases and ways to reduce the risk for tick bites.
The first limitation of our study is that fever was a required symptom for participation, so cases of asymptomatic SFGR could have been missed. We also did not survey all hospitals in the region. We included the reference hospital where the most severe cases are transferred. Those limitations might have resulted in an underestimation of the total number of cases in the region but might also have resulted in an overestimation of cases of severe disease among persons with SFGR. PCR confirmation of the rickettsia species in our study was obtained on the basis of a short sequence of 1 gene (rOmpA), which is commonly used in Rickettsia diagnostics (19). However, relying on a single, short amplicon limits the confidence with which one can make a precise species diagnosis because of the high genetic similarity and conserved nature of Rickettsia genomes. We could not identify species of Rickettsia for 6 rickettsioses and 37 ticks (SFGR without species identification), which might have been related to the sequencing methodology of only using the short sequence of 1 gene. The methods used could possibly have contributed to misclassification or missed diagnoses of species. Sequencing of 2 or 3 genes would have improved the accuracy and reliability of species differentiation. Last, we collected ticks only from 2 districts, which could have resulted in incomplete identification of ticks and Rickettsia species diversity (40).
In conclusion, we identified rickettsia-positive humans in at least 85% of the Pavlodar region districts and positive ticks in all 2 investigated districts. The predominant species identified among persons hospitalized with rickettsioses were R. sibirica and R. raoultii. Among the collected ticks, Rickettsia prevalence was ≈9%, and 5% carried R. raoultii. Almost 80% of persons with Rickettsia had a rash, half had an eschar, and 13% had no SFGR cutaneous signs or lymphadenopathy. More than half of participants removed ticks with bare hands, and almost 70% reported not using any protection against tick bites. Enhanced routine testing in the region is key to improving clinical management and disease surveillance. Increased awareness among clinicians and the general population about rickettsial symptoms and risk for tick bites could help reduce disease transmission and improve medical care.
Dr. Bumburidi graduated from the Belarus State Medical University and the Field Epidemiology Training Program (CDC, Kazakhstan). She works at the US Centers for Disease Control and Prevention office in Kazakhstan to improve the public health surveillance of tickborne diseases (Crimean-Congo hemorrhagic fever, rickettsioses, tickborne encephalitis), respiratory diseases, and COVID-19.
