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Laboratory-acquired Scrub Typhus and Murine Typhus Infections: The Argument for a Risk-based Approach to Biosafety Requirements for Orientia tsutsugamushi and Rickettsia typhi Laboratory Activities
research-article
Author(s):
Stuart D Blacksell
1
,
2
,
3 ,
Matthew T Robinson
2
,
3 ,
Paul N Newton
2
,
3 ,
Nicholas P J Day
1
,
3
Publication date (Electronic):
10 August 2018
Journal:
Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America
Publisher:
Oxford University Press
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Abstract
The highest-risk activities for scrub/murine typhus laboratory-acquired infections were working with infectious laboratory animals. Eight scrub typhus deaths occurred during the preantibiotic era. Risk-based biosafety approaches would improve efficiencies of in vitro/in vivo growth of scrub/murine typhus.
Abstract
This study examined the literature on laboratory-acquired infections (LAIs) associated with scrub typhus ( Orientia tsutsugamushi) and murine typhus ( Rickettsia typhi) research to provide an evidence base for biosafety and biocontainment. Scrub typhus LAIs were documented in 25 individuals, from 1931 to 2000 with 8 (32%) deaths during the preantibiotic era. There were 35 murine typhus LAI reports and no deaths. Results indicated that the highest-risk activities were working with infectious laboratory animals involving significant aerosol exposures, accidental self-inoculation, or bite-related infections. A risk-based biosafety approach for in vitro and in vivo culture of O. tsutsugamushi and R. typhi would require that only high-risk activities (animal work or large culture volumes) be performed in high-containment biosafety level (BSL) 3 laboratories. We argue that relatively low-risk activities including inoculation of cell cultures or the early stages of in vitro growth using low volumes/low concentrations of infectious materials can be performed safely in BSL-2 laboratories within a biological safety cabinet.
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Most cited references44
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Isolation of a novel Orientia species (O. chuto sp. nov.) from a patient infected in Dubai.
Alexander L. Richards, Richard Fuller, Daniel Paris … (2010)
In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patient's acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene (rrs), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name "Orientia chuto," and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.
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Rickettsial Infections and Fever, Vientiane, Laos
The Lao People's Democratic Republic (Laos) is situated mostly east of the Mekong River and borders Thailand, Cambodia, Burma (Myanmar), China, and Vietnam. Most (83%) of the population of 5.2 million are rural rice farmers, the per capita income is US $326/year, and life expectancy is 54 years ( 1 ). Although more data have been obtained in wealthier countries in Asia, minimal information exists on the clinical epidemiology of infectious disease in Laos. The etiology of fever in Laos usually remains obscure because of limited laboratory diagnostic facilities. In 2000, the main differential diagnoses for adults admitted with fever to the hospital in Vientiane, the capital, were slide-positive malaria or slide-negative syndrôme paludéen, or malaria syndrome: both were treated with antimalarial drugs and the latter with additional antimicrobial drugs (unpub. data). Rickettsial diseases, caused by Orientia tsutsugamushi (scrub typhus), Rickettsia typhi (murine typhus), and members of the spotted fever group (SFG), cause fever in Thailand, Malaysia, China, and Vietnam ( 2 – 5 ), and their public health consequences have recently been emphasized in Sri Lanka ( 6 ) and Nepal ( 7 ). Indonesian peacekeeping troops seroconverted to O. tsutsugamushi and R. typhi during their residence in Cambodia ( 8 ), but acute, symptomatic infections with rickettsia have not been described there since the 1930s ( 9 ). No studies that examined the causes of fever in Laos, which has economic, cultural, and geographic differences from adjoining countries, have been published. Such information is crucial in developing appropriate diagnostic tests and guidelines, determining empiric treatment for nonmalarious fever, and planning public health interventions. The mite vectors of scrub typhus have been described from Laos ( 10 ), but no rickettsial disease has been described from the country, apart from the seroconversion of US troops to O. tsutsugamushi ( 11 ). Therefore, we conducted a 2-year prospective study of the causes of fever among adults admitted to Mahosot Hospital, who were both blood-culture and malaria-smear negative, to determine the causes of syndrôme paludéen. We describe the serologic test results for rickettsiae. Methods Study Site and Patients The study was conducted at Mahosot Hospital, Vientiane, a 365-bed primary- to tertiary-care hospital that specializes in internal medicine, which has ≈1,200 admissions per month. This hospital, along with 4 other major hospitals (1,210 beds total) and local provincial and district hospitals, serves a population of ≈900,000 people, including the urban population of Vientiane City and surrounding farming communities of Vientiane Province, and less frequently, outlying provinces. We recruited patients admitted from November 2001 to October 2003 on all 4 adult medical wards (including an adult intensive care unit), making up 91 beds. Ethical clearance was granted by the Faculty of Medical Sciences Ethical Review Committee, National University of Laos. Clinical Procedures All adults (>15 years of age) admitted with fever had blood cultures taken if community-acquired septicemia was suspected and they gave verbal informed consent. If the patient came from an area of Laos with endemic malaria, Giemsa-stained malaria thick and thin films were examined. If the blood culture showed no clinically meaningful growth after 3 days of incubation, the malaria film was negative, and the patient gave verbal informed consent, a 5-mL whole blood sample was taken for serum analysis. An additional 5-mL convalescent-phase venous blood sample was collected ≈1 week later. The presence of eschars was not recorded systematically, since without evidence of rickettsial infection they were not routinely looked for. Patients' conditions were further investigated and treated according to local hospital practice. Laboratory Procedures Serum samples were stored at –80°C until analysis. Specific microimunofluorescence (IFA) assays were performed in Marseille, France, by using whole-cell antigens of O. tsutsugamushi serotypes Karp, Kato, Gilliam, and Kawasaki ( 12 ) and with Bartonella henselae, Coxiella burnetii, R. conorii subsp. indica, R. felis, R. heilongjiangensis, R. helvetica, R. honei, R. japonica, Rickettsia "ATI," R. slovaca, and R. typhi ( 13 – 15 ). An IFA result was considered positive if any of the following were detected: 1) positive antibody titers >1:128 for immunoglobulin G (IgG) and >1:64 for IgM, 2) seroconversion, or 3) >4-fold increase in titers between acute- and the convalescent-phase serum ( 5 , 13 ). Western immunoblotting was performed on samples positive for Rickettsia spp. both before and after cross-absorption with relevant antigens ( 12 , 13 ). Full blood counts (n = 364) and serum biochemical test results (n = 352) were analyzed on Abx MICROSOT (Abx Hematologie, Montpellier, France) and Cobas Integra (Roche Co. & Tegimenta Ltd, Rotkreuz, Switzerland) analyzers, respectively. Statistical Analysis Analysis was performed by using Stata v. 8 (StataCorp LP, College Station, TX, USA). Categoric variables were compared with Fisher exact test and continuous variables by Student t test and Mann-Whitney U test as appropriate. Multivariate logistic regression (backwards) was performed to evaluate variables associated with serologic diagnoses. Results Serology During the 2 years of the study, 466 adults were recruited; clinical and laboratory data, including rickettsial serology, were available for 427. Forty-five patients (12.6%) did not have a prior blood culture, and 218 (51%) had a convalescent-phase serum sample taken (median 5 [range 1–50] days after the admission sample). Of 427 patients, serologic evidence for acute rickettsial infections were found in 115 (26.9%): O. tsutsugamushi in 63 (14.8%), R. typhi in 41 (9.6%), and SFG rickettsiae in 11 (2.6% [8 R. helvetica, 1 Rickettsia "AT1," 1 R. felis, and 1 R. conorii subsp. indica]). No serologic evidence was found for acute B. henselae, C. burnetii, R. heilongjiangensis, R. honei, R. japonica, or R. slovaca infection. Of the 63 patients with serologic evidence of infection with O. tsutsugamushi, the highest titers were with the Gilliam serotype for 9 patients, the Gilliam or Kawasaki serotype in 9, the Gilliam or Kato serotype in 6, and all 3 serotypes in 39. Clinical Features Patients with scrub typhus could not be distinguished reliably from those with murine typhus at the bedside or in retrospective review of all clinical and laboratory details (Table 1). Patients with scrub typhus had a higher frequency of lymphadenopathy and abnormal chest examination than patients with murine typhus (p 90 IU/L) serum creatinine kinase concentrations were found in 273 (63.9%) of patients in the serologic study: 57% with scrub typhus, 63% with murine typhus, and 50% with positive R. helvetica serologic test results. Patients with rickettsioses who had myalgia on admission had significantly higher serum creatinine kinase (geometric mean 119 IU/L, 95% confidence interval [CI] 90–158) concentrations than those who did not (geometric mean 47 IU/L, 95% CI 26–87) (p = 0.02). Table 1 Admission clinical features of 104 Lao adults with serologic evidence of acute murine and scrub typhus* Variable Murine typhus (n = 41)† Scrub typhus (n = 63)† p value Reference range Age, (y)‡ 40 (17–70) 31 (16–73) 0.5 No. (%) male 26 (63) 40 (63) 0.6 No. days ill‡ 11 (3–30) 10 (2–42) 0.2 Headache (%) 38 (95) (n = 40) 60 (95) 1.0 Abdominal pain (%) 17 (43) (n = 40) 22 (35) 0.5 Nausea (%) 18 (45) (n = 40) 39 (62) 0.09 Vomiting (%) 11 (28) (n = 40) 25 (40) 0.3 Diarrhea (%) 7 (18) (n = 40) 22 (35) 0.07 Cough (%) 14 (35) (n = 40) 24 (38) 0.8 Sputum (%) 8 (20) (n = 40) 13 (21) 1.0 Dyspnea (%) 5 (13) (n = 40) 7 (11) 1.0 Chest pain (%) 3 (8) (n = 40) 13 (21) 0.1 Back pain (%) 15 (38) (n = 40) 19 (30) 0.5 Dysuria (%) 3 (8) (n = 40) 2 (3) 0.4 Arthralgia (%) 10 (25) (n = 40) 13 (21) (n = 62) 0.6 Myalgia (%) 34 (85) (n = 40) 59 (95) (n = 62) 0.1 Sore throat (%) 3 (8) (n = 40) 12 (19) 0.2 Lymphadenopathy (%) 1 (3) (n = 38) 27 (46) (n = 59) 50 μmol/L (%) 2 (6) (n = 32) 4 (8) (n = 52) 1.0 No. patients serum AST >105 IU/L (%) 11 (36) (n = 31) 18 (35) (n = 52) 1.0 No. patients serum ALT >105 IU/L (%) 6 (19) (n = 31) 5 (10) (n = 52) 0.3 Deaths (%) 0 1 (1.5) *AST, aspartate aminotransferase; ALT, alanine aminotransferase; CI, confidence interval.
†The available sample size is given in parentheses where the entire sample was not available for a given variable.
‡Median (range).
§Mean (95% CI).
¶Geometric mean (95% CI). Seventeen patients with scrub typhus (27.0%) had evidence for severe organ dysfunction; 7 (11.9%) of 59 had meningismus, 7 (11.1%) of 63 had dyspnea, and 7 (13.2%) of 53 had a serum creatinine level >133 μmol/L. While 4 (7.7%) of 52 patients had a total serum bilirubin level >50 μmol/L, 18 (34.6%) of 52 had a serum aspartate aminotransferase (AST) level >3 times the upper limit of the reference range. Nine patients with murine typhus and severe organ dysfunction (22.5% of 40 patients with data) were also encountered; 2 (5.1%) of 39 had meningism, 5 (12.5%) of 40 had dyspnea, and 2 (6.3%) of 32 had a serum creatinine level >133 μmol/L. Of the 8 patients with serologic evidence of acute R. helvetica infection, 6 had headache, 4 had vomiting, 1 had diarrhea, 2 had cough, 2 had dyspnea, 7 had myalgia, 4 had a palpable liver, and none had palpable lymphadenopathy or splenomegaly (Tables 2 and 3). One had a petechial rash at admission, and rash developed in 1 patient 2 days after admission. The median (range) serum biochemistry results for patients with R. helvetica infection were creatinine 85 (67–142) μmol/L, AST 84 (35–118) IU/L, alanine aminotransferase (ALT) 50 (14–87) IU/L, albumin 39 (23–45) g/L, creatinine kinase 49 (16–125) IU/L, alkaline phosphatase 115 (96–217) IU/L, direct bilirubin 4.8 (3.7–7.3) μmol/L, and total bilirubin 9.5 (8.8–16.8) μmol/L. None of the 8 patients had a bilirubin level >50 μmol/L or an ALT level >3 times the upper limit of the reference range, but 2 patients had an AST level >3 times the upper limit of the reference range. Table 2 Clinical features of patients with serologic evidence for acute spotted fever rickettsioses admitted to Mahosot Hospital* Patient no. Age (y), sex Occupation Month of onset of illness Clinical features Home 45 30, male Construction worker March 18-day fever, myalgia, nausea, epistaxis, vomiting, abdominal pain, petechial rash on trunk and legs; liver and spleen not palpable; treated with ampicillin and gentamicin Vientiane City 72 35, female Teacher April 13-day fever, chills, headache, nausea, myalgia, vomiting, conjunctival suffusion, dyspnea, 12-cm liver; treated with ofloxacin Vientiane City 86 25, male Health worker May 11-day fever, headache, nausea, vomiting, abdominal pain, 10-cm liver Vientiane City 114 18, male Student June 14-day fever, chill, headache, arthralgia, myalgia, rash developed 2 days after admission, 12-cm liver; treated with ofloxacin Vientiane Province 198 50, male Government official September 24-day fever, headache, arthralgia, myalgia, vertigo, epistaxis, diarrhea; abdominal CT suggested hepatic carcinoma; no antimicrobial drug Xieng Khuang Province 237 64, male Government official September 21-day fever, myalgia, arthralgia, abdominal pain, sore throat, cough, dyspnea; chest exam abnormal Vientiane City 290 24, female Construction worker March 7-day fever, headache, vomiting, myalgia, unproductive cough, diarrhea; treated with doxycycline Vientiane City 362 23, female Student June 10-day fever, myalgia, headache, conjunctival suffusion, 8-cm liver; treated with doxycycline Vientiane City 297 43, female Housewife March 14-day fever, headache, jaundice, RUQ pain, myalgia, 8-cm hepatomegaly; abdominal CT suggested tumor of intrahepatic bile ducts (cholangiocarcinoma?); treated with ampicillin and gentamicin Xieng Khuang Province 55 34, female Housewife April 7-day fever, chills, headache, myalgia, diarrhea, abdominal pain, nausea, vomiting, rash on arms and abdomen; treated with oral chloramphenicol Vientiane Province 239 46, male Merchant November 6-day fever, headache, myalgia, arthralgia, nausea, abdominal pain, diarrhea, dyspnea, dry cough, and sore throat; treated with doxycycline Vientiane City *CT, computed tomographic scan; RUQ, right upper quadrant. Table 3 Serologic results of patients with serologic evidence for acute spotted fever rickettsioses admitted to Mahosot Hospital Patient no. Immunofluorescence results (IgG/IgM admission sample, IgG/IgM convalescent-phase sample)* Rickettsia japonica R. helvetica R. heilongjiangensis R. slovaca R. felis R. honei R. conorii† "AT1"‡ 45 0/0,
1:256/1:128 0/0,
1:1,024/1:256 0/0,
1:1,024/1:128 0/0,
1:1,024/1:128 0/0,
1:256/1:256 0/0,
1:256/1:256 0/0,
0/1:256 0/0,
1:256/1:256 72 1:64/1:32 1:128/1:32 1:64/1:32 1:128/1:32 0/0 0/0 0/0 0/0 86 0/0,
1:64/0 0/0,
1:64/1:128 0/0,
1:64/0 0/0,
1:64/1:128 0/1:128,
0/1:128 0/0,
0/1:32 0/0,
0/1:32 1:128/0,
1:128/1:32 114 1:128/1:64 1:256/1:512 1:128/1:64 1:256/1:512 0/1:32 0/0 0/0 0/0 198 0/0 1:128/1:64 0/0 0/1:32 0/0 1:128/0 1:64/0 1:256/0 237 1:128/0 1:256/1:32 1:128/0 1:256/1:32 0/0 0/1:32 0/1:32 0/1:64 290 0/1:32,
0/1:32 1:64/1:32,
1:64/1:32 0/0,
0/0 0/0,
0/0 0/0,
0/0 0/0,
0/0 0/0,
0/0 0/1:32,
0/1:32 362 0/0,
0/0 1:16/1:16,
1:32/1:32 0/0,
0/0 0/0,
0/0 1:16/1:16,
1:16/1:32 0/0,
0/0 0/0,
0/0 0/0,
1:32/1:32 297 0/0,
0/1:64 0/0,
0/1:64 0/0,
0/1:64 0/0,
0/1:64 0/0,
0/0 0/0,
0/1:64 0/0,
0/0 0/0,
0/1:64 55 0/0,
1:64/1:32 0/1:64,
1:64/1:128 0/0,
1:64/1:32 0/1:64,
1:64/1:128 0/0,
1:256/1:128 0/0,
1:256/0 0/0,
1:64/0 0/0,
1:256/0 239 0/0 0/0 0/0 0/0 1:64/0 1:64/1:32 1:64/1:32 1:64/1:32 *Titers in boldface indicate the pathogen considered to be responsible for the serologic response.
†R. conorii subsp. indica.
‡Rickettsia "AT1" from Japan. Geographic Distribution Districts in which patients lived were recorded for 417 (98%) patients in the serologic study; 73% lived in Vientiane City, and 22% Vientiane Province. The proportion of patients with a home address in Vientiane City was 71% for scrub typhus and 55% for murine typhus patients. Outside Vientiane City and Province, patients with scrub typhus came from Houaphanh and Borikhamxay Provinces, and patients with murine typhus came from Borikhamxay and Luang Prabang Provinces. Of the 11 patients with serologic evidence of spotted fever rickettsiosis, 7 were from Vientiane City, 2 from Vientiane Province, and 2 from Xieng Khuang Province. Outcome Of 63 patients with scrub typhus for whom outcome is known, 1 (1.6%) died in the hospital. This 23-year-old housewife died 14 days after delivering a healthy girl at home; she had gone to the hospital with a 1-week history of fever before parturition. Pneumonia, vaginal bleeding from retained placenta, and hypotension developed; her Glasgow Coma Score was 7 of 15. In the hospital, she underwent uterine curettage and received ampicillin, gentamicin, azithromycin, ceftriaxone, and metronidazole. Fever developed in the daughter, and she died 4 days after her mother. The death rate among adults with serologic evidence of an acute rickettsiosis was therefore 1 in 115 (0.9%). Discussion These serologic data suggest that scrub typhus and murine typhus are underrecognized causes of fever among adults in Vientiane. A wide diversity of rickettsiae were identified for the first time in Laos. Scrub typhus was the most common rickettsiosis identified. The patients tended to be young adult males presenting with fever, headache, nausea, myalgia, lymphadenopathy, and a palpable liver. Seventeen (27%) patients with scrub typhus had severe disease, and 18 (34.6%) had a liver biochemistry profile consistent with that of hepatitis. In a recent series of 462 patients with scrub typhus from Japan, lymphadenopathy, headache, myalgia, hepatomegaly, and eschar were recorded in 52%, 46%, 16%, 3%, and 87% of patients, respectively. Elevated serum AST and ALT levels were also common (87% and 77%, respectively) among these Japanese patients ( 16 ). In comparison to Lao patients, Japanese patients had a substantially lower prevalence of myalgia and hepatomegaly. The clinical importance of acute scrub typhus in the death of the Lao patient who also had retained placenta and probable intrauterine infection remains uncertain. Her infant may have died of neonatal scrub typhus ( 17 ). Of 12 case reports of scrub typhus in pregnancy ( 17 – 19 ), 8 recorded stillbirth, miscarriage, neonatal scrub typhus, or neonatal death, but all the mothers survived. During the 2 years of this study, patients with scrub typhus became ill in the late hot weather and monsoon, similar to observations made 60 years ago in Burma ( 20 ), but different from the geographically variable epidemiologic features noted in Japan ( 16 ). Recent clinical observations suggest that the prevalence of eschars in Lao patients with serologically confirmed scrub typhus when the entire skin surface is examined is ≈52% (unpub. data) and 0% in patients with confirmed murine typhus. Therefore, a thorough search for eschars will help with the diagnosis of scrub typhus. Patients with murine typhus also tended to be young adult males with a clinical profile similar to those with scrub typhus but with a strikingly lower frequency of lymphadenopathy (3% vs. 46%). Similar proportions of patients with murine typhus and scrub typhus had raised serum bilirubin and AST levels. In a series of 137 patients with murine typhus in southern Thailand ( 21 ), 20% had skin rash, 24% had hepatomegaly, and 5% had splenomegaly. In contrast, among 83 Cretans, 80% had a rash, perhaps because it was easier to detect on fairer skin ( 22 ). A relatively low frequency of lymphadenopathy in patients with murine typhus has been described from Crete (4% [22]), Texas (16% of children [23]), and Spain (2% [24]). In the Lao series, cough was present in 35% of patients with murine typhus. Respiratory symptoms and signs have been reported among murine typhus patients with cough present in 59% ( 25 ), 15% (children [23]), 28% ( 21 ), and 25% ( 24 ) of patients. No concurrent comparisons have been made of clinical features of scrub and murine typhus at 1 site, but the Lao data suggest that the presence of peripheral lymphadenopathy, chest signs, and eschars are clinically useful signs that suggest scrub, rather than murine, typhus. We also found serologic evidence for 4 SFG species. Although Western blotting and cross-absorbance studies were performed, evidence for rickettsiae in Laos is based on serologic methods and therefore, especially for SFG, needs to be confirmed by genetic analysis ( 4 ). Human SFG Rickettsia infections have been described in Thailand, China, Korea, Malaysia, and Japan ( 3 , 4 , 26 , 27 ) but not in Laos, Vietnam, Burma or Cambodia. Evidence for human R. helvetica infections has been found in Europe ( 14 , 28 , 29 ), Thailand ( 5 ), and possibly Australia or Japan ( 30 ). One of the Lao patients with apparent R. helvetica infection had a rash, unlike the 8 patients described previously with R. helvetica infection ( 5 , 28 ). Evidence for acute human infection with R. felis has been found in North and South America, Europe, and the Thailand/Burma border ( 5 , 31 ). The clinical symptoms of the patient described from the Thailand/Burma border were similar to those of our Lao patient, and neither had a rash. Evidence for R. conorii has been found in India ( 4 , 32 ) and on the Thailand/Burma border ( 5 ). Rickettsia "AT1" was originally isolated from Japanese Amblyomma ticks, and its genotype is most closely related to rickettsiae from Slovakian Ixodes ticks ( 33 ). The relevance of Rickettsia "AT1" to human disease remains uncertain. Although no acute C. burnetii infections were found in this series, Q fever has recently been described from northeast Thailand ( 34 ). Raised serum creatinine kinase levels have been described in patients with scrub typhus ( 35 ) and as an apparently nonspecific result of febrile illness ( 36 ). In a series of patients with fever in Israel, an elevated creatinine kinase level was associated with increased blood urea, low serum phosphate, reduced consciousness, tremor, and muscle tenderness. Alcoholism and high body temperature may also be associated factors ( 36 ). In Laos, a rise in creatinine kinase level may also have been a consequence of the common practice of administering intramuscular injections before hospital (unpub. data). Because serum creatinine kinase concentrations are higher in patients with rickettsioses who have myalgia than in those without, muscle pain is likely to be associated with mild muscle damage. This study is of similar design to a recent investigation of the causes of fever in adults living in and around another tropical capital city, Kathmandu, although the Nepalese study included outpatients and sampled 4 months of 1 year ( 7 ). The frequency of rickettsioses was lower in patients in Kathmandu, with serologic evidence of acute infection with murine typhus in 11% and scrub typhus in 3%. The high incidence of patients in Vientiane who have diseases for which the vectors, such as chiggers and ticks, are likely to be predominantly rural is not surprising. Many inhabitants of the city visit farms in rural areas, and persons with occupations that would not conventionally be regarded as of high risk for rickettsioses may be exposed. In addition, suburban scrub typhus has been described ( 37 ). The decision to enter a particular patient into the study was the responsibility of many doctors, and some infected patients may not have been recruited. Only 11% of the Lao population live in the relatively urbanized areas of Vientiane City, and the results of this study are unlikely to be applicable to the rest of the country, which is diverse in geography and ethnicity. A hospital-based study such as this will tend to underestimate the incidence of disease, and infections, such as scrub typhus, which tend to affect farmers, will be more common in rural Laos. Additional limitations of the study are that we did not perform serologic analysis on all patients who did not have a clinically meaningful blood culture during the study period, that the median interval between acute- and convalescent-phase serum samples was relatively short (5 days), and that 49% of patients did not have a convalescent-phase sample. These data have affected local clinical practice. With the realization that scrub typhus is an important disease, patients' skin surfaces are now routinely completely examined for eschars, and doxycycline therapy is added at an earlier stage for patients with headache, fever, and myalgia. The drugs usually administered for syndrôme paludéen were ampicillin or cotrimoxazole, both of which are ineffective against rickettsiae. These results suggest that an antirickettsial agent, such as doxycycline, should be included in the empiric treatment of Lao adults with fevers whose clinical features are consistent with a rickettsiosis. However, analysis of the clinical features of patients in this study with rickettsiosis, leptospirosis, dengue, and typhoid (unpub. data) suggest that these diseases are difficult to distinguish reliably on clinical examination and that rapid, inexpensive diagnostic tests will help guide therapy. An oral drug with high efficacy against uncomplicated rickettsiosis, leptospirosis, and typhoid could be of considerable use. Azithromycin is effective in treating uncomplicated typhoid fever in Vietnam ( 38 ) and scrub typhus in Korea ( 39 ), and it may be effective against leptospires in vitro ( 40 ). In parallel with the adoption of effective artemisinin-based combination therapy for malaria in rural Laos, the need is urgent to develop rapid and inexpensive tests to diagnose alternative causes of fever and to improve the treatment of common nonmalarious fevers.
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Endemic Scrub Typhus–like Illness, Chile
M. Balcells, Ricardo Rabagliati, Patricia García … (2011)
The primary hosts for Rickettsia species are arthropods that can also act as disease vectors for humans and other vertebrates. Ticks are vectors for most rickettsioses caused by spotted fever group rickettsiae. Alternative vectors for rickettsiae are well known, including fleas as vectors for murine typhus (R. typhi) and flea-borne spotted fever (R. felis), mites as vectors of rickettsialpox (R. akari), and scrub typhus (Orientia tsutsugamushi), and lice as vectors for epidemic typhus (R. prowazekii) ( 1 ). In Chile, the last outbreak of epidemic typhus began in 1933 and continued through 1939 ( 2 ). In the following years, effective control and sanitary measures were developed and implemented. No new cases of rickettsial disease have been reported in this country since 1976 ( 3 ). Scrub typhus, caused by O. tsutsugamushi, which is usually transmitted by trombiculid mites in Asia, northern Australia, and the western Pacific region has never been described in Chile ( 4 ). Although sporadic cases of scrub typhus have been reported well outside the traditionally endemic regions ( 5 , 6 ), no reports are known of scrub typhus being acquired in the Western Hemisphere ( 4 ). In addition, no human case of rickettsial spotted fever has been documented in Chile, although there is evidence of rickettsial infections in dogs ( 7 ) and of the presence of R. felis in cats and cat fleas ( 8 ). We report a case of scrub typhus–like illness in Chile. Materials and Methods The patient was a previously healthy 54-year-old man who recalled having been bitten by terrestrial leeches on several occasions but not by ticks. He was hospitalized after symptoms including a high-grade fever developed. During treatment, a black eschar with an erythematous halo on the left leg was found. A biopsy sample from the leg eschar was submitted to the laboratory for histopathologic analysis and subjected to microscopy. A routine blood chemistry panel was analyzed. In addition, an ELISA to detect O. tsutsugamushi–specific immunoglobulin G was performed with acute-phase and convalescent-phase serum samples ( 9 ). The same skin biopsy samples of the eschar and rash were submitted for molecular biology analysis. DNA was extracted from the skin biopsy samples by using the QIAamp Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The prokaryotic 16S rRNA gene was amplified and sequenced by using described primers ( 10 ). The PCR products were purified with the GFX DNA gel band purification kit (GE Healthcare, Piscataway, NJ, USA) and sequenced by using the BigDye Terminator version 3.1 Cycle Sequencing Kit and a 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences obtained from the leg and arm samples were assembled by using the Sequencher DNA Software (Gene Codes Corporation, Ann Arbor, MI, USA) and were judged to be identical. The sequences were initially compared with other 16S rRNA sequences in GenBank by using the National Center for Biotechnology Information BLAST network service software ( 11 ). The sequence of the biopsy sample (referred to as the Chiloé Island sample) has been deposited in GenBank under accession no. HM155110. Sequence differences between the Chiloé Island sample and isolates of O. tsutsugamushi were determined after aligning 16S rRNA sequences, using ClustalX in MEGA4 ( 12 ). The patient had been involved in ecological studies at a university field camp at the southern end of Chiloé Island, in southern Chile. The region is rainy and has abundant natural vegetation and evergreen forests. During January 2006, the patient spent 3 weeks on a field study (sleeping in a log cabin) with daily forest incursions. He recalled having been bitten by terrestrial leeches on several occasions but not by ticks. A week after returning to the capital, Santiago, and 6 days before his admission to the hospital, a high-grade fever, headache, myalgias, and scanty dry cough developed. Four days later, a rash appeared in the abdominal region that progressed to his face and limbs. At admission, the patient had an axillary temperature of 39ºC, pulse 101 beats/min, blood pressure 110/75 mm Hg, and bilateral conjunctival suffusion. He had an extensive rash on his face, trunk, and limbs but not on the palms and soles, with a microvesicular center in some of the lesions (Figure 1, panel A). A black eschar with an erythematous halo on the left leg was found. He recalled having been bitten by a leech ≈3 weeks before (Figure 1, panel B). Figure 1 Evidence of acute infection of the skin and subcutaneous tissue in patient admitted for treatment of scrub typhus-like symptoms in Chile. A) Rash on admission, left arm. B) Necrotizing eschar with erythematous halo over the left leg. Results Dermis and subcutaneous fat showed a necrotizing leukocytoclastic vasculitis, perivascular infiltrates with lymphocytes and macrophages, and extravasation of erythrocytes (Figure 2, panel A). Gram, Giemsa, and Warthin-Starry silver stains did not show any microorganisms. A tissue sample recovered from a paraffin-embedded sample for electron microscopy, showed round and oval rickettsia-like microorganisms, maximum diameter 0.2–0.5 μm, inside the cytoplasm endothelial cells (Figure 2, panel B). Figure 2 Results of biopsy analysis of tissue sample from eschar on the left leg of patient admitted for treatment of scrub typhus–like symptoms, Chile. A) Leukocytoclastic vasculitis. Hematoxylin and eosin stained; original magnification ×200, inset ×400. B) Endothelial cell, showing nucleus (N) within the cytoplasm (C, inset). Arrows show similar round and oval organisms, electron-dense, surrounded by electron-lucent halo of rickettsial type microorganisms. Electron microscopy; original magnification ×15,000, inset ×20,000. Blood tests showed a leukocyte count of 9,200 cells/mm3, with 28% immature forms and a slight elevation of hepatic aminotransferase levels (aspartate aminotransferase 198 U/L [reference range 10–40 U/L], alanine aminotransferase 256 U/L [reference range 10–55 U/L], and alkaline phosphatase 338 U/L [reference range 45–115 U/L]) with normal bilirubin level. Blood cultures (2 sets) were negative as were serologic test results for measles, varicella, leptospirosis, and HIV. A spotted fever rickettsiosis was suspected and doxycycline (100 mg 2×/d) was started on the day after admission. ELISA to detect O. tsutsugamushi specific immunoglobulin G on the acute-phase sample had no reactivity (titer 17% ( 5 ). Given that the Chiloé Island sample shows almost twice as much divergence from O. tsutsugamushi for the 16S rRNA sequence compared with O. chuto sp. nov., it is not unreasonable that substitutions in the PCR primer sites of the Chilean sample exist, explaining the negative results that were found in our study. Discussion We describe a case of rickettsiosis acquired in Chiloé Island, where the local population is mostly of the Huilliche ethnic background. One of the ancient local legends refers to a disease developing in persons who penetrate the jungle, with the development of high fever and red spots all over the body. However, no scientific medical report had confirmed this finding. Even though the existence of scrub typhus has never been recorded in Chile, its vector, the trombiculid mite (Acari: Trombiculidae), has been recently described in wetlands from a distant region of southern Chile, although not on Chiloé Island ( 18 ). Our patient recalled specifically having been bitten by a leech in the site where an eschar later developed. Terrestrial leeches are common on Chiloé Island vegetation. These include members mainly from the family Mesobdellidae, including the species Mesobdella gematta and Nesophilaemon skottsbergi ( 19 ). The leeches live among trees, ferns, bushes, and fallen leaves. All are sanguivorous parasites of vertebrate animals, and local persons are frequently exposed to leech bites on the island. Rickettsiae have been reported in leeches in Japan ( 13 , 14 ). In those studies, the glossiphoniid leech species harbored bacteria of the genus Rickettsia, as assessed by electron microscopy and PCR analysis. The results of analysis of the 16S rRNA gene sequence suggest that the sample reported represents a previously unreported, divergent form (species) of Orientia spp.–like bacteria. The degree of sequence differentiation from isolates of Orientia spp. previously studied in Asia and the Middle East indicates that the Chiloé Island sample is not simply a transplanted form from Asia that happened to be discovered in Chile, but rather it represents a long divergent lineage and may be indicative that other Orientia spp.–like pathogens are to be found outside southern and eastern Asia or northern Australia. The difficulty of obtaining PCR amplification of additional sequences, such as the GroEL and 47-kD protein genes, would be consistent with the identification of a new lineage divergent from Asian forms of O. tsutsugamushi. Moreover, the reactivities of the serum samples to O. tsutsugamushi Karp, Kato, Gilliam ELISA antigens (titer 400) suggest that the cross-reactivity of assay antigens to those of the new sample may exist but be limited, again consistent with a divergent lineage. Future steps following the case presented will involve investigating whether Chiloé Island’s leeches carry rickettsiae and whether these rickettsiae, according to additional DNA sequence analysis, are closely related to members of O. tsutsugamushi or if they represent a new lineage within or closely related to the known forms of Orientia. If no related rickettsiae are identified from leeches, an alternative possibility is that trombiculid mites are present on Chiloé Island and that these are the vectors of the pathogen. However, the observation that the eschar developed at the site of leech attachment would appear to argue against an alternative vector. Nevertheless, chiggers, the proven vector free-living stage in the mite life cycle that feeds on the vertebrate hosts, are small and easily overlooked. Thus, a mite cannot be positively excluded as the vector in this case. Whether other sporadic cases of human rickettsial illness may have occurred in that area should also be the subject of future investigation.
Author and article information
Journal
Journal ID (nlm-ta): Clin Infect Dis
Journal ID (iso-abbrev): Clin. Infect. Dis
Journal ID (publisher-id): cid
Title:
Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society
of America
Publisher:
Oxford University Press
(US
)
ISSN
(Print):
1058-4838
ISSN
(Electronic):
1537-6591
Publication date
(Print):
15
April
2019
Publication date
(Electronic):
10
August
2018
Publication date PMC-release: 10
August
2018
Volume: 68
Issue: 8
Pages: 1413-1419
Affiliations
[1
]Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol
University, Bangkok, Thailand
Author notes
Correspondence: S. D. Blacksell, Mahidol-Oxford Tropical Medicine Research Unit, Faculty
of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand (
stuart@
123456tropmedres.ac
).
Author information
Stuart D Blacksell
http://orcid.org/0000-0001-6576-726X
Article
Publisher ID: ciy675DOI: 10.1093/cid/ciy675
PMC ID: 6451999
PubMed ID: 30107504
SO-VID: c44c377d-50c4-4ffe-8244-670ce756cf7a
Copyright © © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases
Society of America.
License:
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
History
Date
received
: 30
April
2018
Date
accepted
: 09
August
2018
Page count
Pages: 7
Funding
Funded by:
Wellcome Trust
10.13039/100010269
Categories
Subject:
Viewpoints
ScienceOpen disciplines: Infectious disease & Microbiology