Comparative evaluation of the indirect immunoperoxidase test for the serodiagnosis of rickettsial disease

An indirect immunoperoxidase test was compared with an indirect fluorescent antibody test and the Weil-Felix OXK test for serodiagnosis of scrub typhus by measuring the rickettsial antigen specific activity of IgG, IgM, and whole globulin. Acute and convalescent sera from 50 Rickettsia tsutsugamushi isolate-positive scrub typhus patients and from 45 febrile patients diagnosed as having diseases other than scrub typhus were tested. The receiver operating characteristic for each test showed that the indirect immunoperoxidase and indirect fluorescent antibody tests were more sensitive and specific than the Weil-Felix test using convalescent and acute as well as paired sera. The indirect immunoperoxidase test showed no cross-reactivity when R. tsutsugamushi antigen was tested against sera collected from patients living outside the scrub typhus-endemic area with diseases other than scrub typhus. The indirect immunoperoxidase and indirect fluorescent antibody tests were comparable in measured response to R. tsutsugamushi, R. typhi, and TT-118 (spotted fever group) antigen. Thus the indirect immunoperoxidase test represents a sensitive, specific, reproducible, and practical semiquantitative test for rickettsial disease diagnosis.     

Enzyme immunoassay of antibody to Rochalimaea quintana: diagnosis of trench fever and serologic cross-reactions among other rickettsiae.

Enzyme immunoassay (EIA) tests were used to diagnose trench fever and to determine cross-reactions of Rochalimaea quintana with other rickettsiae. The results were compared with those obtained by counterimmunoelectrophoresis (CIE). All sera from cases of primary or relapsed forms of trench fever were positive both in EIA, with serum antibody titers of 1:20-1:640, and in CIE, giving one to three precipitin lines. Sera from patients with other rickettsial infections were also tested for reactivity with R. quintana antigen: typhus group (Rickettsia prowazekii, Ricketsia mooseri), 15 sera; spotted fever group (Ricketsia ricketsii, Rickettsia akari), eight sera; and scrub typhus (Rickettsia tsutsugamushi), six sera. Strong reactions occurred with four sera from patients with scrub typhus, giving one or two lines in CIE and EIA titers of 1:40-1:160; these results were extended to guinea pig antisera to R. tsutsugamushi. About 50% of typhus group sera reacted with a single line in CIE and had antibody titers of 1:20-1:80 by EIA. The results show that EIA is accurate for the diagnosis of trench fever and, with the results obtained by CIE, suggest that R. quintana is antigenically related to R. tsutsugamushi and possibly to rickettsiae in the typhus group as well.     

Detection of rickettsioses and Q fever in Sri Lanka

Current serological evidence suggests the presence of scrub typhus and spotted fever group (SFG) rickettsiosis in Sri Lanka. Our objective was to identify rickettsial agents/Q fever as aetiological causes for patients who were presumed having rickettsioses by the presence of an eschar or a rash. Sera from patients with unknown origin fever from Matara were tested by immunofluorescence for SFG rickettsial antigens, typhus group rickettsiae, Orientia tsutsugamushi, and Coxiella burnetii antigens. Thirteen (7.3%) of the patients presented with a rash, 11 (6.1%) had an inoculation eschar, and 16 patients recalled a tick or flea bite. We found that 25 (14%) patients had scrub typhus, 6 (3%) SFG rickettsioses, 3 (1.6%) acute Q fever, 3 (1.6%) murine typhus, and 3 (1.6%) were infected by Rickettsia felis. In addition to already described scrub and murine typhus, we found that R. felis and C. burnetii infections should be considered in Sri Lanka.     

Rickettsia serosurvey in Kimberley, Western Australia.

To determine if antibodies to rickettsiae (scrub typhus, spotted fever, and typhus group rickettsiae) occur among persons living in the Kimberley (northern tropical) region of Western Australia, 920 sera collected in a non-random manner in 1996 from patients in Kununurra, Broome, Fitzroy Crossing, Wyndham, Derby, and Halls Creek were tested by micro-immunofluorescence for antibodies to a panel of rickettsial antigens. Of 920 sera examined, 52 (5.6%) were positive for antibodies to one or more of the three groups of rickettsial microorganisms. The largest group of sera (24; 2.6%) were positive for scrub typhus (Orientia tsutsugamushi). Eleven other sera (1.2%) were positive for scrub typhus and spotted fever group rickettsiae and four (0.4%) were positive for scrub typhus, spotted fever group, and typhus group rickettsiae. In addition 13 sera (1.4%) were positive only for spotted fever group rickettsiae. In this study, only titers > or = 1:256 were considered significant. Thus, there is serologic evidence for scrub typhus and spotted fever group rickettsial infections in the Kimberley region of Western Australia. Because of the method of serum collection, it is not possible to determine the prevalence of seropositivity, but the data support the need for a proper epidemiologic study of rickettsial diseases in this region of Australia.     

The use of the indirect immunoperoxidase test for the serodiagnosis of rickettsial diseases in Malaysia

The indirect immunoperoxidase (HP) test has been used extensively in most government hospitals in Malaysia for the serodiagnosis of scrub typhus, murine typhus and tick typhus during the 1990s. The test was used to determine the IgG and IgM antibody titers in patients' sera for three rickettsial species, ie Orientia tsutsugamushi OT; the causative agent of scrub typhus), Rickettsia typhi (RT; the causative agent of murine typhus), and TT118 spotted fever group rickettsiae (TT; the causative agent of tick typhus). The serological findings obtained from Malaysian hospitals using the IIP test (1994-1999) were analyzed. During the six-year period, a total of 61,501 patients' sera were tested, of which 9.6%, 10.5%, and 12.9% had antibody (IgG and/or IgM of > or = 1:50) for OT, RT and TT respectively. A total of 8.6%, 9.8%, and 9.7% of sera had IgG antibody of > or = 1:50 for OT, RT, and TT respectively, indicating past infection. A total of 3.4%, 3.8%, and 6.4 % of sera had IgM antibody of > or = 1:50 for OT, RT, and TT respectively, indicating recent infection. A total of 2,986 (4.9%), 1,882 (3.1%), and 1,574 (2.6%) of sera had IgG and/or IgM antibody titers of > or = 1:400 for OT, RT, and TT respectively, suggesting active rickettsial infection. The seropositivity rates of OT, RT and TT varied according to geographical locations. While the seropositivity of OT remained constant during the six-year period, a reduction in the seropositivity of both RT and TT was noted during recent years. The serological findings reflect the endemicity of rickettsial diseases, including tick typhus, and endemic typhus in various parts of Malaysia. Awareness of these diseases by health and medical staff and by the general public is important if the mortality and morbidity associated with scrub typhus, tick typhus, and murine typhus in Malaysia, are to be reduced.     

Orientia tsutsugamushi in peripheral white blood cells of patients with acute scrub typhus.

Scrub typhus, caused by Orientia tsutsugamushi, is an acute illness that occurs in many parts of Asia. Clinical manifestations range from inapparent to organ failure. Organisms disseminate from the skin to target organs, suggesting that they may enter the peripheral circulation. Here, peripheral blood cell smears from patients with acute scrub typhus were obtained before treatment and for 2 days after treatment and reacted with antibodies specific for O. tsutsugamushi. White blood cells from 3 of 7 patients with acute scrub typhus stained positively for O. tsutsugamushi. Cells containing O. tsutsugamushi were mononuclear and were detected on each day of sampling. The presence of O. tsutsugamushi in peripheral white blood cells of patients with acute scrub typhus is a new finding with clinical and pathogenic implications.     

Imported rickettsial disease: clinical and epidemiologic features

PURPOSE AND METHODS: The rickettsioses continue to constitute major health problems in many parts of the world. With increasing international travel, recognition of rickettsial diseases by physicians is becoming more important. The clinical features of four cases of rickettsial disease imported into Canada over a five-year period are presented; two patients with tick typhus (Rickettsia conorii), one patient with scrub typhus (R. tsutsugamushi), and one patient with murine typhus (R. typhi). We also present the North American data over the past 10 years from the Centers for Disease Control (CDC) (Atlanta). RESULTS: Since 1983 in the United States, three cases of imported scrub typhus, all after travel to India, were confirmed, as well as six cases of murine typhus after travel to southeast Asia. At the CDC, 67 imported cases of tick typhus have been confirmed by indirect fluorescent antibody test since 1976; most illnesses occurred after travel to Africa. CONCLUSION: Rickettsial diseases are underrecognized by physicians, who should consider these diagnoses in travelers returning from endemic areas. Since effective treatment is available, prompt diagnosis and treatment are important. In all cases, specific serologic confirmation should be obtained.     

Historical epidemics of scrub typhus in Queensland and Papua New Guinea

Undifferentiated febrile diseases (e.g., Mossman fever) from northern Queensland were eventually partially attributed to mite-transmitted rickettsial infections known as scrub typhus or tsutsugamushi fever. Scrub typhus became a major medical threat to military operations in Papua New Guinea during the Second World War and killed more Australian soldiers than malaria in the pre-antibiotic era. Further investigations showed scrub typhus to be an occupational disease of rural workers in north Queensland especially around Cairns and Innisfail. Occasional small epidemics of scrub typhus still occur during military exercises in Queensland, but as scrub typhus is not a reportable disease, its presence in the civilian community is largely unknown. Increased use of serological testing in patients with fever and rash illnesses after exposure in northern Queensland is likely to show that scrub typhus is a modern infection that remains treatable with antibiotics once it is identified.     

Epidemiology and ecology of rickettsial diseases in the People's Republic of China

Since 1949, information on rickettsial diseases in the People's Republic of China has been virtually nonexistent in the West. This is the first comprehensive review of the ecology and epidemiology of Chinese rickettsial diseases to be published outside the People's Republic. At least five rickettsioses exist in China: scrub typhus, murine typhus, epidemic typhus, Q fever, and one or more spotted fever-group (SFG) rickettsioses. Although epidemic typhus has been controlled and scrub typhus has abated in many areas, murine typhus, Q fever, and SFG rickettsiosis are important public health problems. Serologic surveys indicate high prevalences of antibodies to Coxiella burnetii, Rickettsia tsutsugamushi, and SFG rickettsiae in some regions; these rickettsiae have been isolated from humans, arthropods, and animals. Doxycycline has emerged as the best treatment for murine typhus, epidemic typhus, and scrub typhus. China offers both opportunities and challenges for the investigation and alleviation of the problems of rickettsial diseases.     

Tsutsugamushi disease during the last seven years in the past in Hamamatsu City, Shizuoka Prefecture--including evaluation of hyponatremia in scrub typhus

Six cases of scrub typhus (tsutsugamushi disease) were reported to the Shizuoka Prefecture Hamamatsu City public health center during the seven years from 2001 to 2007. The content of the clinical record of the five cases were investigated. High serum titers of antibody to Gilliam-type Orientia tsutsugamushi were detected by immunofluorescense assay in most of these patients. Fever, rash, headache and relative bradycardia seen at a high frequency. On peripheral blood smear examination, atypical lymphocytes were detected in 3 cases. Serum electrolyte examination revealed hyponatremia in 4 (80%) patients; SIADH was suspected in one of these cases. All the patients improved promptly following start of therapy with intravenous or oral minocycline.     

Rickettsial infections and their clinical presentations in the Western Province of Sri Lanka: a hospital-based study.

BACKGROUND: Rickettsial infections are re-emerging. A study of the geographical distribution of rickettsial infections, their clinical manifestations, and their complications would facilitate early diagnosis. METHODS: Thirty-one selected patients from the Western Province of Sri Lanka were studied for rickettsial species, clinical manifestations, and complications. RESULTS: Of 31 patients with possible rickettsioses, 29 (94%) fell into the categories of confirmed, presumptive, or exposed cases of acute rickettsial infections (scrub typhus was diagnosed in 19 (66%), spotted fever group in eight (28%)). Early acute infection or past exposure was suggested in two (7%) cases; cross-reactivity of antigens or past exposure to one or more species was suggested in nine (31%). Seventeen out of 19 (89%) patients with scrub typhus had eschars. Nine out of 29 (32%) patients had a discrete erythematous papular rash: seven caused by spotted fever group, two by scrub typhus. Severe complications were pneumonitis in eight (28%), myocarditis in five (17%), deafness in four (14%), and tinnitus in two (7%). The mean duration of illness before onset of complications was 12.0 (SD 1.4) days. All patients except one made a good clinical recovery with doxycycline or a combination of doxycycline and chloramphenicol. CONCLUSIONS: In a region representing the low country wet zone of Sri Lanka, the main rickettsial agent seems to be Orientia tsutsugamushi. Delay in diagnosis may result in complications. All species responded well to current treatment.     

Short report: detection of Orientia tsutsugamushi in clinical samples by quantitative real-time polymerase chain reaction

Orientia tsutsugamushi infection causes scrub typhus, a common zoonosis of rural Asia. Orientia tsutsugamushi was recently detected by a real-time quantitative polymerase chain reaction (qPCR) assay in animal specimens. We evaluated the same qPCR assay in specimens obtained from patients with serologically proven scrub typhus infections. The 47-kDa qPCR assay was more sensitive than was mouse inoculation; it was reactive in whole blood specimens from all 10 isolate-positive patients and in 7 of 17 isolate-negative individuals (P = 0.003, Fisher's two-tailed exact test). As few as 1,076 O. tsutsugamushi copies/microL were detected in whole blood. Four of 7 sera from isolate-proven scrub typhus infections were also reactive by qPCR. The assay was unreactive in all 12 individuals without scrub typhus infection. This is the first demonstration of a sensitive and specific real-time qPCR assay for human scrub typhus infection.     

The laboratory diagnosis of rickettsial diseases in Hong Kong.

Indirect immunoperoxidase (IP) tests against antigens of Rickettsia typhi, R. sibirica and R. tsutsugamushi were carried out on serum samples from 10 patients known to have high titres against one or more Proteus OX antigens in the Weil-Felix (WF) test. The IP test confirmed rickettsial infection in nine of the 10 patients, giving unequivocal indications of the grouping of the infecting Rickettsia species and, in one instance, showing a diagnostic IgM titre in a specimen taken on day 3 of the patient's illness. IP tests confirmed four cases of spotted fever group rickettsial infection, three cases of urban or murine typhus and two of scrub typhus.     

Identification of strain-specific and group-reactive antigenic determinants on the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi

Hybridoma antibodies (Hab) were prepared against the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi and were examined for homologous and heterologous reactivity using an indirect immunofluorescence assay. Strain-specific Hab demonstrated homologous IFA titers ranging from 1/320 to 1/1,280 and did not react (less than 1/10) with the heterologous strains. The cross-reactive Hab generally reacted equally with all three strains in the scrub typhus group; however, there were some Hab that reacted with only one of the two heterologous strains tested. The Hab also were examined in enzyme-linked immunosorbent assays with scrub typhus antigens eluted from SDS-polyacrylamide gels. Most Hab reacted with either one or several of the six eluted antigens detected with a polyclonal immune serum. It was also observed that strain-specific and cross-reactive Hab sometimes reacted with the same antigen, suggesting the existence of multiple antigenic determinants in one electrophoretic peak. The data suggest that strain-specific Hab can be used in the indirect immunofluorescence assay to identify isolates of R. tsutsugamushi without the cross-reactions usually observed with polyclonal antisera, and that they are useful probes for detection and analysis of rickettsial antigens.     

Patient and sample-related factors that effect the success of in vitro isolation of Orientia tsutsugamushi

Orientia tsutsugamushi is the causative agent of scrub typhus infection, a major cause of human disease in rural areas of Southeast Asia. Twenty-six blood samples collected from patients with serologically proven scrub typhus during a six month period were sent to Bangkok (535 km from the clinical site) by road at ambient temperature (average daily temperature range: 27.1-29.1 degrees C) for attempted in vitro isolation in Vero cells. O. tsutsugamushi was isolated from 12 samples (sensitivity 46.7%) with the time to isolation ranging from 16 to 37 days [median 27 days, inter-quartile range (IQR) 22.5-33.5 days]. Patient factors such as days of fever and O. tsutsugamushi IgM antibody titer, transport factors such as transit time, and isolate genotype (Karp and Gilliam/Kawasaki) were assessed to determine their influence on the outcome of in vitro isolation. None of the factors significantly influenced the isolation outcome. This study demonstrates that O. tsutsugamushi can often be isolated in vitro from the blood of scrub typhus patients when transported at ambient tropical temperatures for many days.     

Murine typhus identified as a major cause of febrile illness in a camp for displaced Khmers in Thailand

Scrub and murine typhus have been identified as causes of illness among the 238,000 displaced Khmer people residing in temporary settlements on the Thai side of the Thai-Cambodian border. Still, the true extent of the problem and the relative frequency of infection with scrub typhus as compared to murine typhus are unknown. We evaluated consecutive patients with unexplained pyrexia (documented fever, no exclusionary diagnosis, and constitutional symptoms) in 1 temporary settlement over 1 month. Laboratory studies included culture of blood and assay of paired sera for rickettsial IgM and IgG antibody, for dengue IgM and IgG antibody, and for leptospiral IgM and IgG antibody. Among 37 patients (27 adults and 10 children), 28 (75%) had a rickettsiosis (26 cases of murine typhus and 2 cases of scrub typhus). No case of enteric fever, dengue, or leptospirosis was diagnosed. The illnesses of 9 patients were not identified. Signs and symptoms did not distinguish confirmed rickettsial infections from undiagnosed illnesses. The 1 month attack rate of rickettsial infection was 29/100,000 for children and 185/100,000 for adults. Murine typhus was a major cause of febrile illness in this settlement.     

Immunohistochemical study of scrub typhus: a report of two cases

Scrub typhus is a zoonotic disease caused by Orientia tsutsugamushi, which is transmitted by chiggers. The target cells of this rickettsia are poorly defined in humans. Immunohistochemical staining of tissue sections of patients with scrub typhus is helpful in investigating the target cells of this rickettsia in different organs. We studied two autopsy specimens by immunohistochemical staining using a specific antibody against O. tsutsugamushi. Rickettsiae were located in endothelial cells in all of the organs evaluated, namely heart, lung, brain, kidney, appendix and skin, within cardiac muscle cells and renal tubular epithelial cells, and in macrophages located in the lymph node, liver and spleen. In conclusion, O. tsutsugamushi may disseminate into multiple organs through endothelial cells and macrophages, resulting in the development of fatal complications.     

Laboratory diagnosis of two scrub typhus outbreaks at Camp Fuji,Japan in 2000 and 2001 by enzyme-linked immunosorbent assay,rapid flow assay,and Western blot assay using outer membrane 56-kD recombinant proteins

Two scrub typhus outbreaks occurred among U.S. Marines training at Camp Fuji, Japan, between October 25 and November 3, 2000 and October 17 and November 30, 2001. Nine cases in approximately 800 Marines in 2000 and eight cases in approximately 900 Marines in 2001 (approximate attack rates = 1.1% and 0.9%, respectively) reported with signs and symptoms of fever, rash, headache, lymphadenopathy, myalgia, and eschar. Serologies and rapid response to doxycycline treatment indicated they had scrub typhus. Sixty-four convalescent serum samples (18 suspected cases and 46 negative controls) from U.S. Marines training at Camp Fuji during the outbreaks were assessed by enzyme-linked immunosorbent assay (ELISA), rapid flow assay (RFA), and Western blot assay for evidence of infection with Orientia tsutsugamushi, the causative agent of scrub typhus. All but one suspected case had serologic evidence of scrub typhus and all 46 control sera were non-reactive to O. tsutsugamushi antigens. The recombinant 56-kD antigen (r56) from the Karp, Kato and Gilliam strains of O. tsutsugamushi in an ELISA format provided better results than Karp r56 alone (ELISA and RFA) or whole cell antigen preparation from Karp, Kato and Gilliam (ELISA).     

Possible acute coinfections in Thai malaria patients

We conducted serodiagnostic testing for dengue virus infection, murine typhus, scrub typhus and leptospirosis in Plasmodium falciparum-infected individuals in Thailand. Sera from 194 malaria patients with a median age of 24 years were tested. No antibody titers diagnostic of dengue virus infection were demonstrated, but 29 (15%) of patients had serological evidence of scrub typhus, 45 (23.2 %) patients had evidence of murine typhus, and 15 (7.7%) sera tested positive for leptospirosis. Our serological results suggested that duel infections are not uncommon in malaria that is acquired in Thailand. However, our results must be confirmed by prospective studies aimed at describing the causative organisms. Mixed infections would have multiple implications for clinicians, including unexpected clinical findings and apparent poor responses to antimalarial treatment in patients thought only to have malaria.