We compared a commercially available dot-blot immunoassay system with the indirect immunofluorescence assay (IFA) in tests of known negative and known positive sera from scrub typhus cases. Using a panel of 100 sera from patients with various rickettsial and nonrickettsial infections, we observed that the IFA was 99% specific and the dipstick assay was 98% specific. In tests of 91 sera (30 negative and 61 positive for scrub typhus antibodies) from a study of febrile patients in Malaysia, using the standard of an IFA titer < 1:64 as negative, an IFA titer > 1:128 as positive, and an IFA titer = 1:64 as either positive or negative (supported by clinical records), dipsticks were 83% specific and 90% sensitive. The quantitative correlation of the dipsticks to IFA titers was confirmed by significant differences in geometric means of inverse IFA titers corresponding to the number of positive dipstick spots (no dots = 8.5, one dot = 43.3, two dots = 206.7, and three dots = 676.9). The assay would enable physicians and public health workers who deal with patients to quickly diagnose and appropriately treat most cases of the disease, especially in areas of high prevalence where the proportion of false-positive results to true-positive results would be low.
The seroprevalence of Orientia tsutsugamushi, Rickettsia typhi, and TT118 spotted fever group (SFG) rickettsiae in 300 rubber estate workers in Slim River, Malaysia was determined in December 1996 and March 1997. In December, which was the wet season, 23.3%, 3.0%, and 57.3% of the population had antibodies detected against the three rickettsiae, respectively. The highest seropositive rate of 40% was detected for single infection with SFG rickettsiae, followed by a rate of 15.3% for both O. tsutsugamushi and SFG rickettsiae among the rubber estate workers. Subjects less than 21 years old had a lower seroprevalence of SFG rickettsiae compared with the other age groups. Indians had a higher seroprevalence of O. tsutsugamushi compared with other ethnic groups. Rubber tappers had a higher seroprevalence of SFG rickettsiae compared with other occupational groups. During the dry season in March 1997, there was a significant increase in the seroprevalence of R. typhi. The seroconversion rates for IgM against O. tsutsugamushi, R. typhi, and SFG rickettsiae were 5.7%, 12.3%, and 15.1%, respectively, during the four-month period. Significant variations of antibody titers towards the three rickettsiae was noted among subjects who were bled twice. This suggests a significant and continual exposure of rubber estate workers to the three rickettsiae.
A seroepidemiological survey of 837 people and 383 febrile patients was performed in rural areas of Sabah. We determined that the rickettsial diseases scrub typhus and endemic typhus were uncommon causes of febrile illness, as was tick typhus, except in forest dwelling peoples. The rate of occurrence of SFGR specific antibody was 16.5% among 412 forest dwellers, indicating that tick typhus may be a frequent cause of illness in this population.
Limited data is available on the current status of scrub typhus infection in the aboriginal population in Malaysia. This study was aimed to provide recent data on the degree of exposure of 280 individuals from seven aboriginal subgroups to Orientia tsutsugamushi (causative agent of scrub typhus) in West Malaysia. The environment, socioeconomic and behavioural risk factors associated with the disease were also investigated.
The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.
The seroprevalence of various Orientia tsutsugamushi (OT) strains among Malaysian patients with suspected scrub typhus infections was determined using an indirect immunoperoxidase (IIP) assay. IgG against a single OT strain were detected in six sera (3 Karp, 1 Gilliam and 2 TC586), whereas IgM antibodies against a single OT strain (Gilliam) were noted in 3 sera (Gilliam). IgG reactive to all OT strains were present in 33 (47.1%) of the 70 sera and IgM reactive to all OT strains were present in 22 (78.6%) of the 28 sera. The fact that most sera were reactive to multiple OT strains suggests that group-specific antigens are involved in scrub typhus infections, whereas very few were due to strain-specific epitopes present on these strains. Peak IgG and IgM titers were noted more frequently against Gilliam, Karp, and TA763 strains: this suggests that these strains may be the commonest infecting strains among Malaysian patients. Two predominant OT polypeptides consistently reacted with patients' sera were the 70 kDa and 56 kDa proteins.
In this study, recombinant proteins that encompassed the AD I-AD III regions of 56 kDa immunodominant gene of 2 Orientia tsutsugamushi (OT) serotypes; Gilliam and TA763 were expressed in Escherichia coli. Both recombinant proteins exhibited serologic cross-reactivity with the rabbit antisera against various OT serotypes, as evaluated by enzyme-linked immunosorbent assay (ELISA), but not against other rickettsial species, including Rickettsia typhi, R. prowazekii and TT118 SFG rickettsiae. The feasibility of using the recombinant proteins as a diagnostic reagent was further evaluated by ELISA using sera from blood donors and scrub typhus patients. The results suggested a higher affinity of the antihuman IgM than IgG with both recombinant proteins. The IgM ELISA findings were agreeable with the results of indirect immunoperoxidase (IIP) assay especially with sera of high antibody (1:1600). However, more than one antigen are probably needed for development of an effective assay for serodiagnosis of scrub typhus in endemic areas.
A serosurvey was conducted in 1995-97 among 1596 febrile patients from 8 health centres in Malaysia for antibodies against Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) by using an indirect immunoperoxidase assay. A total of 51.4% patients had antibody against at least 1 of those rickettsiae. Antibody to SFGR was most prevalent (42.5%), followed by RT (28.1%) and OT (24.9%). The seroprevalences of antibodies to SFGR, RT or OT alone were 12.4, 3.6 and 4.3%, respectively. Antibodies against more than 1 species of rickettsiae were presence in 31.1% of the patients, suggesting the possibility of co-infection, previous exposures or serological cross-reactivities. Seroprevalence of the various rickettsiae varied according to locality, with SFGR antibodies being the most prevalent in most areas. There was no significant association of prevalence of rickettsial antibody with gender. The seroprevalence of OT, SFGR and RT increased with patient age but an increase of antibody titre with age was not significant. Those working in the agricultural sectors had significantly higher seroprevalence of OT, SFGR and RT than those not related with agricultural activities. Scrub typhus remains a public health problem with an estimated annual attack rate of 18.5%. Tick typhus and murine typhus as shown in this serosurvey appear much more widespread than scrub typhus in this country.
The seroprevalence of Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) among blood donors and febrile Malaysian patients in the urban areas was determined. Of the 240 blood donors, 5.4%, 9.2% and 1.7% had either present or previous exposure to OT, RT and SFG rickettsiae, respectively. Patients admitted to an urban hospital had high seroprevalences of OT (43.5%) and RT (22.9%), as compared to SFGR (11.6%). Antibody levels suggestive of recent infections of scrub typhus, murine typhus and tick typhus were detected in 16.8%, 12.7% and 8.2% of patients respectively. No significant difference was noted in the distribution of rickettsial antibodies among urban patients from 2 geographical locations. However, the serologic patterns of rickettsial infection in the urban areas were different form those of rural areas.
Fifty-one Rickettsia tsutsugamushi isolates from small mammals collected in central Peninsular Malaysia serologically characterized by direct immunofluorescence using eight prototype strains. Karp-related (TA763, TA686, TA716) antigens were found in 90.2% of the isolates.
One hundred and fourteen Rickettsia tsutsugamushi isolates, recovered from febrile patients in central Peninsular Malaysia, were antigenically analyzed by direct immunofluorescence using eight prototype strains. Twenty-nine antigenic types were detected. The TA763, TA716, Karp and TA686 strains were the most common and occurred singly or in combination with each other or other strains in 86% of the isolates.
An epidemiological study in a mature oil palm estate in Peninsular Malaysia has demonstrated a low prevalence of R. tsutsugamushi infection in small mammals. The direct fluorescent antibody technique for assaying infections in chiggers proved more sensitive than mouse inoculation. Most infections in both chiggers and rodents were caused by the Karp strain.
Serological surveillance for up to two years of 114 patients with laboratory confirmed scrub typhus showed that antibody to Rickettsia tsutsugamushi as demonstrated by the indirect fluorescent antibody test is short-lived. The mean reversion time from mean peak titre (1:499) was 48.9 weeks and the calculated annual reversion rate to a titre less than 1:50 was 61%. This can be used to estimate attack rates based on point prevalence of antibody. The relationship between antibody prevalence and attack rates observed by other workers was confirmed using this model. The possible uses of the finding and its implications in Malaysia are briefly discussed.
Based on the prevalence of antibody, an estimated 3% of the population of rural Malaysia is infected with Rickettsia tsutsugamushi each year, resulting in positive antibody rates in focal areas of 6 to 69%. Most of these infections do not appear to produce clinical scrub typhus. A wide range of seropositivity rates was found in areas otherwise resembling each other in predominant occupation, terrain, and nearby habitat. The prevalence rates however were significantly higher in people who worked in forested areas and significantly lower in people with urban occupations.
Scrub typhus is an endemic problem in Malaysia. Yet its diagnosis appears to depend heavily on the Wetl-Felix test as the more sophisticated diagnostic procedures are not available routinely. We therefore reviewed our experience with scrub typhus patients treated at the Melaka General Hospital from 1983 to April 1986, to identify those clinical features which are diagnostic of this rickettsial illness. Based on the clinical presentation of our patients and the dramatic response of scrub typhus to Doxycycline, we propose a clinical approach to diagnosis until more specific and cheap diagnostic procedures become available in our laboratories. Otherwise, this rickettsial illness will continue to be under-recognised.
The optimal conditions for the determination of exposure to scrub typhus by the whole blood lymphocyte transformation assay was 7 days culture of 10% blood in RPMI 1640 medium supplemented with 10% human AB-negative serum and L-glutamine with 50-200 micrograms protein/ml of Karp, Kato, or Gilliam strain membrane antigen. A simple exponentially decaying linear model shows the decrease in lymphocyte viability, the ability of sensitized cells to be stimulated with PHA mitogen, and the corresponding decrease in stimulation by scrub typhus antigens with increasing time of preincubation on ice. The lower limit of stimulation index for the detection of scrub typhus by whole blood lymphocyte transformation assay was 4.0 with a type I error of 1%.
Binding studies of 160 overlapping, synthetic octapeptides from the hydrophilic regions of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi with sera from patients with scrub typhus revealed 15 immunodominant peptides which are recognized by all the sera tested. Further analysis of the specificity of peptide binding with five of these peptides indicated that the peptides showed significantly stronger binding to scrub typhus patients' sera than they did to sera from patients with other febrile illnesses common in the region, i.e., malaria, dengue fever, typhoid fever, and leptospirosis. The main antibody class binding to these peptides appears to be immunoglobulin M, and there appears to be little correlation between reactivity with peptides and antibody titers measured by the indirect immunoperoxidase test.