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.
Malaysian, British and New Zealand soldiers were tested for evidence of infection with Rickettsia tsutsugamushi after several weeks' exposure to the infection during field exercises in Malaysia. 39 (5.0%) of 787 British and New Zealand soldiers developed immunofluorescent antibody (IFA) to R. tsutsugamushi to a titre of 1:50 and two (0.3%) to a titre of 1:100. 11 (1.5%) of 751 Malaysian soldiers also developed low titres less than or equal to 1:100. These low antibody levels were not correlated with clinical disease, and their significance is unknown. Seven (0.9%) of the Malaysians showed an IFA rise to greater than or equal to 1:200, and three of these experienced febrile illnesses, one lasting two weeks. An additional eight Malaysian soldiers had an IFA titre of greater than or equal to 1:400 when first tested and six of these also had a Proteus OXK agglutinin titre of greater than or equal to 1:160, indicating infection shortly before the study.
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.
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.
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.
Using an indirect immunofluorescence technique, sera from 113 cynomolgus monkeys (Macaca fascicularis), trapped in Peninsular Malaysia, were screened for the presence of antibody to six prototype strains of Rickettsia tsutsugamushi combined into three polyvalent groupings: I--Karp, TA716, and TA763; II--Gilliam; and III--TA678 and TH1817. Fifteen percent (17/113) of the monkeys had antibody titers greater than or equal to 1:50 to one or more of the antigenic groups. Although a titer greater than or equal to 1:150 is generally considered indicative or prior Rickettsia tsutsugamushi infection, we selected a less than 1:25 titer as a conservative standard to insure non-infected animals. Using this criterion, 62 (55%) of the 113 monkeys were accepted for use in scrub typhus studies. The high prevalence of antibody to scrub typhus in the semi-arboreal cynomolgus monkey is in marked contrast to the low prevalence reported in the strictly arboreal silvered leaf monkeys (Presbytis cristatus). The results of this study indicate that cynomolgus monkeys should be rigorously screened for evidence of prior infection before they are included in experimental scrub typhus studies.
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.
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.
Two communities of Orang Asli (aborigines) in Peninsular Malaysia were observed for evidence of Rickettsia tsutsugamushi infection over periods of 1-8 mo. Sequential sera were examined for antibody by the indirect immunofluorescence test. The incidence of infection in the two self-selected populations in the two communities was calculated to be 3.9% per month and 3.2% per month.
An explanation was sought for the disparity between the low reported incidence of scrub typhus and the high prevalence of antibody to Rickettsia tsutsugamushi in the rural population of Malaysia. A combination of isolation of the organism, titration of antibody by indirect immunofluorescence, and the Weil-Felix test was used to confirm infections. Scrub typhus was found to be very common, causing 23% of all febrile illnesses at one hospital. The infection was particularly prevalent in oil-palm workers, causing an estimated 400 cases annually in a population of 10,000 people living on one plantation. The clinical syndrome, whether mild or severe, was difficult to distinguish from that due to other infections. Eschars, rashes and adenopathy were uncommon. When used to examine early sera, the Weil-Felix test failed to confirm the diagnosis in most infections.20
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%.
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.
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 sensitivities and specificities of the indirect microimmunofluorescent antibody (IFA) and Weil-Felix (OXK) tests for scrub typhus were established for a range of titers using groups of diseased and control (other febrile illnesses) patients diagnosed by other methods. At a cut-off point of greater than or equal to 1:400, the IFA test was 0.96 specific, and at greater than or equal to 1:320, the OXK was 0.97 specific. Using either these highly specific levels of antibody or other rigorous diagnostic criteria (isolation or 4-fold rising titers), the prevalence of scrub typhus infection was determined to be 0.22 in an unselected population of febrile patients in a rural Malaysian hospital. Probability values (Pr) for the correct diagnosis of scrub typhus were then calculated from the specificity, sensitivity and prevalence determination for a range of titers. The Pr for an OXK titer of greater than or equal to 1:320 was 0.79, and the Pr for an IFA titer of greater than or equal to 1:400 was 0.78. When both these titers were present in a single specimen, the Pr increased to 0.96.
Scrub typhus is a major cause of febrile illness throughout the Asia-Pacific region. It is commonly undiagnosed, partly because of the lack of a simple, reliable diagnostic test which can be used in clinical laboratories. The indirect immunoperoxidase technique, configured into a test kit, was provided to technicians who were trained in its use. They used the kit during a 2 year field trial in their respective clinical hospital laboratories throughout Malaysia. In an evaluation using 1,722 consecutive sera tested in those laboratories, the kit was found to have a median sensitivity for IgG detection of 0.85 (range 0.33-0.95), a median specificity of 0.94 (range 0.88-1.00), reproducibility of 0.86, and efficiency of 0.92 when compared to the reference laboratory. In a proficiency survey in which 10 laboratories received 3 coded test samples, all but 2 laboratories had results within 1 dilution of the reference laboratory in quantitating specific IgG, whereas 7 laboratories were within 1 dilution in quantitating IgM. The shelf life of the kit was at least 1 year at 4 degrees C.
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.
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.