Seroprevalence of scrub typhus infection in patients with pyrexia at some malaria clinics in three western provinces of Thailand

In Thailand, the epidemiological data on scrub typhus infection represents only "the tip of an iceberg" especially in malaria clinics where patients come to seek attention because of other febrile illnesses that may have initial clinical signs that are indistinguishable from malaria. The objectives of this study were to determine the prevalence of antibody titers to Orientia tsutsugamushi, and its various strains, among patients at some malaria clinics in three western provinces of Thailand. The sample was represented by 200 patients from 6 malaria clinics in Ratchaburi, Petchaburi and Kanchanaburi provinces between June and November, 1994. Blood specimens were collected with their consent. Immunofluorescent antibody assays (IFA) were used for measuring IgM and IgG antibody titers for scrub typhus infection. The results showed that the prevalence rate for scrub typhus infection (IgM and/or IgG titer > or = 50) was 59.50% (119 cases). The immunofluorescent antibody response to various strains of O. tsutsugamushi showed that co-infections with the Karp, the Gilliam and the Kato strains were the most common (found in 68.10% of cases). Geometric mean antibody titers (GMT) were highest for the Karp strain, followed by the Gilliam then Kato strains. In conclusion, this study indicates that the prevalence rate of scrub typhus is not rare in these areas.     

Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56

Although the 56-kDa protein of Rickettsia tsutsugamushi has been presumed to play important roles in generating protective immunity against scrub typhus, studies of this protein have been impeded. We used the recombinant 56-kDa protein of R. tsutsugamushi Boryong fused with the maltose-binding protein of Escherichia coli (MBP-Bor56) to analyze its ability to induce protective immunity in a C3H/HeDub murine model. Intraperitoneal immunization of mice with MBP-Bor56 resulted in an increase in the 50% minimal lethal dose of more than 160 times compared with that for the control mice. Splenic mononuclear cells from the mice immunized with MBP-Bor56 showed a dose-dependent pattern of lymphocyte proliferation response and secreted gamma interferon and interleukin-2 when stimulated with irradiated R. tsutsugamushi Boryong, which is a cytokine profile of Th1 cells. High titers of antibody to R. tsutsugamushi were also demonstrated by indirect immunofluorescent-antibody testing. These findings suggest that the 56-kDa protein of R. tsutsugamushi is one of the candidates for a vaccine against scrub typhus.     

Epitope mapping of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi

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.     

Scrub typhus in the Western Pacific region

Scrub typhus is widely endemic in Asia. Man's behaviour and climatic changes greatly influenced the occurrence of the disease. Increasing prevalences of scrub typhus have been reported from some Asian countries and may coincide with the widespread use of beta-lactam antibiotics or to improve diagnostic facilities and/or more urbanisation into rural areas. Many cases acquired in Asia surfaced in Europe and America. The disease probably is overlooked among paediatric patients. Most patients with scrub typhus present with acute fever of unknown origin (acute FUO). Eschars are rare among Southeast Asian patients. Complications usually develop after the first week of illness. The complications include pneumonitis, meningoencephalitis, renal failure and jaundice. Improved serologic and molecular diagnostic tests are now available. Although drug-resistant strain of Orientia tsutsugamushi has been reported, the infection usually responds to simple but unpopular drugs such as doxycycline or chloramphenicol.     

High-level expression of a 56-kilodalton protein gene (bor56) of Rickettsia tsutsugamushi Boryong and its application to enzyme-linked immunosorbent assays

The 56-kDa protein of Rickettsia tsutsugamushi, which is located on the rickettsial surface, has been shown to be an immunodominant antigen. The gene that encodes the 56-kDa protein of R. tsutsugamushi Boryong (bor56) was cloned. Sequencing revealed an open reading frame of 1,602 bp encoding 534 amino acids with a molecular weight of 56,803. The 56-kDa protein of R. tsutsugamushi Boryong (Bor56) was expressed as a fusion protein with the maltose-binding protein of Escherichia coli by deleting 252 bp from the 5' end of the open reading frame and subcloning it into the StuI site of pIH821. The recombinant fusion protein was purified by amylose column chromatography for application in an enzyme-linked immunosorbent assay to evaluate the ability of the method to detect the antibody to R. tsutsugamushi in human patient sera. By using sera from 100 patients with scrub typhus and 70 patients with other febrile diseases, a high diagnostic sensitivity (95%) and a high diagnostic specificity (100%) were demonstrated, suggesting the suitability of the recombinant antigen for use as an immunodiagnostic tool.     

A new ecology for scrub typhus associated with a focus of antibiotic resistance in rice farmers in Thailand

Following the documentation of chloramphenicol-resistant and doxycycline-resistant strains of Orientia tsutsugamushi (Hyashi) in northern Thailand, we conducted ecological and epidemiological studies near the houses of patients hospitalized with antibiotic-resistant infections. New associations between chiggers, rodents, and O. tsutsugamushi in active rice agriculture areas, an ecological habitat not described previously, are reported. Rattus rattus (L.) was the most common species (representing 85.8% of the 1,433 rodents processed), followed by Rattus losea (Swinhoe) (9.4%), Bandicota indica (Bechstein) (3.6%), and Rattus argentiventer (Robinson and Kloss) (1.3%). O. tsutsugamushi was isolated from 30% of the R. rattus and R. losea, 29% of the B. indica, and 33% of the R. argentiventer collected. Mean minimum infection rates were 0.03 in Leptotrombidium chiangraiensis Tanskul & Linthicum, a new species of chigger, and 0.002 in Leptotrombidium imphalum (Vercammen-Grandjean & Langston), a chigger species not previously associated with scrub typhus transmission. Efficient vertical and horizontal transmission of O. tsutsugamushi by L. chiangraiensis and L. imphalum was demonstrated. During a 19-mo period from October 1993 to April 1995, the overall prevalence of human IgM and IgG antibody to O. tsutsugamushi was 25.5 and 47.3%, respectively. L. chiangraiensis and L. imphalum are incriminated as vectors of O. tsutsugamushi in a rice field habitat associated with a focus of antibiotic resistance.     

A new species of Leptotrombidium (Acari:Trombiculidae) collected in active rice fields in northern Thailand

Leptotrombidium (Leptotrombidium) chiangraiensis Tanskul & Linthicum is described and illustrated as new from specimens collected from the rodents Rattus rattus (L., 1758), Rattus argentiventer (Robinson & Kloss, 1916), Rattus losea (Swinhoe, 1870), and Bandicota indica (Bechstein, 1800) in Chiangrai Province northern Thailand. The new species was collected in active rice fields and adjacent fruit plantation areas. The etiological agent of scrub typhus, Orientia (formerly Rickettsia) tsutsugamushi (Hayashi), has been isolated from patients who live and work in the same habitat where L. chiangraiensis is the predominant Leptotrombidium species.     

Detection of southern African human immunodeficiency virus type 1 subtypes by polymerase chain reaction: evaluation of different primer pairs and conditions

The purpose of the study was to develop a specific and sensitive PCR protocol using env, gag and LTR primer pairs to detect HIV-1 subtypes present in the Western Cape, South Africa. Twenty-two virus strains, belonging to HIV-1 subtypes B, C and D, were randomly selected for PCR evaluation. Cell lysates prepared from these virus-infected cultured cells were tested using 5 different primer pairs: gag SK38/SK39; gag 22/SK39; gag a/b, gag c/d (nested); env SK68/SK69 and LTR SK29/SK30. Eight different PCR profiles were evaluated: one profile each for the 3 gag primer pairs, 3 profiles for the env and 2 profiles for the LTR primer pairs. The number of PCR cycles, time per cycle and/or annealing temperature were changed in each profile. The optimum PCR profile for a specific primer pair was defined as that which detected one copy of proviral plasmid DNA after dot-blot hybridisation. Gag primer pairs detected HIV-1 DNA in all 22 samples. With the env primer pair, suboptimal conditions failed to detect most of the HIV-1 subtype C samples. By increasing the number of cycles and time per cycle, a 100% sensitivity was achieved. With the LTR primer pair all samples were detected by decreasing the annealing temperature and increasing the individual cycle times. This confirms that once PCR conditions are optimised, all HIV-1 subtypes in our study could be detected using different PCR primer pairs.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

Antibodies to Orientia tsutsugamushi in soldiers in northeastern Thailand

The prevalence and incidence of antibodies to Orientia tsutsugamushi, the etiologic agent of scrub typhus, in Thai soldiers living and working near the Thai-Cambodian border in Si Sa Ket Province was investigated. The point prevalence of antibodies varied from 0 to 4.1%. The incidence of antibodies, calculated from individuals who seroconverted following a negative result in a previous bleeding 3 to 5 months earlier, was 4.21% (9/214) in January 1992, 0 in April 1992 and 3.76% (8/213) in September 1992. An annual infection rate of 2.66% was estimated.     

Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5' end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P = 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.     

Experience with the two-windows method for mediastinal lymph node dissection in lung cancer

A rapid dipstick test for scrub typhus was prospectively evaluated in Chiangrai, northern Thailand. Sera from 162 patients with fever of unclear etiology were tested by a dot blot immunoassay using two different antigen concentrations. Dipsticks coated with lower concentration of antigen lacked sensitivity compared with the indirect immunoperoxidase test. Dipsticks with higher antigen concentration had increased sensitivity that was equivalent to that of the immunoperoxidase test. By increasing the antigen concentration on the dipstick, sensitivity increased from 67% to 100%, positive predictive value increased from 90% to 93%, and negative predictive value rose from 92% to 100%. The specificity of both antigen concentrations was 98%. This study establishes that scrub typhus can be confirmed serologically by use of a dipstick assay and that serodiagnosis can be effectively tailored to a target population.     

Morphometric variables of developing primary maxillary first molar crowns in humans

Complementary DNA sequences were selected from a resource of tentatively identified clones from a porcine small intestine cDNA library. Forty PCR primer pairs were designed to amplify 101-309 base pairs of the 3' untranslated region of the genes. The PCR conditions were optimized by altering both formamide and magnesium concentrations on samples of pig, mouse, and hamster DNA. Twenty primer pairs that, under stringent conditions, were pig-specific and amplified the expected fragments were chosen for regional assignment in a pig/rodent hybrid cell panel. Furthermore, 22 primer pairs were chosen to amplify DNA from the parental animals of the PiGMaP shared reference families in order to detect possible polymorphisms. Primer pairs that generated polymorphisms were used for genetic mapping. A total of 22 porcine expressed sequence tags (ESTs) were cytogenetically or genetically mapped by this approach. Twelve of the mapped ESTs could be added to the human-porcine comparative map.     

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.     

Detection of false-negative results in nested primer PCR of proviral HIV-1 DNA

A control template for a competitive nested primer PCR of the HIV-1 gag region was constructed. This construct shares the primer recognition sequences with the wild-type template and yields a 97-bp fragment after amplification (wild-type: 115 bp). To provide an internal control for the individual PCR runs, six copies of this nested primer control plasmid were introduced into a reaction tube containing the specific sample (under the PCR conditions used, this copy number reproducibly gave a positive PCR signal). The results of our study show the feasibility of this concept by analyzing a plasmid (pBH10) containing HIV-1 wild-type sequences, and examination of samples from a cohort of HIV-1-seropositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, which without the use of the control template would have yielded a false-negative result.     

Detection of soluble methane monooxygenase producing Methylosinus trichosporium OB3b by polymerase chain reaction

The soluble methane monooxygenase (sMMO) enzyme complex of methanotrophs cometabolizes haloaliphatic compounds such as trichloroethylene. Two 18-mer oligonucleotides as primary primers and a nested primer of the same length were selected to amplify specific DNA sequences of the sMMO gene cluster using polymerase chain reaction (PCR). Two DNA fragments of sizes 270 and 400 base pairs were obtained when purified DNA from the methanotroph Methylosinus trichosporium OB3b was used as template. The primers were specific for sMMO sequences of M. trichosporium, since none of the 13 bacterial isolates screened yielded the expected length of PCR-amplified DNA fragments. The detection limit of the PCR method was 5 x 10(2) cells of M. trichosporium. The sMMO sequences were successfully amplified in groundwater (containing native microbial population) when seeded with M. trichosporium, FP1 sense (5'-ATGTCCAGCGCTCATAAC-3'), RP1 antisense (5'-TCAGATGTCGGTCAGGGC-3'), FP2 sense nested (5'GCCATCATCGGTCAGGGC-3'), and FP2 sense nested (5'-GCCATCATCGAGGACATC-3').     

Is Kaposi's sarcoma--associated herpesvirus ubiquitous in urogenital and prostate tissues?

Controversy exists as to whether Kaposi's sarcoma-associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.     

GeneUp: a program to select short PCR primer pairs that occur in multiple members of sequence lists

A computer program is presented that selects a small set of short primer pairs for PCR to sample all the sequences in a user-specified list of mRNAs. Such primer pairs could be used to increase the probability of sampling mRNAs of particular interest in differential display and to generate simplified hybridization probes for DNA chips or arrays. The program uses simulated PCR to find pairs of primers that sample more than one sequence in the list. A small set of such primer pairs is selected that give maximal coverage of the sequences in the list. Primer pairs are excluded that: (i) generate simulated PCR products of the same size from a number of sequences in the list, (ii) can easily form primer dimers, (iii) are outside a specified range of G + C content or (iv) occur in another list of undesirable sequences, such as rRNAs and Alu repeats. Five lists consisting of from 48-285 cDNA sequences were used to test the program. A small number of pairs of primers, 8-10 bases in length, were selected that fit the above criteria and that generate one or more simulated PCR products in all or most of the cDNAs in each list.     

Sero-negative tsutsugamushi disease (scrub typhus) diagnosed by polymerase chain reaction

We report a case of sero-negative tsutsugamushi disease diagnosed by polymerase chain reaction (PCR). A 54-year-old man who worked in Nagano prefecture presented with flu-like symptoms that did not respond to cephalosporin therapy. On admission to another hospital, chest roentgenography revealed abnormal shadows; liver dysfunction was also present. Despite therapy, the patient's condition gradually worsened and he was transferred to our intensive care unit. Erythema on all extremities and scabs on the right medial femoral region and the dorsum of the left foot suggested a diagnosis of tsutsugamushi disease. We administered minocycline and gave percutaneous cardiopulmonary support for adult respiratory distress syndrome. Despite all efforts, the patient died. Although serologic tests were not positive, Karp strains of R. tsutsugamuschi were identified on PCR amplification. Autopsy revealed evidence of acute hemorrhagic pancreatitis, which has not been reported previously in tsutsugamushi disease. We conclude that PCR techniques may be useful in confirming a diagnosis of early tsutsugamushi disease.