Sensitivity of polymerase chain reaction assay for Rickettsia tsutsugamushi in patients' blood samples.

We developed a nested polymerase chain reaction (PCR) method to detect Rickettsia tsutsugamushi (R. tsutsugamushi) DNA and determined its sensitivity. Primers were selected from the DNA sequence of the 58-kDa group-specific antigen gene of the Karp strain. The target sequence of rickettsial DNA was detectable as the band corresponding to 88 bp in 1.0 microgram of the DNA extracted from BS-C-1 cells infected with R. tsutsugamushi. Rickettsia-specific bands were observed not only for the homologous Karp strain, but also for four heterologous strains: two other reference strains (Gilliam and Kato) and two prototype strains prevalent in Miyazaki district (Irie and Hirano). The minimum copy number detectable by this method was estimated to be five rickettsiae. All of nine peripheral blood mononuclear cell samples from patients with tsutsugamushi disease who were seen 2-11 days after disease onset tested positive for rickettsial DNA. The PCR assay method presented here could be a specific diagnostic tool for tsutsugamushi disease, especially in its early acute stage.

Miliary tuberculosis presenting as fever and jaundice with hepatic failure.

A 58-year-old male patient with miliary tuberculosis presenting as jaundice and hepatic dysfunction was reported. He was admitted to the Miyazaki Medical College Hospital, Miyazaki, Japan, because of fever and jaundice. Chest x-ray revealed a calcified primary affect of tuberculosis in the left upper lung field and miliary shadows throughout both lung fields. Liver function tests showed indications of obstructive jaundice and hepatic dysfunction. He rapidly deteriorated and died 3 days after admission. Autopsy revealed disseminated miliary tuberculosis in all major organs. Many miliary tubercles were densely distributed in the liver, especially in and near the portal tracts. The intestine was free from tuberculous lesions. Miliary tuberculosis with jaundice is rare and its pathogenesis is discussed.

[Infections in hematological malignancies--clinical analysis of septic patients admitted to the Second Department of Miyazaki Medical College Hospital in the past ten years].

Two hundred and sixty-two patients (actual number 162) of hematological malignancies were admitted to our department from November 1977 to December 1986. Fourty-three of them (16.4%) were demonstrated to be accompanied with sepsis by blood culture. In acute non-lymphocytic leukemias (AML, APL, AMoL) the rate of sepsis was 33.8% (27 patients), while in lymphocytic malignancies (ML, HD, ATL) it was 11.7% (16 patients), particularly being 3.0% in ATL. Among the detected pathogenic microorganisms, gram-negative bacilli were 86.2% in the former and 50.0% in the latter. Especially, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli occupied 58.6% of the total in the former. Laboratory examination, when sepsis occurred, revealed peripheral neutropenia in acute non-lymphocytic leukemias (mean 831/cmm) but not in lymphocytic malignancy (mean 4,420/cmm). And 20 of the 27 cases showed remarkable neutropenia of below 500/cmm in the former. On the other hand in the latter, out of 16 only one with ATL was the case. Hypogammaglobulinemia was one of the characteristic features in lymphocytic malignancies but not in acute non-lymphocytic leukemias. Hypogammaglobulinemia in lymphocytic malignancies might be affected by long-term immunodepressant therapy. Immunologic skin reaction was demonstrated to be decreased in lymphocytic malignancies on admission. From the findings mentioned above, affecting factors to infections may be mainly neutropenia in acute non-lymphocytic leukemias and immunodeficiency in lymphocytic malignancies. And sepsis can occur frequently under neutropenic condition. In ATL both of humoral- and cellular-immunologic disturbance were detected before therapy. But peripheral neutrophil count was maintained to be normal and this could be the reason for the low septic incidence in ATL despite of total immunodepression.

[Seroepidemiological analysis of the age specific prevalence of anti HTLV-I in Miyazaki Prefecture].

Antibody for HTLV-I in sera from 11,224 residents in Miyazaki Prefecture, Japan, was determined by indirect immunofluorescent antibody method to compare age- and sex-specific antibody prevalence among three geographically divided areas of the prefecture. There was a significant difference in the positive rate of older age groups among three areas, 9.0% for the northern part, 14.5% for the southern part and 8.4% for Miyazaki City, in spite of little variation in younger age groups. A marked rise of positive rate in the southern part at the age of 40th was observed, which suggests that changes of some conditions for mother-to-child transmission happened in the time of their birth. Six seroconversions were observed by the follow-up study for five years of the 971 residents. They were between 28 and 38 years of age, four men and two women. This may provide another reason for the increase in antibody positive rate by age in the adult. However, it could not be investigated if the seroconversion was caused by a horizontal transmission from their spouse. The possibility of the long latency of the virus in man as in the experimental animal may also have to be considered.

[Liver abscesses successfully treated by intraportal administration of amphotericin B in a case of acute myeloblastic leukemia (M2)].

A 46-year-old male was admitted to our hospital because of relapse of acute myeloblastic leukemia (M2). Remission was successfully reinduced after reinduction chemotherapy consisting of daunorubicin, cytosine arabinoside, etoposide and vincristine, but was complicated by neutropenia. After the therapy, the patient had persistent fever of about 38 degrees C despite broad-spectrum antibiotics therapy and the patient developed pain in the right quadrant of the abdomen. The white blood cell count rose to 23000/mm3. Liver function tests showed abnormal findings mainly consisting of an elevated serum alkaline phosphatase level. Ultrasonography showed multiple hypoechoic lesions in the liver and CT scans also revealed multiple low density areas. Therefore he was suspected of having a complication of liver abscesses. Amphotericin B was administered 75 mg/day intravenously every other day. A percutaneous liver biopsy was performed, but was not diagnostic. Blood cultures were negative for pathogens. Amphotericin B was administered up to a cumulative dosage of 2.3 g, but the patient remained febrile. Then he had an exploratory laparotomy and an open liver biopsy. The liver biopsy samples showed fungal elements proved by PAS staining. A catheter was inserted into the portal vein. Administration of Amphotericin B was started 20 mg daily through the catheter. The temperature fell to normal after institution of this therapy. The abnormal findings in CT scans almost disappeared and the inflammatory findings became negative after he had received intraportal administration of Amphotericin B over three months. Through the analysis of this case study, we confirmed that the intraportal administration of Amphotericin B was effective to the intractable liver abscesses due to fungi.

Suppression of delayed-type hypersensitivity to PPD and PHA in elderly HTLV-I carriers.

In a previous study on immune responsiveness among asymptomatic human T-cell leukemia virus type I (HTLV-I) carriers, we found that carriers had significantly reduced delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD) skin testing. The association was strongest among persons at least 60 years of age. In order to evaluate this finding further, we evaluated the response to both PPD and phytohemagglutinin (PHA) in an elderly population. Fifty-six consecutive hospitalized patients with nonimmunosuppressive diseases were examined. None had a history of tuberculosis nor evidence of the known HTLV-I-associated diseases. The subjects' ages ranged from 62-93 years (median = 75 years); 43 were women and 13 were men. Twenty-two of the subjects were HTLV-I antibody positive. Among the carriers, there was an increased level of nonreactivity to PPD, the relative risk adjusted for age (RR) being 1.9 (95% confidence interval, 0.62-5.8), as well as to PHA of RR = 2.3 (0.60-9.0). When subjects were cross-classified for response to both skin tests, 15 of 17 carriers were nonreactive to either or both antigens compared to 15 of 25 noncarriers [RR = 5.1 (0.99-25.9) (p value, one-sided = 0.026)]. The decline in reactivity to both antigens increased with age, but was consistently lower among the carriers. Among subjects with positive reactions to PPD, the degree of reaction as measured by the size of erythema was reduced among the carriers; however, for PHA responders, the response in carriers appeared to be normal. Among the HTLV-I antibody negative subjects, the size of erythema for both antigens was strongly correlated (p = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

[Clinical and etiological studies of tsutsugamushi disease in Miyazaki district--correlation of serological type of R. tsutsugamushi to clinical feature].

The correlation of pathogenic and immunologic characteristics of Rickettsia tsutsugamushi to clinical findings of patients with tsutsugamushi disease in Miyazaki was investigated. In two immunological types, Hirano type strains seemed to have higher virulence to mice than Irie from the findings during the course of infection and on autopsy. A strain of Hirano type was so virulent as to succumb to the infection. As to clinical findings, incidence of hepatomegaly was slightly higher in Hirano type patients than Irie, which is one of the signs in severe type tsutsugamushi disease. This was supported by the higher mean value and frequent appearance of abnormality in liver function test, sGOT, sGPT and LDH, in this type of patients.

[A study on immunity in tsutsugamushi disease].

The immune response in human tsutsugamushi disease (scrub typhus) was studied. Anti-rickettsial activity of sera, peripheral mononuclear cells and their culture supernatants from patients on in vitro growth of Rickettsia tsutsugamushi proliferating in normal human peripheral macrophages was examined. The results obtained were as follows. 1) Sera from patients at the early convalescent stage, which exhibited high antibody titers against R. tsutsugamushi, effectively inhibited their growth in macrophages. 2) Sera from patients after a long period from the onset showed low antibody titers and did not inhibit rickettsial growth. 3) Mononuclear cells and T cell enriched fractions suppressed rickettsial growth when they were obtained from the patients at the early convalescent stage and even after as long as 3.5 years from the onset. 4) The culture supernatants of the T cell-enriched fractions which were collected from the patients mentioned above, exhibited a similar antirickettsial activity. These findings indicate that sensitized T lymphocytes and macrophages might play a fundamental role in immunological defense mechanism in tsutsugamushi disease. And the results obtained in our experiments are compatible with those previously reported in experimental scrub typhus in laboratory animals such as mice and monkeys.

Suppression of tuberculin skin reaction in healthy HTLV-I carriers from Japan.

Although it is thought that infection with human T-lymphotropic virus type I (HTLV-I) is immunosuppressive, this has not been clearly demonstrated among healthy carriers, and there are no data concerning delayed-type hypersensitivity (DTH). To evaluate this hypothesis, DTH to purified protein derivative (PPD) of tuberculin was measured in 126 healthy adults from an endemic area for HTLV-I infection in southern Japan. Among the 39 HTLV-I carriers, only 15% had detectable induration following PPD exposure, compared to 46% of the 87 non-carriers. In addition, the size of erythema among those carriers with a positive reaction was about 70% of that among non-carriers. Overall, there was a significantly inverse association between the degree of DTH response and prevalence of antibody. In relation to subjects with strong to moderate reaction, those with negative or indefinite reaction were 6 times more likely to be a carrier. This association was much stronger among subjects aged 60 years or older than among younger persons. These findings indicate that there is subclinical immunosuppression among HTLV-I carriers, which increases with age.

Comparative diagnostic assay results for detecting antibody to HTLV-I in Japanese blood donor samples: higher positivity rates by particle agglutination assay [corrected].

Higher positivity rates for prevalence of anti-HTLV-I antibody have been reported for the gelatin particle agglutination (PA) assay when compared to that of the indirect immunofluorescence assay using acetone-fixed HTLV-I producing cells (IF-FA). To evaluate the discrepancy between these two screening methods, PA-positive/IF-FA-negative sera were tested by four additional assays for anti-HTLV-I: indirect immunofluorescence assay using live HUT102 cell membranes (IF-MA), enzyme immunoassay (EIA), radioimmunoprecipitation (RIP), and Western blotting (WB). Sera obtained from 6915 Japanese blood donors were assayed for anti-HTLV-I antibody by PA, and 389 (5.6%) were positive. These 389 sera were re-examined by IF-FA, and 29 (7.5%) were negative. Sufficient material was present for 20 of the 29 PA-positive/IF-FA-negative sera for further evaluation by the IF-MA, EIA, RIP, and WB. Fifteen (75%) of the 20 were positive by IF-MA, but only 6 (30%) were positive by EIA. Both RIP and WB confirmed 17 (85%) of the samples, with each detecting a serum that was negative by the other. Thus, 18 (90%) of the 20 were confirmed by either RIP or WB. The nonconfirmed sera were all positive on PA at low titer. These findings suggest that the PA assay is more sensitive than either IF-FA or EIA.