To evaluate the efficacy of drugs for treatment, we tried to make an animal model of Pneumocystis carinii (P. carinii) pneumonia.
Specific pathogen free (SPF) rats immunosuppressed with corticosteroid were intratracheally inoculated with P. carinii. Six weeks after the inoculation, the lung sections of infected rat lung showed increased numbers of P. carinii in the alveoli and thickening of alveolar septa with mononuclear cell infiltration. From 7 to 9 weeks after inoculation, the intensity of infection became more severe, and some rats died at this period. Then, to evaluate drug efficacy in this model, we finished the drug therapy by the 6th week and used the number of P. carinii in the lung and the inflammation score as an indicator of drug efficacy.
In this P. carinii pneumonia animal model, both sulfamethoxazole-trimethoprim clinically established anti P. carinii drug and interferon-gamma which is one of the lymphokines mainly produced by T lymphocytes indicated therapeutic and synergistic efficacy against P. carinii pneumonia.

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From November 1951 to 1954, 416 patients in Kumayama Town, Okayama Prefecture, suffered from epidemic hepatitis, so-called “Kumayama Hepatitis”. The mortality rate was 13.98% in the first one year, and the rate of progress to chronic hepatitis after 10 years was 24.6%. In the present study surveyed in 1991, 720 residents in this area had 13.6% positive anti-HCV (C100-3), significantly higher than in control areas. The rate of positive anti-HBs and abnormal liver functions in the subjects with positive anti-HCV was 42.9% and 40.8%, respectively. In 29 patients with clinically typical “Kumayama Hepatitis”, the positivity rate of and-HA IgG, anti-HBs, and anti-HCV, and the prevalence of abnormal liver function was 82.8%, 41.4%, 34.5%, and 10.3%, respectively. In conclusion, it is suggested that the hepatitis epidemic in Kumayama Town was caused by HAV and superinfected by HCV and/or HBV, then its clinical manifestations became complicated.

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Staphylococcus aureus and Staphylococus spp. were isolated from healthy students and their drug resistance was investigated. S. aureus was isolated from 17 of 70 persons (24.3%). Fourteen strains of 22 isolates of S. aureus were ampicillin-resistant. Two strains each were cefmetazole-resistant and gentamicin-resistant. None of the strains was found to be methicillin-resistant. Compared with the strains isolated from the hospital ward environment, S. aureus in healthy individuals was relatively sensitive to antibiotics.

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HIV-1 strains were isolated from four patients treated with AZT for more than 6 months and from three patients untreated with AZT. Isolates from patients treated with AZT have been cultured by passage of virus in cell culture in the presence of 1μM AZT. However the isolates from patients untreated with AZT could not be cultured in the presence of AZT.
The RT gene of HIV-1 isolates which had been cultured in the presence of AZT were amplified by PCR and cloned to M13 vector. Four amino acid mutations in RT gene (Asp⁶⁷, Lys⁷⁰, Thr²¹⁵, Lys²¹⁹) associated with resistance to AZT were analysed.
All of the 22 clones obtained from the isolates in the presence of 1μM AZT had mutations at codon 215 (Thr-Tyr or Phe). Some of the 22 clones also had other mutations at codon 67 (Asp-Ser), codon 70 (Lys-Arg) and codon 219 (Lys-Glu or Gln).
Four amino acid residues in RT gene of the isolates which had been cultured in the presence of AZT were compared to that of the isolates cultured in the absence of AZT. The clones of the isolates obtained from the patients (04 or 05) had mutations at only codon 215 (Thr→Tyr) in both the presence and the absence of AZT. All of the clones of the isolates obtained from the patients (06 or 07) had mutations at codon 215 and some of them had other mutations.
These data suggest that the AZT resistant variants emerge in patients on long-term therapy with AZT and that the mutation of codon 215 in RT are essential for AZT resistant variants.

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The effects of nalidixic acid (NA) and various new quinolones, such as norfloxacin (NFLX), ofloxacin (OFLX), tosufloxacin (TFLX), lomefloxacin (LFLX) and temafloxacin (TMFX), on the superoxide production of human neutrophil NADPH oxidase were investigated. In their therapeutic concentrations, NA, NFLX and OFLX had no significant effects on superoxide generation of NADPH oxidase. However, TFLX, LFLX and TMFX increased superoxide production by about 130 percent. Fifty μM NA increased superoxide production by 130 percent. Recently, it has been reported that NA and the new quinolones have toxic side effects on the central nervous system. In this report, relationships between toxic side effects of the new quinolones and superoxide production were discussed.

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The vaginal colonization with Staphylococcus aureus, including strains that have methicillin resistance and production of toxic shock syndrome toxin-1 (TSST-1), enterotoxins and protease was studied in 308 women at the time of delivery.Thirty-six of these women were colonized with S.aureus in the vagina.All of the isolates were methicillin sensitive S.aureus (MSSA), except one woman who had both methicillin resistant S.aureus (MRSA) and MSSA.The strains, included MRSA, did not elaborate the TSST-1 and produced protease.The distribution of protease pattern (types) observed by MSSA strains on standard methods caseinate agar plates was type A, 1 strain; type B, 15 strains;type C, 6 strains;type D, 14 strains. Sixteen of the 36 strains produced non of the enterotoxins and 20 strains produced enterotoxins and the type distribution was enterotoxin type A, 6 strains;type B, 10 strains; type C, 1 strain; A and B, 1 strain; A, B and C, 2 strains. These results provide the possibility of pathogenic S.aureus colonized in vagina transmit to neonates at the time of delivery.

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We attempted to detect cytomegalovirus DNA (CMV-DNA) and Pneumocystis carinii DNA (P. carinii-DNA) in sputum samples of 18 hematological neoplasm patients with pneumonia, using rapid cycle DNA amplification. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-pl samples in this glass capillary tubes. After a total amplification time of 15 min, the amplified products were electrophoresed on agarose gels and visualized with ethidium bromide.
In three cases, CMV-DNA was detected at about the time the pneumonia occurred. These patients were successfully treated with ganciclovir in the early stages of infection and CMV was not detected by the virus culture method. In four other cases, P. carinii-DNA was detected in their sputum samples but not detected by Grocott staining. These four cases of P. carinii were successfully treated with sulfamethoxazole-trimethoprim. For detection of CMV-DNA and P. carinii-DNA using the capillary polymerase chain reaction (PCR), a different temperature setting base on the primer difference was not necessary. Therefore, capillary PCR was performed at the same time for detection of CMV and P. carinii.
We conclude that capillary PCR amplification is a valuable tool for rapid diagnosis and early treatment of pneumonia due to CMV and P. carinii.

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Malaria is absent in Japan, but about sixty imported cases are reported in a year. We think it desirable that all medical care institutions should examine for malaria infection promptly. Diagnosis of malaria, in Japan, depends mostly on the examination of thin blood smears stained by Giemsa. However, we sometimes find atypical changes of infected red blood cells, especially in their size. It was also presumed that infected red cells may differ in their size and other morphology by their geographical origin.
The present study was designed to investigate the influences of malaria infection on the morphology of host red cells. Thin blood smear samples from the cases of a single species plasmodial infection with epidemiological circumstances were chosen for this investigation from the specimens which we examined in our laboratory. Cases with a history of chemoprophylactic or chemotherapeutic use within 1 month prior to the blood examination were excluded. Cases were classified according to species of the infected parasites and the geographical origin; Africa, South-East Asia, and Western Pacific. The distribution of red cell diameters and the ratio of maximum diameter to minimum diameter were determined on blood smears using oil immersion lens. Measurement was completed with 20 or 30 infected red cells for each developmental stage of the parasites and 30 non-infected red cells per slide. The presence of Scht ffner's dots was observed on blood smears from vivax or ovale malaria patients. We examined also for Maurer's dots and fimbriated margin of red cells on falciparum and ovale malaria specimens respectively.
Mean red cell diameters of each specimen ranged as follows: 6.93.8.30μm for non-infected red cells, 7.25-8.40μm for Plasmodium falciparum-infected, 7.47.10.76μm for P. vivax-infected, 8.64-9.36μm for P. ovale-infected, and 6.77-9.45μm for P. malariae-infected ones. Red cell diameters were significantly larger in P. vivax (71.4% of cases for ring form, and all cases for the other developmental stages) and P. ovale (all cases for ring form, trophozoite and gametocyte)-infected ones. No significant differences in the size of infected red cells according to the area of infection were observed.
The percentages of Schaffner's dots, when seen, were 7.5% ring form of P. vivax-infected, 72.9% trophozoite of P. vivax-infected, 95.0% schizont of P. vivax-infected, 90.0% gametocyte of P. vivaxinfected, 90.0% ring form of P. ovale-infected, 95.0% trophozoite of P. ovale-infected, and 100.0% gametocytes of P. ovale-infected red cells. The incidence of fimbriated margin on P. ovale-infected red cells by developmental stages were 20.0%, 27.5% and 40.0% for ring form-infected, trophozoite-infected, and gametocyte-infected ones. It was concluded that, in many cases, we can differentiate the species of Plasmodium by proper staining and investigation of a certain number of infected red cells.

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We investigated the yearly changes of the incidence of Pseudomonas aeruginosa (P. aeruginosa) isolated from chronic lower respiratory tract infections (CLRTI), and also performed a clinical study on CLRTI with P.aeruginosa by transtracheal aspiration (TTA) to clarify the recent trend of P.aeruginosa infection in CLRTI and the predisposing clinical factors to the acute exacerbation.
The isolation rate of P.aeruginosa among the total isolated bacterias in CLRTI between December 1978 and March 1983 was 8.4%, but it increased to 23.1% between April 1988 and March 1993.
In 69 episodes (40 cases) of P.aeruginosa isolated from CLRTI between April 1983 and March 1993, monomicrobial infections of P.aeruginosa were 42 episodes (60.9%) and polymicrobial infections were 27 episodes (39.1%).When the diseases were classified into acute exacerbated and non-exacerbated phases, polymicrobial infections were seen more in the former phase, and the principal organisms detected with P.aeruginosa were Haemophilus influenzae and Streptococcus pneumoniae.In the acute exacerbated cases, predisposing conditions concerning the exacerbation were divided into four patterns: 1.polymicrobial infections with H.influenzae or S.pneumoniae, 2.after acute upper respiratory tract infections due to viral superinfection, 3.early phase from bacterial replacement by P.aeruginosa, 4.immunocompromised states such as adrenal corticosteroid administration or systemic underlying diseases.
These results suggest that the importance of P.aeruginosa in CLRTI is increasing year by year and we must pay attention to the fact that P.aeruginosa alone may also cause acute exacerbation in the latter 2 patterns of the condition.

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It has been reported that antibodies to HIV-1 could be detected in saliva of patients with AIDS. We studied whether saliva is potentially useful for screening of HIV-1 infected person. Pairs of serum and saliva were collected from both 19 HIV-1 seropositive outpatients (CDC type, II: 15, III: 1, IV: 3) of AIDS clinic in our hospital and 4 controls. Multiple saliva collection was done from seropositives periodically for 8 months after the first sampling. Serum and saliva were tested with ELISA and Western blot (WB) methods by using kits of diagnostics Pasteur (ELAVIA MIXT and LAV Blot-1).
All pairs of serum and saliva from controls were clearly negative by ELISA. Nineteen sera of proven seropositive cases and paired 18 saliva samples were confirmed to be positive in ELISA test employed, but O.D. value of 1 saliva sample was below the cut-off level. However, in the follow-up study, samples taken from the same individuals after the first sampling showed positive results. Forty-seven saliva samples from seropositives were served for the WB test. Clearly positive bands were observed in 43 samples. In each of the remaining 5 samples, the final decision was “indeterminate”, although a strong reactive band was observed at GP-160.
The results mentioned above suggested that saliva was useful for screening of antibodies to HIV-1 in epidemiological studies, though it is necessary to improve the sensitivity of ELISA and WB for tests of saliva.

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