[Evaluation of new method for diagnosis of opportunistic fungal infection].

New methods for the possible presence DNA specific for Aspergillus or Trichosporon species were developed. In aspergillus PCR, Aspergillus l8S rRNA encoding gene was amplified from five strains of Aspergillus species by the nested PCR but not from other microorganisms. Results of preliminary investigation of this method demonstrated efficient detections of Aspergillus species in serum samples of rats model of aspergillosis and 29 patients with invasive aspergillosis. In trichosporon PCR, Trichosporon 26S rRNA encoding gene was amplified from two strains of Trichosporon species, and detected by the nested PCR in 64 % of serum samples of patients with trichosporonosis, while glucuronoxylomannan antigen was detected in 55 % of samples. The high sensitivity and specificity of the nested PCR indicate that the assay can provide early diagnosis with sufficient accuracy to be clinically useful for patients with opportunistic fungal infection.

[Recent knowledge allowing diagnosis and treatment of deep-seated trichosporonosis].

Deep-seated trichosporonosis is a lethal opportunistic infection in immunocompromised patients. For the rapid diagnosis of this condition, we developed a novel nested-PCR assay that detects DNA specific for clinically important strains of Trichosporon in serum of patients with disseminated trichosporonosis. In this assay, two sets of oligonucleotide primers were derived from the sequence of 26 S ribosomal RNA genes of T. asahii. The specific fragment was amplified from T. asahii and T. mucoides but not from other microorganisms. In a retrospective study using serum samples of patients with disseminated trichosporonosis, the specific fragment was detected in 64% (7 of 11). To treat this infection, we studied the efficacy of rhG-CSF alone and in combination with antifungal agents against disseminated trichosporonosis in neutropenic mice. The results suggested that rhG-CSF might be a useful immunomodulator against Trichosporon infections and the therapeutic outcome might be better when used in combination with antifungal agents.

[Clinical study on mechanism of Pneumocystis carinii pneumonia by polymerase chain reaction].

Pneumocystis carinii is a human respiratory pathogen which causes fatal pneumonia in patients under immunosuppressed or immune deficient conditions. Recent work have documented the usefulness of the polymerase chain reaction (PCR) method in the detection of P. carinii from clinical samples. Therefore, we described our experience in using PCR method in the detection of P. carinii from respiratory samples. In our study, bronchial washing or BALF were good for diagnosis of P. carinii pneumonia (PCP) by PCR. However, PCR method in the detection of P. carinii from swab or sputum was too sensitive because small numbers of P. carinii organisms might be insignificant in causing the disease. It might reveal colonization or asymptomatic carrier state in the upper respiratory tract. Therefore, our result suggested that colonization or asymptomatic carrier state in the upper respiratory tract could eventually evolve into PCP. This would also facilitate basic progress in the pathology or epidemiology of P. carinii infection. In addition, an usefulness of prophylactic therapy for PCP was documented by PCR.

[Detection of Aspergillus species DNA in serum samples of the patients with non-invasive pulmonary aspergillosis].

We investigated the possible presence of Aspergillus species DNA in serum samples of two patients diagnosed as having non-invasive pulmonary aspergillosis by a nested polymerase chain reaction (PCR) method. The nested PCR results were negative in serum samples of the patients with chronic necrotizing pulmonary aspergillosis and pulmonary aspergilloma. When left pneumothorax happened to the patient with chronic necrotizing pulmonary aspergillosis and bronchial washing was performed to the patient with pulmonary aspergilloma, the nested PCR results turned positive. We consider this method useful for the diagnosis of semi-invasive stage of pulmonary aspergillosis. However, further prospective evaluation with a large clinical sample is required.

[Case report: subcutaneous abscess and thoracic empyema caused by Alcaligenes xylosoxidans].

Alcaligenes xylosoxidans is a glucose-nonfermentative gram-negative rod which usually exists in the environment. This organism while causing pneumonia, sepsis, meningitis and urinary tract infection in the compromised host, rarely causes thoracic empyema. We report a case of thoracic empyema and subcutaneous abscess due to A. xylosoxidans. A 74-year-old male, who had undergone right total pneumonectomy for chronic necrotizing pulmonary aspergillosis a year ago, was admitted to our hospital because of fever. CT scans of the chest revealed a subcutaneous abscess and empyema. Empyema and subcutaneous pus were aspirated. Culture of materials produced A. xylosoxidans. There was no significant change on symptoms and examinations despite therapy with PIPC 4 g/day and thoracic drainage. Finally, surgical treatment was required and the patient was cured.

[A case of tuberculous peritonitis diagnosed by ultrasonography-guide peritoneal biopsy].

The diagnosis of tuberculous peritonitis is quite difficult because the symptoms are not specific for the disease and the incidence of occurrence are relatively rare. We report a case of tuberculous peritonitis diagnosed by ultrasonography-guided peritoneal biopsy. A 64-year-old male was admitted to our hospital because of fever, dyspnea and abdominal pain. Laboratory findings revealed an elevated ESR (53 mm/1 hr.) and positive CRP. The tuberculin skin test was negative. The chest radiograph revealed bilateral pleural effusion. Abdominal ultrasonographic examination and computed tomography showed ascitic fluid, thickening of the mesentery and peritoneum, and inflammatory pseudotumor of the omentum. Ascitic fluid was exudate with a high lymphocyte count and elevated ADA (184 IU/l). Microbiological studies with the fluid were negative. Peritoneal biopsy guided by ultrasonography was performed, and the specimens showed central caseous necrosis surrounded by epitheloid cells and acid-fast bacilli were demonstrated. The size of the pseudotumor, pleural effusion and ascites decreased after antituberculous chemotherapy with corticosteroid was given. Diagnosis of tuberculous peritonitis has often been made by laparotomy or laparoscopy. In a case of this kind, percutaneous peritoneal biopsy guided by ultrasonography is safe and useful.

[Detection of DNA specific for Aspergillus species in serum samples from two patients with invasive pulmonary aspergillosis].

We investigated the possible presence of DNA specific for Aspergillus species in serum samples of two patients who were strongly suspected for invasive pulmonary aspergillosis (IPA) by a nested polymerase chain reaction (PCR) method. Both patients were diagnosed as having acute myelogenous leukemia and treated with induction chemotherapy. During chemotherapy-induced granulocytopenia, they complained of high fever, and the chest X-rays indicated infiltration shadows in their lungs. They were treated with antibiotics intravenously, but no clinical improvement was observed. As the results of the nested PCR were positive at the acute stage of infection, amphotericin B i.v. and granulocyte colony stimulating factor s.c. administrations were started in both cases. In case 1, the infectious disease improved and the nested PCR results turned negative after treatment. In case 2, in spite of the progression of the disease, the nested PCR results turned negative during treatment. Although we consider this method very useful for the diagnosis of IPA, further prospective evaluation with a large clinical population sample is required.

[Clinical characterization of blaIMP positive gram-negative rods isolated cases].

We detected the metallo-beta-lactamase gene blaIMP positive strains of the gram-negative rods (GNR) isolated in Oita Medical University Hospital between 1993 and 1999 and studied the clinical characteristics of patients infected or colonized with blaIMP positive GNR. 25 strains (20 Pseudomonas aeruginosa and 5 Serratia marcescens) were detected and most of them were isolated from urinary samples after 1997. In the studies of antimicrobial susceptibility, some strains had sensitivity to aztreonum or imipenem although most of the strains showed multidrug resistance. When blaIMP positive GNR were isolated from patients, these strains were thought to have caused infection in 88% of the patients. About half of the patients were over 65 years old and had malignant diseases. Most of the patients had inserted urinary tract catheters, intratracheal tube or intravernous catheters. It was suggested that the insertion of the catheters were related to infection of blaIMP positive GNRs. Two patients were not treated with any antibiotics before the isolation of blaIMP positive GNRs although more than half of the patients were administered carbapenems and cephems. Most of strains were isolated in the same department and showed the same genotype by pulsed field gel electrophoresis.

[A case of pulmonary manifestation associated with myelodysplastic syndrome].

A 69-year-old woman with myelodysplastic syndrome (MDS) was admitted to our hospital because of recurrent fever and pulmonary infiltration shadows. On the seventh day of hospitalization, she had an attack of high fever and cough and laboratory tests revealed an elevated leukocyte count and elevated serum C-reactive protein. Chest radiographs showed infiltration shadows in the right middle and lower lung fields. Because a diagnosis of bacterial pneumonia was initially suggested, she was treated with antibiotics. However, the infiltration shadows on the chest radiograph had not improve, so bronchofiberscopy was performed. Analysis of fluid obtained by bronchoalveolar lavage (BAL) showed an increase in the total cell count, predominantly in lymphocytes and neutrophils. A transbronchial biopsy specimen showed infiltration of numerous neutrophils with necrosis under the bronchial epithelium, and edematous septa were infiltrated with numerous neutrophils and lymphocytes. BAL, blood, urine, bone marrow, and sputum cultures were all free of bacteria, mycobacteria and fungi. Interstitial infiltration by numbers of neutrophils associated with MDS was diagnosed and steroid treatment was performed.

[Pulmonary dirofilariasis associated with pleural effusion].

A 61-year-old man was admitted to our hospital because of a long history of productive coughing. A chest roentgenogram and CT scan showed a right-sided pleural effusion. The effusion fluid was blood-stained but showed no cytological evidence of malignancy. Marked eosinophilia was found in blood and in the pleural effusion fluid. Ouchterlony's double-diffusion test done with the patients serum and pleural effusion fluid in agarose showed specific bands toward Dirofilaria immitis antigen, and this specificity was confirmed with an enzyme linked immunosorbent assay inhibition test. The final diagnosis was pulmonary dirofilariasis, and the patient responded to diethylcarbamazine.

Expression of the cell-surface heparan sulfate proteoglycan mRNA in monkey submandibular gland.

We investigated the mRNA expression of cell-surface heparan sulfate proteoglycans, syndecans and glypican, in the adult female monkey submandibular gland using the reverse transcription-polymerase chain reaction (RT-PCR) technique. Agarose gel electrophoresis of the PCR products of the cDNA generated from RNA was carried out to demonstrate the expression of mRNA in syndecan-1, syndecan-2, syndecan-4 and glypican in this study. In order to compare the mRNA expression level among the cell-surface heparan sulfate proteoglycans, we measured changes in the relative intensity of PCR products with increasing thermal cycle number. The expression levels were syndecan-4 > syndecan-1, syndecan-2 > glypican. Considering these results together with our previous report, we found that the cell-surface heparan sulfate proteoglycans, syndecan-1, syndecan-2, syndecan-4 and glypican, are synthesized in the monkey submandibular glands, and that their ectodomains are released into the extracellular matrix. It was speculated that control of the expression patterns of the cell-surface proteoglycans may regulate the cellular function and behavior in the submandibular gland.

Evaluation of PCR for detection of DNA specific for Aspergillus species in sera of patients with various forms of pulmonary aspergillosis.

Pulmonary aspergillosis is classified into invasive, saprophytic, and allergic forms. In this study, we evaluated the usefulness of PCR for differentiating between different forms of aspergillosis or in monitoring disease activity during treatment by detecting DNA specific for Aspergillus species in the serum. Nested PCR was used to detect Aspergillus DNA in the sera of 30 patients with various forms of pulmonary aspergillosis. The results were compared with those of latex agglutination tests for detecting galactomannan antigen. We also examined the serial changes in the results of nested PCR during and after treatment of a subgroup of patients with invasive pulmonary aspergillosis with amphotericin B. The highest proportion of positive nested PCR results were in patients with invasive aspergillosis (10 of 12; 83%), while patients with pulmonary aspergilloma had the lowest frequency of positive tests (1 of 9; 11%). These results suggested that the sensitivity of the nested PCR depends on the extent of invasion by Aspergillus species. Serial assays showed that the results of nested PCR became negative shortly after commencement of antifungal treatment and that such changes did not correlate with clinical responsiveness to treatment. Our results indicate the potential usefulness of nested PCR with serum samples for the diagnosis of invasive aspergillosis and the detection of a shift in the status of infection from a noninvasive type to invasive aspergillosis. However, the results of the nested PCR did not correlate with the response to antifungal treatment.

Comparison of PCR, (1-->3)-beta-D-glucan and galactomannan assays in sera of rats with experimental invasive aspergillosis.

We compared PCR, galactomannan detection assay using a latex agglutination test and (1-->3)-beta-D-glucan detection assay in detecting infection in rats experimentally infected with Aspergillus fumigatus. On day 2 after inoculation, (1-->3)-beta-D-glucan and nested PCR were positive for 80%, while galactomannan detection assay was positive for 60%. In addition, the positive result of nested PCR (87.5%) was higher than those of galactomannan detection assay (75%) and (1-->3)-beta-D-glucan (71.4%) on day 3 after inoculation. The sensitivity of nested PCR was superior to those of galactomannan detection assay and (1-->3)-beta-D-glucan detection assay. The three diagnostic tests were compared with histopathological findings, and the sensitivity of three diagnostic tests was correlated with histopathological changes. In addition, the elevated levels of (1-->3)-beta-D-glucan paralleled the development and progression of pulmonary aspergillosis. Our results indicate that a combination of two or three of these tests seems to provide a rapid diagnosis of invasive aspergillosis and assist in the evaluation of the development and severity of invasive aspergillosis.

Experimental model of progressive disseminated trichosporonosis in mice with latent trichosporonemia.

Trichosporon asahii and Trichosporon mucoides are the most common strains of fungi that cause disseminated trichosporonosis, a severe opportunistic infection in immunocompromised hosts. We have previously established a nested PCR assay using serum samples for detection of both strains. Here we describe a new experimental animal model for investigating the underlying mechanisms of disseminated trichosporonosis. T. asahii (OMU239, a clinical isolate from a patient with acute myelogenous leukemia) and 8-week-old ICR male mice were used in all experiments. A suspension of T. asahii (3 x 10(6) CFU/animal) was injected into the caudal vein of each mouse after immunosuppression with cyclophosphamide (200 mg/kg of body weight/day for 2 days) and prednisolone (30 mg/kg/day for 1 day). Mice were then divided into four subgroups (R0, R1, R2, and R3) based on the time of reimmunosuppression. The latter was performed using the same drugs 1 week (group R1), 2 weeks (group R2), and 3 weeks (group R3) after fungal infection. Reimmunosuppression was not performed in group R0. The 5-week-survival rates of mice after T. asahii infection were 0% for group R1, 50% for group R2, 80% for group R3, and 80% for group R0. There was a significant difference in the survival rates between group R1 and either group R0 or R3 (P < 0.05). Fungal clearance in peripheral blood and various organs of group R1 and R2 was delayed relative to that of group R0 but was similar to the control in group R3 in spite of reimmunosuppression. Our results suggest that the critical period for the development of disseminated trichosporonosis in our model is shorter than 3 weeks after T. asahii infection. We concluded that mice during this critical period were in a state of latent trichosporonemia. Comparison of the survival rates suggests that the nested PCR assay was more useful than blood culture and glucuronoxylomannan antigen assay in the detection of this latent trichosporonemia.

[Endobronchial actinomycosis].

A 63-year-old man was admitted to our hospital with obstructive pneumonia. The chest X-ray film and computed tomogram showed an infiltrative shadow in the right lower lung field. Examination with a fiberoptic bronchoscope showed a mass in the right basal bronchus. These findings suggested the diagnosis of lung cancer with obstructive pneumonia. Histopathological examination of a specimen obtained by transbronchial biopsy revealed sulfur granules with infiltration of neutrophils, which led to the diagnosis of endobronchial actinomycosis. After three months of treatment with penicillin, the mass disappeared. Comparison of bronchoscopic findings before and after penicillin treatment clearly showed the efficacy of therapy.