Thin-section CT findings in Pseudomonas aeruginosa pulmonary infection.

OBJECTIVE: The aim of this study was to assess clinical and pulmonary thin-section CT findings in patients with acute Pseudomonas aeruginosa (PA) pulmonary infection. METHODS: We retrospectively identified 44 patients with acute PA pneumonia who had undergone chest thin-section CT examinations between January 2004 and December 2010. We excluded nine patients with concurrent infections. The final study group comprised 35 patients (21 males, 14 females; age range 30-89 years, mean age 66.9 years) with PA pneumonia. The patients' clinical findings were assessed. Parenchymal abnormalities, enlarged lymph nodes and pleural effusion were evaluated on thin-section CT. RESULTS: Underlying diseases included malignancy (n=13), a smoking habit (n=11) and cardiac disease (n=8). CT scans of all patients revealed abnormal findings, including ground-glass opacity (n=34), bronchial wall thickening (n=31), consolidation (n=23) and cavities (n=5). Pleural effusion was found in 15 patients. CONCLUSION: PA pulmonary infection was observed in patients with underlying diseases such as malignancy or a smoking habit. The CT findings in patients with PA consisted mainly of ground-glass attenuation and bronchial wall thickening. ADVANCES IN KNOWLEDGE: The CT findings consisted mainly of ground-glass attenuation, bronchial wall thickening and cavities. These findings in patients with an underlying disease such as malignancy or a smoking habit may be suggestive of pneumonia caused by PA infection.

Inhibitory effect of statins on inflammatory cytokine production from human bronchial epithelial cells.

Statins are 3-hydroxy-3-methylglutaryl-co-enzyme A reductase inhibitors of cholesterol biosynthesis, and have been reported to exert pleiotropic effects on cellular signalling and cellular functions involved in inflammation. Recent reports have demonstrated that previous statin therapy reduced the risk of pneumonia or increased survival in patients with community-acquired pneumonia. However, the precise mechanisms responsible for these effects are unclear. In the present study, we examined the effects of statins on cytokine production from lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Interleukin (IL)-6 and IL-8 mRNA expression and protein secretion in LPS-stimulated cells were inhibited significantly by the lipophilic statin pitavastatin and the hydrophilic statin pravastatin. As these inhibitory effects of statin were negated by adding mevalonate, the anti-inflammatory effects of statins appear to be exerted via the mevalonic cascade. In addition, the activation levels of Ras homologue gene family A (RhoA) in BEAS-2B cells cultured with pitavastatin were significantly lower than those without the statin. These results suggest that statins have anti-inflammatory effects by reducing cytokine production through inhibition of the mevalonic cascade followed by RhoA activation in the lung.

[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 ankylosing spondylitis associated with pulmonary aspergillosis and amyloidosis].

A 56-year-old man was admitted because of diarrhea, cough, weight loss, and disturbance of consciousness. He had been diagnosed as having ankylosing spondylitis at 18-years old. The spondylitis progressed until there was complete rigidity of the spine including the neck, hip and knee joints. Human leukocyte antigen (HLA) B27, which has been characteristic of ankylosing spondylitis, was also present in this case. A chest radiograph showed pleural thickness and a cavity in the right upper lobe; and a soft tissue mass and fluid level was found in the cavity. Aspergillus fumigatus was detected in the sputum and pulmonary aspergillosis was diagnosed. Biopsy of the colon revealed that a large interstitial amyloidosis. Despite the treatment of the patient's malnutrition and lung aspergillosis using amphotericin B, the clinical course was rapidly progressive and the patient died of respiratory failure due to lung aspergillosis. It is important to be aware of these rare complications, which are correlated with the prognosis in cases of ankylosing spondylitis.

[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.

Immunization of rhesus monkeys with a recombinant of modified vaccinia virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge.

Dengue epidemics increasingly pose a public health problem in most countries of the tropical and subtropical areas. Despite decades of research, development of a safe and effective live dengue virus vaccine is still at the experimental stage. To explore an alternative vaccine strategy, we employed the highly attenuated, replication-deficient modified vaccinia Ankara (MVA) as a vector to construct recombinants for expression of the major envelope glycoprotein of one or more dengue virus serotypes. MVA recombinants expressing the highly immunogenic C-terminally truncated dengue type 2 virus (DEN2) or dengue type 4 virus (DEN4) envelope protein (E), approx. 80% of the full-length, were evaluated for their protective immunity in animal models. Each of these recombinants elicited an elevated antibody response to DEN2 or DEN4 E in mice following the booster inoculation, as detected by radio-immunoprecipitation. Recombinant MVA-DEN2 80%E, but not MVA-DEN4 80%E, induced a neutralizing antibody response. The MVA-DEN2 80%E recombinant was chosen to further evaluate its ability to induce resistance to wild type DEN2 challenge in monkeys. Monkeys immunized twice with recombinant MVA-DEN2 80%E developed a low to moderate antibody response and were partially protected against DEN2 challenge, as determined by the viremia pattern. Importantly, the subsequent study showed that all four monkeys immunized with the recombinant in a three dose schedule developed an increased level of antibodies and were completely protected against DEN2 challenge. The potential efficacy of recombinant MVA-DEN2 80%E to protect primates against dengue infection suggests that construction and evaluation of MVA recombinants expressing other serotypes of dengue virus E for use in a tetravalent vaccine strategy might be warranted.

[Anti-influenza A viral drug--amantadine].

Amantadine, an inhibitor of M2 ion channel of influenza A virus, is an oral antiviral drug which specifically inhibits the uncoating and viral replication of the influenza A virus. Studies have shown that amantadine treatment within 48 hours of acute infection of influenza A reduces fever within 24 hours and shortens the course of illness. Amantadine has been found to have an efficacy of 50 to 90% prevention of illness. However amantadine-resistant viruses have been recovered approximately 30% patients treated with amantadine, as early as 2-3 days into treatment. Side effects of insomnia, decreased concentration and dizziness have been reported in 5 to 33% of amantadine recipients. Therefore amantadine should be only used for influenza A infected high risk persons.

PCR detection of DNA specific for Trichosporon species in serum of patients with disseminated trichosporonosis.

Deep-seated trichosporonosis is a lethal opportunistic infection that disseminates rapidly and widely in immunocompromised patients, and early diagnosis is crucial for the treatment of this infection. We developed a novel nested-PCR assay that detects DNA specific for clinically important strains of Trichosporon in serum samples from patients with disseminated trichosporonosis. In this assay, two sets of oligonucleotide primers were derived from the sequence of 26S rRNA genes of Trichosporon asahii. The specific fragment was amplified from T. asahii and T. mucoides, but not from other microorganisms, including some other basidiomycetous fungi (Cryptococcus, Malassezia, Rhodotorula, and Sporobolomyces). Target DNA was detected by the nested PCR with as little as 5 fg of the extracted DNA of T. asahii. In a study using 11 clinical samples, the specific fragment was detected by the nested PCR in 64% (7 of 11) of sera from patients with histologically diagnosed disseminated trichosporonosis, while glucuronoxylomannan antigen was detected in only 54% (6 of 11) of the samples. Our new nested-PCR assay using serum samples can be performed repeatedly throughout the course of the disease. In addition, not only can it be used for early diagnosis of trichosporonosis, but it may also be beneficial for monitoring its progress or response to therapy.

[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.

Evaluation of molecular strategies to develop a live dengue vaccine.

BACKGROUND: Millions of individuals are estimated to become infected with dengue virus each year, particularly in tropical and subtropical regions. Mortality is low but infection can lead to a severe form of dengue, characterised by haemorrhage and shock. A safe and effective vaccine against dengue is still not available. OBJECTIVE: To use the successful construction of dengue type 4 virus (DEN4) cDNA, which yields infectious RNA transcripts, to provide a new approach to the development of safe and effective dengue vaccines. STUDY DESIGN: The 3' and 5' noncoding (NC) regions of the genome were targeted to construct DEN4 deletion mutants, because the sequences in these regions are thought to play an important role in the regulation of viral replication. DEN4 cDNA was also employed to construct a viable chimeric virus with dengue type 1, 2 or 3 antigenicity, by substitution of heterotypic structural protein genes. RESULTS: Most viable mutants, recovered from the cDNA constructs, were partially restricted for growth in simian cells as analysed by plaque morphology assay and viral yield analysis. Several 3' NC deletion mutants which exhibited a range of growth restriction in cell culture were further evaluated for infectivity and immunogenicity in rhesus monkeys. Occurrence and duration of viraemia were reduced for these deletion mutants, compared to the wild type DEN4. Analysis of antibody response to infection in rhesus monkeys also indicated that some of these mutants were attenuated. These DEN4 deletion mutants represent promising live dengue vaccine candidates that merit further clinical evaluation. Chimera DEN1/DEN4 or DEN2/DEN4 which expresses DEN1 or DEN2 antigenicity were also used to infect monkeys. Most monkeys immunised with these chimeric viruses, singly or in combination, developed high titres of neutralising antibodies and were protected against homotypic wild type DEN1 or DEN2 challenge. CONCLUSIONS: DEN4 and its derived chimeric viruses of other three dengue serotype specificity, that contain appropriate attenuating mutations, have a potential use in a tetravalent live vaccine against dengue.

Genetic determinants responsible for acquisition of dengue type 2 virus mouse neurovirulence.

Studies conducted some 50 years ago showed that serial intracerebral passage of dengue viruses in mice selected for neurovirulent mutants that also exhibited significant attenuation for humans. We investigated the genetic basis of mouse neurovirulence of dengue virus because it might be directly or indirectly associated with attenuation for humans. Analysis of the sequence in the C-PreM-E-NS1 region of the parental dengue type 2 virus (DEN2) New Guinea C (NGC) strain and its mouse-adapted, neurovirulent mutant revealed that 10 nucleotide changes occurred during serial passage in mice. Seven of these changes resulted in amino acid substitutions, i.e., Leu55-Phe and Arg57-Lys in PreM, Glu71-Asp, Glu126-Lys, Phe402-Ile, and Thr454-Ile in E, and Arg105-Gln in NS1. The sequence of C was fully conserved between the parental and mutant DEN2. We constructed intertypic chimeric dengue viruses that contained the PreM-E genes or only the NS1 gene of neurovirulent DEN2 NGC substituting for the corresponding genes of DEN4. The DEN2 (PreM-E)/DEN4 chimera was neurovirulent for mice, whereas DEN2 (NS1)/DEN4 was not. The mutations present in the neurovirulent DEN2 PreM-E genes were then substituted singly or in combination into the sequence of the nonneurovirulent, parental DEN2. Intracerebral titration of the various mutant chimeras so produced identified two amino acid changes, namely, Glu71-Asp and Glu126-Lys, in DEN2 E as being responsible for mouse neurovirulence. The conservative amino acid change of Gu71-Asp probably had a minor effect, if any. The Glu126-Lys substitution in DEN2 E, representing a change from a negatively charged amino acid to a positively charged amino acid, most likely plays an important role in conferring mouse neurovirulence.

[A pathological study of cytomegalovirus infections in autopsied cases with adult T-cell leukemia].

In this study, cytomegalovirus (CMV) infection was found in eleven of 21 autopsied cases (52.4%) with adult T-cell leukemia (ATL). Seven cases (63.6%) revealed disseminated infection in more than three organs. The lungs were involved in all eleven cases (100%), adrenal glands in eight cases (72.7%), esophagus in four cases (36.4%), and stomach, small intestine and urinary bladder in three cases (27.3%). Histopathological findings suggested that lung involvement was the cause of death in five of the 11 cases, the small intestine were involved in two of the 3 cases, and the adrenal glands were involved in one of the 8 cases. In summary, CMV infection was found to be the main cause of death in five (45.5%) of the 11 ATL patients.

PCR detection of DNA specific for Aspergillus species in serum of patients with invasive aspergillosis.

We investigated the possible presence of DNA specific for Aspergillus species in serum samples of patients with invasive aspergillosis (IA) by the nested PCR method. Fourteen strains of fungi including 5 strains of Aspergillus species and 10 strains of common bacteria were used for examination of specificity and sensitivity of the nested PCR. Two sets of oligonucleotide primers were derived from the sequence of the variable regions V7 to V9 of the 18S rRNA genes of Aspergillus fumigatus. The specific fragment was amplified from five strains of Aspergillus species in the single and nested PCR but not from other microorganisms. Target DNA was detected by the nested PCR with as little as 50 fg of the extracted DNA of A. fumigatus. We investigated the detection of DNA specific for Aspergillus species in serum samples of a murine model of aspergillosis and 20 patients with IA. The specific fragment was detected by the nested PCR in 71% of serum samples of infected mice and 70% of serum samples of patients with IA, while galactomannan antigen was detected in 43 and 60% of samples, respectively. 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 immunocompromised patients with IA.

[Detection and quantitation of cytomegalovirus DNA in plasma from patients with cytomegalovirus pneumonia].

Detection and semiquantitation of cytomegalovirus (CMV) DNA in plasma from 17 immunocompromised patients with CMV pneumonia diagnosed histopathologically, 15 CMV seropositive patients without CMV pneumonia and 24 CMV-seropositive healthy volunteers were evaluated, using the polymerase chain reaction (PCR). CMV DNA was detected in plasma from all of 17 patients with CMV pneumonia, from 1 of 15 patients without CMV disease, but from none of healthy volunteers. One patient without CMV disease exhibited positive CMV DNA by PCR 2 days before death. Plasma CMV DNA was negative at the time of admission in all patients, however, it became positive 1-28 days (mean, 14 days) before the onset of CMV pneumonia in 16 patients. The amount of viral DNA in plasma were 10(3) - 10(5) copies/ml (mean, 10 (4.0) copies/ml) when first detected by PCR. At the onset of CMV pneumonia, they were 10(4)-10(6)(mean, 10(5.3) copies/ml), and increased with disease progression and decreased with disease improvement because of treatment with antiviral agents. We succeeded in detection of CMV DNA in plasma before the development of CMV pneumonia, and showed the amount of viral DNA reflected the extent of active CMV pneumonia. Thus, PCR amplification of CMV DNA in plasma is a useful tool for early diagnosis and monitoring of immunocompromised patients.

[Microbiological and clinical study of fungemia between 1981 and 1992].

Fungi were isolated from 113 (14.2%) of 789 patients with positive blood cultures at Oita Medical University Hospital between 1981 and 1992. The rates of fungemia increased in recent years, 13.9% (1981-1985), 12.1% (1986-1988) and 16.9% (1989-1992). The isolated fungi were Candida parapsilosis (25.7%), C. albicans (24.8%), C. tropicalis (14.2%), Trichosporon beigelii (10.6%), C. glabrata (8.0%) and so on. The major fungi were T. beigelii and C. glabrata in patients with hematologic malignancies, whereas they were C. albicans and C. parapsilosis in patients with non-hematologic diseases and C. glabrata increased in both groups. Prophylactic or emiric administration of antifungal agents probably influenced the difference of the causative organisms in the two groups.

Early diagnosis and monitoring of human cytomegalovirus pneumonia in patients with adult T-cell leukemia by DNA amplification in serum.

A nested polymerase chain reaction (PCR) was used to detect human cytomegalovirus (HCMV) DNA in serum of patients with adult T-cell leukemia (ATL). Serum samples were collected consecutively from 11 patients with HCMV pneumonia diagnosed histopathologically and 7 HCMV-seropositive patients without HCMV disease. Serum samples obtained from 24 HCMV-seropositive healthy volunteers were used as controls. The HCMV DNA was detected in serum a mean of 14 days before the onset of HCMV pneumonia, which suggests that DNAemia exists prior to the development of HCMV pneumonia. The amount of viral DNA in serum increased with disease progression and decreased with disease improvement. Thus, the detection of HCMV DNA in serum by nested PCR is useful for monitoring and the early diagnosis of HCMV pneumonia in patients with ATL. In addition, quantitation of HCMV DNA may be useful for monitoring HCMV infection, because it appears to correlate with the activity of the disease.

[Problems concerned with adult T-cell leukemia. 1) Causes of death and early diagnosis of cytomegalovirus pneumonia in adult T-cell leukemia. 2) Clinicopathological features of CD30 positive adult T-cell leukemia].

1) We studied the causes of death confirmed by autopsy or necropsy in 23 adult T-cell leukemia (ATL) patients. Of them eight showed involvement of tumor (35%), eleven infectious disease (47%) (nine cytomegalovirus (CMV) pneumonia, one varicella-zostervirus pneumonia, one pseudomonas aeruginosa pneumonia), and four others (17%). In seven recent cases treated with a new chemotherapy regimen in combination with G-CSF administration, survival was longer than in previous cases and tumor involvement as a cause of death decreased (one case, 14%). However, CMV pneumonia inclined to increase (six cases, 86%). Therefore, we tried to retrospectively detect CMV DNA in the serum of ATL patients using a nested polymerase chain reaction (PCR). CMV pneumonia was reliably diagnosed in eleven ATL patients, whereas CMV DNA was detected in all patients at the time of clinical onset of pneumonia and CMV DNA was detected only in eight patients from 7-35 days before the onset of pneumonia. These findings suggest that the nested PCR assay is a useful tool to early diagnosis CMV pneumonia in ATL patients. 2) Recently, several cases of ATL with CD30 antigen have been reported, but its clinical relevance remains unknown. Accordingly, we studied CD30 antigen expression in 36 ATL patients who had monoclonal integration of HTLV-I provirus in the tumor cells and demonstrated the immunohistochemical and clinical characteristics of these patients. CD30 antigen expression was evident in seven of these 36 patients (19.4%). A comparison of ATL cases with and without CD30 antigen expression revealed significantly large numbers of abnormal lymphocytes in the peripheral blood and lower serum calcium levels in CD30 antigen positive ATL.