Implications of nasopharynx-associated lymphoid tissue (NALT) in the development of allergic responses in an allergic rhinitis mouse model.

BACKGROUND: Nasopharynx-associated lymphoid tissue (NALT) serves as an important inductive site for mucosal immunity in the upper respiratory tract. Despite its importance in the mucosal immune system, little is known regarding the role of NALT in airway allergic immune responses. We aimed to elucidate the role of NALT in the induction of upper airway allergic responses in a mouse model. METHODS: Inhibitor of DNA binding/differentiation 2 (Id2)(-/-) and Id2(+/-) mice was exposed to the ovalbumin (OVA)-induced allergic rhinitis model, because the former resulted in the NALT deficiency. The allergic parameters, such as allergic symptoms, serum OVA-specific immunoglobulin E (IgE) levels, eosinophil infiltration, and cytokine profiles in the nasal mucosa, were compared between Id2(-/-) and Id2(+/-) groups. RESULTS: NALT-null, Id2(-/-) mice displayed significantly lower allergic responses compared with Id2(+/-) mice, as demonstrated by lower levels of allergic symptoms, serum OVA-specific IgE, eosinophilic infiltration, and local Th2 cytokine transcriptions. To determine which of two factors, that is, the absence of NALT or the alteration of immunocompetent cell populations caused by the Id2 deficiency, has a larger effect on the attenuated allergic immune responses in Id2(-/-) mice, lethally irradiated Id2(-/-) mice were engrafted with C57BL/6 wild-type bone marrow cells and showed still significantly lower allergic immune responses compared with equally treated Id2(+/-) mice. In addition, IgE class switch recombination-associated molecules, such as ε immunoglobulin heavy-chain germline gene transcript, ε mRNA, and activation-induced cytidine deaminase mRNA, were detected in NALT from OVA-sensitized wild-type mice. CONCLUSION: These results show the critical role of NALT for the induction of allergic responses in the upper airway at least in part by means of class switching to IgE in situ.

Addition of isovitalex in chocolate agar for the isolation of Haemophilus influenzae.

BACKGROUND & OBJECTIVE: The reason for lack of data on burden of Haemophilus influenzae type b (Hib) in developing countries was mainly failure of detection of this fastidious organism in laboratories. Use of isovitalex (IVX) was suggested as an essential supplement for growing this organism. This study was carried out to investigate the impact of IVX supplementation to chocolate agar for detection of Hib. METHODS: Chocolate agar with and without supplementation of IVX was prepared. Clinical samples as well as reference strains of Hib were simultaneously cultured on both the media. RESULTS: H. influenzae isolates (N=194) were simultaneously grown on chocolate agar (CA) with and without isovitalex (IVX). Average colony size of H. influenzae on CA with IVX (CA-IVX) was larger only by 0.10 cm (range 0.05 to 0.16 cm) compared to CA alone. Addition of IVX to CA increased the cost of media by 2.1-fold. INTERPRETATION & CONCLUSION: Isovitalex is not essential for the isolation and growth of H. influenzae almost halving the cost.

Short report: antimicrobial susceptibility and genetic characteristics of Haemophilus influenzae isolated from patients with respiratory tract infections between 1987 and 2000, including beta-lactamase-negative ampicillin-resistant strains.

The minimum inhibitory concentration (MIC) of five antibiotics and the presence of resistance genes was determined in 163 Haemophilus influenzae isolates collected over 13 years (1987-2000) in four two-yearly sampling periods from patients with respiratory tract infections. The prevalence of beta-lactamase-negative ampicillin-susceptible strains was approximately 80% over the sampling period although fewer strains (65.9%) were recovered in the period 1995-1997. TEM-1 type beta-lactamase-producing strains were less frequent starting at 15.6% and declining to 2.2% in the final sampling period. Low-beta-lactamase-negative ampicillin-resistant (BLNAR) strains were uncommon in 1987-1989 (2.2%), peaked to 19.5% in 1995-1997, but fell back to 11.1% by 2000. Fully BLNAR strains were not detected until the last sampling period (6.7%). The MICs of ampicillin, levofloxacin, cefditoren and ceftriaxone remained stable but there was an eight-fold increase in the MIC of cefdinir over the sampling period. Pulsed-field gel electrophoresis of DNA digests showed that three representative BLNAR strains were genetically distinct and 11 DNA profiles were identified among 17 low-BLNAR strains. These data suggest that the number of genetically altered BLNAR and low-BLNAR strains are increasing in Japan.

Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae isolated from patients with community-acquired pneumonia and molecular analysis of multidrug-resistant serotype 19F and 23F strains in Japan.

A nationwide study was undertaken to determine the susceptibility to penicillin and serotypes of Streptococcus pneumoniae in Japan. S. pneumoniae was isolated from 114 adult patients with community-acquired pneumonia over 22 months at 20 hospitals and medical centres in different regions in Japan. All but five isolates were from sputum. Forty-eight isolates (42.1%) were susceptible, 40 (35.1%) showed intermediate resistance (MIC, 0.12-1.0 microg/ml) and 26 (22.8%) were resistant (MIC, >or=2.0 microg/ml) to penicillin G. All isolates were susceptible to ceftriaxone (breakpoint 1 microg/ml), imipenem (4 microg/ml) and vancomycin (4 microg/ml). Most were resistant to erythromycin, clarithromycin and azithromycin; only two were resistant to levofloxacin. Differences were found in the distribution of serotypes among isolates showing susceptibility to penicillin (predominant types 3, 6B, and 19F), intermediate resistance (6B, 14, 19F, and 23F) and full resistance (19F and 23F). PFGE typing showed that 14 of the 25 strains of serotype 19F had a single DNA profile, pattern A, a pattern closely similar to that of the Taiwan multidrug-resistant 19F clone. Twelve pattern A strains were not susceptible to penicillin but carried the macrolide resistance gene mef(A). The DNA profiles of the 15 strains of 23F were also heterogeneous but six were highly similar (pattern b) yet distinct from the Spanish multidrug-resistant 23F clone although possibly related to the Taiwan multidrug-resistant 23F clone. The pattern b strains were not susceptible to penicillin and also harboured either mef(A) or erm(B). Our results indicate that multidrug-resistant pneumococci are spreading rapidly in Japan. Efforts to prevent the spread of the pandemic multidrug-resistant serotypes should be intensified.

Association of increased platelet-associated immunoglobulins with thrombocytopenia and the severity of disease in secondary dengue virus infections.

Severe thrombocytopenia and increased vascular permeability are two major characteristics of dengue haemorrhagic fever (DHF). To develop a better understanding of the roles of platelet-associated IgG (PAIgG) and IgM (PAIgM) in inducing thrombocytopenia and its severity of disease in patients with secondary dengue virus infection, the relationship between the PAIgG or PAIgM levels and disease severity as well as thrombocytopenia was examined in 78 patients with acute phase secondary infection in a prospective hospital-based study. The decrease in platelet count during the acute phase recovered significantly during the convalescent phase. In contrast, the increased levels of PAIgG or PAIgM that occurred during the acute phase of these patients decreased significantly during the convalescent phase. An inverse correlation between platelet count and PAIgG or PAIgM levels was found in these patients. Anti-dengue virus IgG and IgM activity was found in platelet eluates from 10 patients in an acute phase of secondary infection. Increased levels of PAIgG or PAIgM were significantly higher in DHF than those in dengue fever (DF). An increased level of PAIgM was associated independently with the development of DHF, representing a possible predictor of DHF with a high specificity. Our present data suggest that platelet-associated immunoglobulins involving antidengue virus activity play a pivotal role in the induction of thrombocytopenia and the severity of the disease in secondary dengue virus infections.

Evaluation of PCR-based methods for the diagnosis of tuberculosis by identification of mycobacterial DNA in urine samples.

SETTING: The Chest Clinic and the JICA (Japan International Cooperation Agency) Molecular Laboratories, University Teaching Hospital, Lusaka, Zambia, and the Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan. OBJECTIVE: To evaluate the polymerase chain reaction (PCR) as a laboratory test for the rapid diagnosis of pulmonary tuberculosis in the African situation by identifying mycobacterial DNA in urine samples using two commonly described molecular methods. DESIGN: Prospective collection and laboratory analysis of urine samples from adult Zambian patients with culture-confirmed pulmonary tuberculosis and healthy controls. METHODS: Urine was obtained from 63 patients with culture-confirmed active pulmonary tuberculosis and 63 'healthy' control patients with no active tuberculosis. DNA was isolated from urine sediment and subjected to analyses by two well-described PCR-based methods, 'the Sechi method' and 'the Githui method', for the identification of Mycobacterium tuberculosis DNA. The sensitivity and specificity of the two tests were determined. RESULTS: The sensitivity and specificity of the Githui method were 55.6% (35/63) and 98.4% (62/63), respectively. The sensitivity and specificity of the Sechi method were 28.6% (18/63) and 98.4% (62/63), respectively. Of the 63 patients, 50 (79%) were HIV sero-positive and the frequency of positive PCR urines using the Githui method was greater in HIV-positive patients than in HIV-negative patients (32/50 = 64% vs. 3/13 = 23%; P = 0.05). CONCLUSIONS: Neither the Githui method nor the Sechi method was sensitive enough to be recommended for routine use in clinical practice. PCR-based assays for the detection of M. tuberculosis DNA in urine will require further refinement before they can be recommended for use in clinical practice in Africa. The presence of mycobacterial DNA in urine samples of patients with pulmonary tuberculosis also requires further study.

Enterocolitis caused by methicillin-resistant Staphylococcus aureus: molecular characterization of respiratory and digestive tract isolates.

We investigated the mechanism of outbreak of enterocolitis caused by methicillin-resistant Staphylococcus aureus (MRSA). Five epidemiological markers [coagulase type, enterotoxin type, toxic shock syndrome toxin-1 (TSST-1) production, beta-lactamase production and pulsed-field gel electrophoresis (PFGE)] of 45 strains of MRSA isolated simultaneously from the respiratory tract (nasal cavity and/or pharynx and/or sputum) and stool (plus one sample of gastric juice) in 13 patients (8 males and 5 females, mean age, 77.1 years) were compared retrospectively. Forty-four of the 45 isolates of MRSA were positive for enterotoxin C and TSST-1 production, and the remaining isolate was positive for enterotoxin A and negative for TSST-1 production. All isolates were coagulase type II, and 27 showed beta-lactamase production. The patterns of coagulase type, enterotoxin type, TSST-1 and beta-lactamase production of MRSA isolated from the respiratory tract were similar to those of MRSA isolated from the intestine in 12 of 13 patients. Molecular typing by PFGE demonstrated that the pattern of respiratory tract isolates was identical to those of stool isolates in 9 (69.2%), similar in 3 (23.1 %), and different in 1 (7.7%). The data suggested that enterocolitis might be caused by the MRSA colonized in the respiratory tract and incorporated into the digestive tracts. Therefore, we propose that early eradication of MRSA in the respiratory tract is important for protection of patients against the development of enterocolitis, particularly in susceptible patients, e.g., immunocompromised or pre-operated patients with digestive diseases, especially malignant disease.

Dexamethasone impairs pulmonary defence against Pseudomonas aeruginosa through suppressing iNOS gene expression and peroxynitrite production in mice.

To elucidate the in vivo mechanisms involved in the impairment in pulmonary defence as the result of treatment with glucocorticoids, we established fatal pneumonia with bacteraemia in dexamethasone (DEX)-treated mice by means of an intratracheal challenge of Pseudomonas aeruginosa. An increased neutrophil influx was observed in bronchoalveolar lavage (BAL) fluids from both untreated and DEX-treated mice. The complete suppression of an inducible isoform of nitric oxide synthase (iNOS) mRNA expression and tumour necrosis factor alpha (TNF-alpha) production during the early phase of pneumonia, but not CXC chemokine production, were found in the case of the DEX-treated mice. An immunohistochemical study with a specific antibody also revealed negative staining for nitrotyrosine in the lung tissue of DEX-treated mice, while the formation of nitrotyrosine, which indirectly indicates the generation of peroxynitrite with a potent bactericidal activity, was detected clearly in the bronchial epithelium as well as alveolar phagocytic cells of lung tissue from untreated mice. Furthermore, an intraperitoneal administration of S-methyl-isothiourea (SMT), a potent inhibitor of NOS, significantly decreased the survival and increased bacterial density in the case of untreated mice. In contrast, no significant effects on the survival and bacterial density in the lung and blood were found as the result of treatment with SMT in DEX-treated mice. Collectively, a complete repression of iNOS gene expression and a lack of the generation of peroxynitrite as well as an inhibition of TNF-alpha production in the lung appeared to be responsible for the progression of the fatal pneumonia due to P. aeruginosa in DEX-treated mice.

Rapid identification and antibiotic susceptibility testing of Salmonella enterica serovar Typhi isolated from blood: implications for therapy.

The turnaround time (TAT) for Salmonella enterica serovar Typhi identification and reporting of the antibiotic susceptibility profile was determined for 391 cases of typhoid fever, using the lysis direct plating or lysis centrifugation method of blood culture along with rapid antimicrobial susceptibility testing. The TAT was more rapid (TAT for 90% of the patients [TAT(90)] = 30 h; TAT(100)

Low concentrations of mupirocin in the pharynx following intranasal application may contribute to mupirocin resistance in methicillin-resistant Staphylococcus aureus.

We describe a patient with methicillin-resistant Staphylococcus aureus (MRSA) colonizing the pharynx. The MIC of mupirocin was 0.25 microg/ml before treatment and increased after treatment to 8 microg/ml. Using pulsed-field gel electrophoresis, we confirmed that the genotypes of MRSA that colonized the pharynx before and after the use of mupirocin were identical. We measured the delivery of mupirocin to the pharynx in three normal volunteers and two patients. Low concentrations of mupirocin were present in the pharynx in all cases 10 min to 3 days after intranasal application. Our data suggested that low concentrations of the drug in the pharynx after intranasal application of mupirocin ointment might explain the selection of mupirocin resistance in MRSA.

Community-acquired pneumonia in Ugandan adults: short-term parenteral ampicillin therapy for bacterial pneumonia.

A hospital-based prospective study of 99 patients with community-acquired pneumonia (CAP) was carried out in Kampala, Uganda. We evaluated microbiological etiologies, clinical features and effectiveness of short-term parenteral ampicillin followed by oral amoxicillin for these patients in relation to HIV-status. We demonstrated a very high prevalence (75%) of HIV-1 infection. No significant difference was observed with respect to age, gender, prior antibiotic usage, symptoms, laboratory data or bacterial etiology between HIV-1-infected and HIV-uninfected CAP patients. Most strains of Streptococcus pneumoniae (n = 19) and Haemophilus influenzae (n = 8) isolated from HIV-1-infected patients were penicillin-resistant (95%) and beta-lactamase producing (75%) strains, respectively. A high percentage of good clinical response was found in both HIV-1-infected (81%) and HIV-uninfected (86%) among 39 patients with CAP due to a defined bacterial pathogen. These data support the use of short-term parenteral ampicillin for patients with bacterial CAP irrespective of HIV-status.

Salmonella enteritidis FliC (flagella filament protein) induces human beta-defensin-2 mRNA production by Caco-2 cells.

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a approximately 2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-kappaB (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-kappaB activation and thus plays an important role in up-regulation of the innate immune response.

Typhoid fever in Bangladesh: implications for vaccination policy.

OBJECTIVE: To describe the age-specific distribution of typhoid fever including the degree of Salmonella typhi bacteremia among patients evaluated at a large private diagnostic center in Bangladesh, a highly endemic area. METHODS: We conducted a prospective-, passive- and laboratory-based study to identify patients with S. typhi bacteremia. Subjects (n = 4,650) from whom blood cultures were obtained during 16-month period were enrolled from private clinics and hospitals throughout Dhaka. Isolation and quantification of S. typhi from blood cultures were performed by the lysis direct plating/ centrifugation method. RESULTS: Bacterial pathogens were recovered from blood of 538 of 4,650 patients (11.6%) evaluated. S. typhi was the single most common pathogen recovered, comprising nearly three-fourths of isolates (72.7%; 391 of 538). Isolation rate of S. typhi was highest in monsoon and summer seasons and lowest in winter months. The majority (54.5%; 213 of 391) of S. typhi isolates were from children who were younger than 5 years, and 27% (105 of 391) were from children in the first 2 years of life. The isolation rate was highest (17.4%, 68 of 486) in the second year of life. The number of bacteria in blood on the basis of colony-forming units per ml of blood by age group was inversely related to age. CONCLUSIONS: Detection of S. typhi bacteremia in young children in Dhaka, Bangladesh, was considerably higher than previously appreciated, with a peak detection rate in children < or =2 years of age, indicating the need to reassess the age-specific burden of typhoid fever in the community on a regional basis. Contrary to current recommendations this study suggests that development of new vaccines should target infants and young children.

Alveolar macrophages that phagocytose apoptotic neutrophils produce hepatocyte growth factor during bacterial pneumonia in mice.

Hepatocyte growth factor (HGF) is postulated to play an important role in the repair of pulmonary epithelium in acute lung injury. To evaluate the role of HGF in bacterial pneumonia, the kinetics of HGF production and the cellular sources of HGF have been examined in the lungs of mice that had been intratracheally challenged with Pseudomonas aeruginosa. Neutrophil accumulation in the airway occurred immediately, reached a peak at 36 h, and then progressively declined by 14 d after infection. We found a biphasic pattern of HGF messenger RNA expression and protein synthesis in the lung after bacterial infection. The first peak for HGF production was found at 6 h after infection, and the primary source of HGF was shown to be bronchial epithelial cells. Interestingly, the second peak for HGF production, which was found around 48 to 72 h after infection, was closely associated with the increase in the percentage of alveolar macrophages (AMs) that became positive for myeloperoxidase, indicating phagocytosis of apoptotic neutrophils. The cellular source of the second peak was found to be AMs. Further, murine AMs which phagocytosed apoptotic neutrophils induced higher levels of HGF production in vitro. These results strongly indicate a novel mechanism of HGF production by AMs, which are phagocytosing apoptotic neutrophils, and the pivotal role of AMs in the healing and repair of damaged pulmonary epithelium through the production of HGF.

Emergence and spread of low-level mupirocin resistance in methicillin-resistant Staphylococcus aureus isolated from a community hospital in Japan.

The objective of this study was to investigate the state of mupirocin resistance in methicillin-resistant Staphylococcus aureus (MRSA) in a community hospital in Japan. Ninety strains of MRSA were isolated from the respiratory tract of 56 patients (group I, Jun 1990-Aug 1996) before introduction of mupirocin in Japan, which were compared with 168 strains from 48 patients (group II, Sept 1996-Jan 1998) and 146 strains from 85 patients (group III, Feb 1999-Dec 1999) isolated after introduction of mupirocin. Comparisons were made by determining the minimum inhibitory concentrations (MIC) against nine antibiotics. Fifty-five MRSA isolates from 27 patients [13 (27.1%) of 48 in group II and 14 (16.5%) of 85 in group III] after introduction of mupirocin showed low-level resistance to mupirocin (MIC, 6.25 to 50 microg/ml) but the remaining isolates were sensitive to mupirocin (MIC < or =3.13 microg/ml). Most patients colonized with low-level mupirocin-resistant MRSA were elderly (> or =65 years of age), on total parenteral nutrition or nasal feeding and had other underlying diseases. The proportion of patients colonized with low-level mupirocin-resistant MRSA following repeated use of mupirocin was higher in patients of group II than those of group III. Molecular typing by pulsed-field gel electrophoresis (PFGE) demonstrated that the pattern of 13 MRSA isolates from 13 patients of group II consisted of three patterns (A, B, C) with predominance of pattern A, while the pattern of 13 MRSA isolates from 13 patients of group III consisted of three patterns (A, C, D) with predominance of patterns A and D. Our results indicated that resistance of MRSA to mupirocin remains at a low level at present in Japan. However, we should be aware of the possible emergence of MRSA highly resistant to mupirocin in the future.

[A case of primary racemose hemangioma of bronchial artery with recurrent hemoptysis].

A 42-year old man was admitted to our hospital because of hemoptysis. Bronchial arteriography revealed a tortuous and dilated left bronchial artery with a shunt formation between the bronchial and pulmonary arteries. Bronchial artery embolization using a sponge was performed three times to treat the hemoptysis, but all attempts failed. The patient therefore underwent left lower lobectomy, after which no hemoptysis was observed. Histopathologically, the resected tissue showed no inflammatory change. Interestingly, abnormal vessels resembling arteriovenous malformations were also found. Although the embolization therapy was effective in several reported cases, we concluded that surgery was required for this patient with persistent hemoptysis because of the development of collaterals and a bronchial-pulmonary artery shunt.

Significant reduction of methicillin-resistant Staphylococcus aureus bacteremia in geriatric wards after introduction of infection control measures against nosocomial infections.

OBJECTIVES: (1) To investigate the efficacy of infection control measures against methicillin-resistant Staphylococcus aureus (MRSA) bacteremias in geriatric wards. (2) To identify predisposing risk factors for MRSA bacteremia. METHODS: Cases with nosocomial bacteremias were retrospectively analyzed between January 1991 and March 1995. The study period was divided into four annual periods and the period 1, January to December 1991, was applied as the control. MATERIALS: We investigated patients with nosocomial bacteremias in geriatric wards (190 beds) of AINO Memorial Hospital, affiliated with Nagasaki University. RESULTS: A significant reduction in cases with MRSA-induced nosocomial bacteremia was observed after the introduction of a stringent infection control and prevention program (period 1 vs. periods 2, 3, and 4: p<0.00833, p<0.00167, and p<0.00167, respectively). The major source of bacteremia included urinary tract infections, intravenous catheter-related infections, and infected decubitus ulcers. Improvement of decubitus ulcer was associated with a significant reduction in MRSA bacteremia (period 1 vs. periods 2 and 3: p<0.00017 and p<0.00833). CONCLUSION: Stringent infection control programs, including prevention and treatment of decubitus ulcers, are necessary in geriatric wards to reduce and prevent MRSA bacteremia.