Kinetics and effect of integrin expression on human CD34(+) cells during murine leukemia virus-derived retroviral transduction with recombinant fibronectin for stem cell gene therapy.

The CH-296 recombinant fragment of human fibronectin is essential for murine leukemia virus (MLV)-derived retroviral transduction of CD34(+) cells for the purpose of stem cell gene therapy. Although the major effect of CH-296 is colocalization of the MLV-derived retrovirus and target cells at specific adhesion domains of CH-296 mediated by integrins expressed on CD34(+) cells, the precise roles of the integrins are unclear. We examined the kinetics of integrin expression on CD34(+) cells during the course of MLV-derived retrovirus-mediated gene transduction with CH-296. Flow cytometry revealed that the levels of both very late activation protein (VLA)-4 and VLA-5 on CD34(+) cells freshly isolated from cord blood were insufficient for effective MLV-derived retroviral transduction. However, increases were achieved during culture for preinduction and MLV-derived retrovirus-mediated gene transduction in the presence of a cocktail of cytokines. In addition, we confirmed by using specific antibodies that inhibition of the cell adhesion mediated by the integrins significantly reduced transduction efficiency, indicating that integrin expression is indeed important for CH-296-based MLV-derived retroviral transduction. Only a few cytokines are capable of inducing integrin expression, and stem cell factor plus thrombopoietin was found to be the minimal combination that was sufficient for effective transduction of an MLV-derived retrovirus based on CH-296. Our findings should be useful for improving the culture conditions for CH-296-based MLV-derived retroviral transduction in stem cell gene therapy.

Risk factors for SARS infection within hospitals in Hanoi, Vietnam.

We investigated a nosocomial infection of severe acute respiratory syndrome (SARS) in Vietnam in 2003 and attempted to identify risk factors for SARS infection. Of the 146 subjects who came into contact with SARS patients at Hospital A, 43 (29.5%) developed SARS, and an additional 16 (11%) were asymptomatic but SARS-coronavirus (CoV) seropositive. The asymptomatic infection rate accounted for 15.5% of the total number of infected patients at Hospital A, which was higher than that of 6.5% observed at Hospital B, to where all patients from Hospital A were eventually transported (P<0.05). At Hospital A, the risk for developing SARS was 12.6 times higher in individuals not using a mask than in those using a mask. The SARS epidemic in Vietnam resulted in numerous secondary infections due to its unknown etiology and delayed recognition at the beginning of the epidemic. The consistent and proper use of a mask was shown to be crucial for constant protection against infection with SARS.

Molecular epidemiology of outbreaks and containment of drug-resistant Pseudomonas aeruginosa in a Tokyo hospital.

We witnessed outbreaks of multidrug-resistant (MDR) and drug-resistant Pseudomonas aeruginosa at a hospital in Tokyo, Japan, during the period September 2004 through May 2005. The first outbreak occurred in September and October 2004. Three isolates of MDR P. aeruginosa were identified from urine samples obtained from three nonambulatory immunodeficient patients in one ward. After 3 weeks, another outbreak of P. aeruginosa occurred in the hematology ward on the same floor of the hospital. During the outbreaks, environmental surveys were conducted twice in each of the two wards, at 2-week intervals, to identify the sources of the pathogens. A total of 23 P. aeruginosa isolates, including 11 from environmental sources, were analyzed for chromosomal DNA typing by pulsed-field gel electrophoresis, for O-antigen serotyping, and for other typing. Results revealed two causative clones, as well as environmental contamination by P. aeruginosa clones on the surfaces of urine volume-measuring devices in rooms where urine is handled, which may have been sources of the pathogens during the outbreaks. To prevent further outbreaks, we performed the following: (a) environmental surface monitoring for drug-resistant P. aeruginosa, (b) active surveillance of specimens, (c) strict isolation of infected patients or carriers of MDR P. aeruginosa, (d) rigorous contact precautions, and (e) disinfection with 70% alcohol on the surfaces of apparatuses contaminated by MDR or drug-resistant P. aeruginosa and in the rooms where urine is handled. As a result, the outbreaks were contained.

Detection of multidrug resistance in Mycobacterium tuberculosis.

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.

Outbreaks of multidrug-resistant Pseudomonas aeruginosa in community hospitals in Japan.

We previously reported an outbreak in a neurosurgery ward of catheter-associated urinary tract infection with multidrug-resistant (MDR) Pseudomonas aeruginosa strain IMCJ2.S1, carrying the 6'-N-aminoglycoside acetyltransferase gene [aac(6')-Iae]. For further epidemiologic studies, 214 clinical isolates of MDR P. aeruginosa showing resistance to imipenem (MIC >or= 16 microg/ml), amikacin (MIC >or= 64 microg/ml), and ciprofloxacin (MIC >or= 4 microg/ml) were collected from 13 hospitals in the same prefecture in Japan. We also collected 70 clinical isolates of P. aeruginosa that were sensitive to one or more of these antibiotics and compared their characteristics with those of the MDR P. aeruginosa isolates. Of the 214 MDR P. aeruginosa isolates, 212 (99%) were serotype O11. We developed a loop-mediated isothermal amplification (LAMP) assay and a slide agglutination test for detection of the aac(6')-Iae gene and the AAC(6')-Iae protein, respectively. Of the 212 MDR P. aeruginosa isolates, 212 (100%) and 207 (98%) were positive in the LAMP assay and in the agglutination test, respectively. Mutations of gyrA and parC genes resulting in amino acid substitutions were detected in 213 of the 214 MDR P. aeruginosa isolates (99%). Of the 214 MDR P. aeruginosa isolates, 212 showed pulsed-field gel electrophoresis patterns with >or=70% similarity to that of IMCJ2.S1 and 83 showed a pattern identical to that of IMCJ2.S1, indicating that clonal expansion of MDR P. aeruginosa occurred in community hospitals in this area. The methods developed in this study to detect aac(6')-Iae were rapid and effective in diagnosing infections caused by various MDR P. aeruginosa clones.

"Syndromic surveillance within a hospital" for the early detection of a nosocomial outbreak of acute respiratory infection.

We have performed intra-hospital syndromic surveillance to rapidly detect nosocomial acute respiratory infection outbreaks in both inpatients and health care workers in a hospital. Syndromic surveillance allows the rapid detection of sudden outbreaks, including infections caused by unknown pathogens. This approach depends on the identification of specific "symptoms" as signs of a possible outbreak, with no need for specific diagnoses. Moreover, syndromic surveillance is quick, easy, and inexpensive. Nosocomial infection surveillance is usually performed on inpatients only. However, during the outbreaks of SARS and seasonal influenza, for example, many hospital personnel were infected. In cases of this kind, in order to quickly detect the prevalence of such infections, a surveillance system that includes hospital personnel is essential. This surveillance is promising as a strategy to prepare for re-outbreaks of SARS and the emergence of novel influenza pandemics.

Emergence of rifampicin resistance in methicillin-resistant Staphylococcus aureus in tuberculosis wards.

To assess whether the occurrence of rifampicin (RFP) resistance in methicillin-resistant Staphylococcus aureus (MRSA) is related to treatment of tuberculosis, we determined the RFP susceptibility of MRSA isolates obtained from tuberculosis patients and screened for mutation(s) in the rpoB gene of these isolates. The MICs of RFP for 84 MRSA isolates obtained from two hospitals in Japan were determined. DNA was sequenced in the region 1318-1602 nucleotides (nt) of the rpoB gene, which includes RFP resistance-determining clusters I (1384-1464 nt, 462-488 amino acids). The majority of MRSA isolates from tuberculosis wards, i.e., 48 of 51 (94%) [33 of 34 in a Tokyo hospital (97%) and 15 of 17 in a Chubu hospital (88%)], were resistant to RFP. Meanwhile, no isolates of 33 from the other wards were resistant to RFP. All RFP-resistant MRSA isolates had a mutation(s), including novel mutation(s) such as Val453-->AEPhe, Asp471-->AEAsn, and Ile527-->AELeu, in rpoB. An emergence of RFP-resistant MRSAs in tuberculosis wards in Japan was strongly suggested.

Association of rpoB mutations with rifampicin resistance in Mycobacterium avium.

The susceptibility of clinical isolates of Mycobacterium avium to rifampicin (RIF) was examined. All 32 clinical isolates tested, including 18 from Japan, 13 from Poland and 1 from Thailand, were resistant to RIF (minimum inhibitory concentrations (MICs) > or =32 microg/mL for 17 isolates and 2-16 microg/mL for 15 isolates), whereas the type strain of M. avium ATCC 25291 was susceptible to RIF (MIC < or = 0.03 microg/mL). Mutations in nucleotides 1276-1356 of the rpoB gene, termed the 81 bp core region, are associated with RIF resistance in Mycobacterium tuberculosis. No mutations were found in this region in any of the M. avium clinical isolates tested. However, mutation of G-->A to give a Gly544-->Asp substitution was identified within the rpoB gene downstream of the 81 bp region in all clinical isolates. A RIF-resistant strain (ATCC 25291 Rif(r); MIC> or =32 microg/mL) obtained by culturing the type strain in RIF-containing broth possessed a mutation C-->T to give a His445-->Tyr substitution within the 81 bp region. When the rpoB gene of the ATCC 25291 Rif(r) strain and of a clinical isolate were inserted into Mycobacterium smegmatis, organisms with the ATCC 25291 Rif(r) sequence, but not those with the clinical isolate sequence, showed resistance to RIF. These results suggest that mutations of the 81 bp region of rpoB, as well as factors other than rpoB mutation, confer RIF resistance in M. avium.

Survey of human immunodeficiency virus (HIV)-seropositive patients with mycobacterial infection in Japan.

OBJECTIVE: To assess DNA polymorphisms in mycobacterial isolates obtained from human immunodeficiency virus (HIV)-seropositive patients with tuberculosis in Japan from 1996 to 2003. METHODS: Restriction fragment length polymorphisms (RFLP) from Mycobacterium tuberculosis and Mycobacterium avium isolates obtained from individual seropositive patients with tuberculosis (n=78) were analysed with the use of IS6110 and (CGG)(5) or IS1245 and IS1311, respectively, as markers. As a control, the same procedures were applied to isolates from HIV-seronegative tuberculosis patients (n=87). RESULTS: Of 86 mycobacterial strains, M. tuberculosis, M. avium and Mycobacterium chelonae were identified in 48 (55.8%), 36 (41.9%) and 2 (2.3%) isolates, respectively. The obtained RFLP patterns of M. tuberculosis isolates from both the HIV-seropositive and -seronegative groups were variable, suggesting no obvious clustering among the isolates. Similar results were obtained in isolates of M. avium. CONCLUSIONS: This is the first report on the molecular epidemiology of Mycobacterium spp. isolated from HIV-seropositive patients in Japan. The results indicate that no particular clones of M. tuberculosis or M. avium prevail in HIV-seropositive patients in Japan. Further monitoring of mycobacterial infection associated with HIV infection in Japan should be continued.

Trial surveillance of cases with acute respiratory symptoms at IMCJ Hospital.

We have developed a surveillance system that can detect a severe acute respiratory syndrome (SARS) outbreak in a hospital as quickly as possible using the "SARS alert" strategy proposed by the World Health Organization (WHO). Our research examined hospital staff and in-patients during the winter of 2003/2004. We defined patients with a fever of over 38 degrees C and respiratory symptoms as "cases with acute respiratory symptoms." During the study period, 215 such cases (78% in-patients; 22% hospital staff members) were reported. A rapid diagnostic test for influenza was performed on 131 individuals, with 52 having positive results. There were no cases fulfilling the definition of SARS provided by the WHO in their SARS alert. The present surveillance system will be of use in the early detection of a SARS epidemic in a hospital as well as in early detection of similar illnesses accompanied by acute respiratory symptoms, such as influenza.

Multidrug-resistant Pseudomonas aeruginosa strain that caused an outbreak in a neurosurgery ward and its aac(6')-Iae gene cassette encoding a novel aminoglycoside acetyltransferase.

We characterized multidrug-resistant Pseudomonas aeruginosa strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a P. aeruginosa strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, beta-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of P. aeruginosa, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of bla(IMP-1), a novel aminoglycoside resistance gene, and the aadA1 gene. The aminoglycoside resistance gene, designated aac(6')-Iae, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6')-Iq. Recombinant AAC(6')-Iae protein showed aminoglycoside 6'-N-acetyltransferase activity by thin-layer chromatography. Escherichia coli expressing exogenous aac(6')-Iae showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of gyrA and parC at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that P. aeruginosa IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the aac(6')-I family and mutations in drug resistance genes.

Cloning and characterization of a novel trimethoprim-resistant dihydrofolate reductase from a nosocomial isolate of Staphylococcus aureus CM.S2 (IMCJ1454).

A novel gene, dfrG, encoding a trimethoprim (TMP)-resistant dihydrofolate reductase (DHFR, designated S3DHFR) was cloned from a clinical isolate of methicillin-resistant Staphylococcus aureus. Escherichia coli expressing dfrG was highly resistant to TMP. Recombinant S3DHFR exhibited DHFR activity that was not inhibited by TMP.

Rapid awareness and transmission of severe acute respiratory syndrome in Hanoi French Hospital, Vietnam.

A case-control study was conducted to examine the relationship between severe acute respiratory syndrome (SARS) and the time-dependent precautionary behaviors taken during an outbreak of SARS in Hanoi French Hospital (HFH), Vietnam. Masks (odds ratio [OR] = 0.3; 95% confidence interval [CI]: 0.1, 0.7) and gowns (OR = 0.2; 95% CI: 0.0, 0.8) appeared to prevent SARS transmission. The proportion of doctors and nurses who undertook each measure significantly improved (chi(2) = 9.8551, P = 0.043) after the onset of secondary cases. The impact of individual behaviors on an outbreak was investigated through mathematical approaches. The reproduction number decreased from 4.1 to 0.7 after notification. The basic reproduction number was estimated, and the use of masks alone was shown to be insufficient in containing an epidemic. Intuitive results obtained by means of stochastic individual-based simulations showed that rapid improvements in behavior and isolation would increase the probability of extinction.

Identification of an alternative 5'-untranslated exon and new polymorphisms of angiotensin-converting enzyme 2 gene: lack of association with SARS in the Vietnamese population.

We analyzed genetic variations of angiotensin-converting enzyme 2 (ACE2), considering that it might influence patients' susceptibility to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) or development of SARS as a functional receptor. By cloning of the full-length cDNA of the ACE2 gene in the lung, where replication occurs on SARS-CoV, it was shown that there are different splicing sites. All exons including the new alternative exon, exon-intron boundaries, and the corresponding 5'-flanking region of the gene were investigated and 19 single nucleotide polymorphisms (SNPs) were found. Out of these, 13 SNPs including one non-synonymous substitution and three 3'-UTR polymorphisms were newly identified. A case control study involving 44 SARS cases, 16 anti-SARS-CoV antibody-positive contacts, 87 antibody-negative contacts, and 50 non-contacts in Vietnam, failed to obtain any evidence that the ACE2 gene polymorphisms are involved in the disease process in the population. Nevertheless, identification of new 5'-untranslated exon and new SNPs is considered helpful in investigating regulation of ACE2 gene expression in the future.

Polymorphisms of interferon-inducible genes OAS-1 and MxA associated with SARS in the Vietnamese population.

We hypothesized that host antiviral genes induced by type I interferons might affect the natural course of severe acute respiratory syndrome (SARS). We analyzed single nucleotide polymorphisms (SNPs) of 2',5'-oligoadenylate synthetase 1 (OAS-1), myxovirus resistance-A (MxA), and double-stranded RNA-dependent protein kinase in 44 Vietnamese SARS patients with 103 controls. The G-allele of non-synonymous A/G SNP in exon 3 of OAS-1 gene showed association with SARS (p=0.0090). The G-allele in exon 3 of OAS-1 and the one in exon 6 were in strong linkage disequilibrium and both of them were associated with SARS infection. The GG genotype and G-allele of G/T SNP at position -88 in the MxA gene promoter were found more frequently in hypoxemic group than in non-hypoxemic group of SARS (p=0.0195). Our findings suggest that polymorphisms of two IFN-inducible genes OAS-1 and MxA might affect susceptibility to the disease and progression of SARS at each level.

ACE1 polymorphism and progression of SARS.

We have hypothesized that genetic predisposition influences the progression of SARS. Angiotensin converting enzyme (ACE1) insertion/deletion (I/D) polymorphism was previously reported to show association with the adult respiratory distress syndrome, which is also thought to play a key role in damaging the lung tissues in SARS cases. This time, the polymorphism was genotyped in 44 Vietnamese SARS cases, with 103 healthy controls who had had a contact with the SARS patients and 50 controls without any contact history. SARS cases were divided into either non-hypoxemic or hypoxemic groups. Despite the small sample size, the frequency of the D allele was significantly higher in the hypoxemic group than in the non-hypoxemic group (p=0.013), whereas there was no significant difference between the SARS cases and controls, irrespective of a contact history. ACE1 might be one of the candidate genes that influence the progression of pneumonia in SARS.