Pyrazinamide-monoresistant Mycobacterium tuberculosis in the United States

Mycobacterium bovis is naturally resistant to the antituberculosis drug pyrazinamide (PZA). To determine whether all Mycobacterium tuberculosis complex isolates demonstrating PZA monoresistance were truly M. bovis, we examined the phenotype and genotype of isolates reported as PZA monoresistant in five counties in California from January 1996 through June 1999. Isolates reported by local laboratories to be PZA monoresistant were sent to the state reference laboratory for repeat susceptibility testing using the BACTEC radiometric method and to the Centers for Disease Control and Prevention for pncA sequencing and PCR-restriction fragment length polymorphism (RFLP) analysis of the oxyR gene. Of 1,916 isolates, 14 were reported as PZA monoresistant and 11 were available for retesting. On repeat testing, 6 of the 11 isolates were identified as PZA-susceptible M. tuberculosis, 1 was identified as PZA-monoresistant M. bovis, and 1 was identified as M. bovis BCG. The three remaining isolates were identified as PZA-monoresistant M. tuberculosis. Sequencing of the pncA and oxyR genes genotypically confirmed the two M. bovis and the six susceptible M. tuberculosis species. Each of the three PZA-monoresistant M. tuberculosis isolates had different, previously unreported, pncA gene mutations: a 24-bp deletion in frame after codon 88, a base substitution at codon 104 (Ser104Cys), and a base substitution at codon 90 (Ile90Ser). This study demonstrates that PZA monoresistance is not an absolute marker of M. bovis species but may also occur in M. tuberculosis, associated with a number of different mutational events in the pncA gene. It is the first report of PZA-monoresistant M. tuberculosis in the United States.     

Detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmB gene region

A restriction fragment length polymorphism (RFLP) assay was developed to identify and differentiate Old World, African-Eurasian orthopoxviruses (OPV): variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and taterapox viruses. The test uses amplicons produced from virus genome DNA by PCR with a consensus primer pair designed from sequences determined for the cytokine response modifier B (crmB) gene of 43 different OPV strains of known taxonomic origin. The primer pair amplified a single specific product from each of the 115 OPV samples tested. Size-specific amplicons identified and differentiated ectromelia and vaccinia virus strains, which contain a truncated crmB gene, and enabled their differentiation from other OPV species. Restriction digests of amplified products allowed the identification and differentiation of variola, monkeypox, camelpox, vaccinia, and cowpox virus species and strains.     

一种朊蛋白错误折叠循环扩增方法的建立

目的建立一种类似于PCR的蛋白质扩增方法-蛋白错误折叠循环扩增技术(PMCA),用于朊病毒病脑组织中PrPSc的检测。方法将不同浓度的羊瘙痒因子263K毒株原液与正常仓鼠脑组织匀浆混合,经反复孵育/超声,共10~15个循环。WesternBlot检测扩增产物中蛋白酶K抗性PrPSc信号。结果在本研究试验体系下,263K毒株可以利用仓鼠脑组织为基质在体外迅速复制。所建立的PrPSc-PMCA技术可检测到10-5稀释的毒株原液中的PrPSc。与常规的脑组织免疫印记方法相比,敏感度提高了105~106倍。研究还显示PrPSc还可利用小脑和脑干为基质进行体外扩增复制。结论成功建立了PrPSc-PMCA技术,为朊病毒病的早期诊断和朊病毒生物学特性的研究提供了一种新的手段。     

Acute and long-term humoral immunity following active immunization of rabbits with inacctivated spores of various Encephalitozoon species

Microsporidia of the genus Encephalitozoon are increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. hellem, or E. intestinalis is capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoon antigens was determined by IIF and immunogold electron microscopy; however, only the E. hellem immune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinalis were fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections. Received: 10 April 2000 / Accepted: 18 July 2000     

New Calicivirus isolated from walrus

The nucleotide sequence and genome organization of a new member of Caliciviridae was determined. Cell culture inoculated with fecal matter from walrus was used to recover fragments of a new virus by Suppression Subtractive Hybridization (SSH). The isolate was identified as a member of the Vesivirus genus of Caliciviridae and designated the name Walrus Calicivirus (WCV). Sets of PCR primers spanning the entire putative genome were designed using known sequences of other vesiviruses. The assembled genome was 8289 nucleotides (nt) long and shared no more than 87% identity with sequences of the other members of the genus Vesivirus. The largest open reading frame (ORF1) between positions 4-5646 encoded a polyprotein. ORF2, found at position 5652-7778, encoded a putative capsid protein. ORF3 overlapped ORF2 and encoded a small basic protein. Comparative analysis of multiple caliciviral capsid proteins was performed to propose a uniform capsid structural organization for this viral family.     

Ehrlichia chaffeensis: a prototypical emerging pathogen

Ehrlichia chaffeensis is an obligately intracellular, tick-transmitted bacterium that is maintained in nature in a cycle involving at least one and perhaps several vertebrate reservoir hosts. The moderate to severe disease caused by E. chaffeensis in humans, first identified in 1986 and reported for more than 1,000 patients through 2000, represents a prototypical "emerging infection." Knowledge of the biology and natural history of E. chaffeensis, and of the epidemiology, clinical features, and laboratory diagnosis of the zoonotic disease it causes (commonly referred to as human monocytic ehrlichiosis [HME]) has expanded considerably in the period since its discovery. In this review, we summarize briefly the current understanding of the microbiology, pathogenesis, and clinical manifestations associated with this pathogen but focus primarily on discussing various ecological factors responsible for the recent recognition of this important and potentially life-threatening tick-borne disease. Perhaps the most pivotal element in the emergence of HME has been the staggering increases in white-tailed deer populations in the eastern United States during the 20th century. This animal serves as a keystone host for all life stages of the principal tick vector (Amblyomma americanum) and is perhaps the most important vertebrate reservoir host for E. chaffeensis. The contributions of other components, including expansion of susceptible human populations, growth and broadening geographical distributions of other potential reservoir species and A. americanum, and improvements in confirmatory diagnostic methods, are also explored.     

Extensive transmission of Mycobacterium tuberculosis from a child

BACKGROUND AND METHODS: Young children rarely transmit tuberculosis. In July 1998, infectious tuberculosis was identified in a nine-year-old boy in North Dakota who was screened because extrapulmonary tuberculosis had been diagnosed in his female guardian. The child, who had come from the Republic of the Marshall Islands in 1996, had bilateral cavitary tuberculosis. Because he was the only known possible source for his female guardian's tuberculosis, an investigation of the child's contacts was undertaken. We identified family, school, day-care, and other social contacts and notified these people of their exposure. We asked the contacts to complete a questionnaire and performed tuberculin skin tests. RESULTS: Of the 276 contacts of the child whom we tested, 56 (20 percent) had a positive tuberculin skin test (induration of at least 10 mm), including 3 of the child's 4 household members, 16 of his 24 classroom contacts, 10 of 32 school-bus riders, and 9 of 61 day-care contacts. A total of 118 persons received preventive therapy, including 56 young children who were prescribed preventive therapy until skin tests performed at least 12 weeks after exposure were negative. The one additional case identified was in the twin brother of the nine-year-old patient. The twin was not considered infectious on the basis of a sputum smear that was negative on microscopical examination. CONCLUSIONS: This investigation showed that a young child can transmit Mycobacterium tuberculosis to a large number of contacts. Children with tuberculosis, especially cavitary or laryngeal tuberculosis, should be considered potentially infectious, and screening of their contacts for infection with M. tuberculosis or active tuberculosis may be required.     

从人分离出两株甲型流感H9N2亚型毒株内部基因特性的研究

目的 了解2株从人分离出的H9N2亚型毒株内部基因特性，并弄清其来源。方法 用RT-PCR扩增目的基因，用P^CEM-T Vector(美国Promega公司)，4℃过夜连接，重组质粒转入dH5a细菌，筛选阳性菌落，酶切鉴定，送六合通公司自动测序。然后进行进化树分析。结果 2株测定毒株内部基因均为G9基因系，它们相互间除PA基因有差异外，其余5个基因均相同。结论 2株测定毒株的基因组均为G9基因系，它们是由携带不同基因特性H9N2毒株的禽群分别直接感染人，而不是来自同一禽的H9N2亚型流感病毒。     

Age-related differences in cell-specific cytokine production by acutely ill Malawian patients

Age-related changes in human cell-specific cytokine responses to acute illness have not been well examined. We therefore evaluated age-related differences in T, B and natural killer (NK) peripheral blood lymphocyte cytokine responses of 309 acutely ill hospitalized people in Malawi, Africa, < 1 month-61 years of age. We used four-colour flow cytometry and performed Wilcoxon rank sum and Kruskal-Wallis tests, Pearson (rp) and Spearman (rs) correlations, and linear and logistic regression analyses to control for human immunodeficiency virus infection (HIV) status, the percentages of lymphocytes expressing CD4, and the nature of the acute infection. The percentages of CD8- and CD8+ T cells producing induced IL-8 decreased with age (rs = -0.44 and -0.53). The percentages of T cells producing TNF-alpha were higher, and the percentages producing IL-10 were lower, in those > or =13 than those < 13 years old (medians: 17.7 versus 10.5 and 1.4 versus 3.0, respectively). The percentages of CD8- T cells producing IFN-gamma were higher and stable in those > or =1 year old compared to infants (medians: 23.5 versus 10.4); the percentages of NK producing IFN-gamma were higher post-infancy and then declined to relatively low levels with increasing age. The percentages of T cells producing IL-2 were highest in those 5- <31 years old (median 5.6) and lowest in those > or =31 years old (median 1.9). The ratios of the percentages of T cells producing IL-4 to those producing IL-8 and to those producing IL-10 both increased with age. These data suggest that innate immunity, represented by NK IFN-gamma production, dominates in early life. A number of shifts occur after infancy and before adolescence, including a proinflammatory shift from IL-8 to TNF-gamma and a type 2 shift from IL-10 to IL-4 dominance. These findings suggest distinct age-related differences in the human response to acute illness and may be useful in directing future efforts at immunomodulatory therapies.