Development of a single-tube, cell lysis-based, genus-specific PCR method for rapid identification of mycobacteria: optimization of cell lysis, PCR primers and conditions, and restriction pattern analysis

A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 h without conventional DNA isolation. The following three steps were optimized or developed: (i). a simple, 6-min direct cell lysis protocol as a PCR prestep for generation of DNA-template, (ii). an improved Mycobacterium-specific PCR amplification protocol with a broader species specificity using newly designed primers targeting a 228-bp region of the 65-kDa heat shock protein (hsp) gene and optimal PCR amplification conditions, and (iii). a genus-specific restriction analysis of the PCR product for conclusive identification of the unknown NTM isolates.     

Genotypic analysis of Mycobacterium tuberculosis in Bangladesh and prevalence of the Beijing strain

Genotypic analysis was performed on 48 Mycobacterium tuberculosis complex strains collected from a hospital in Dhaka city. Deletion analysis showed that the isolates were all M. tuberculosis; 13 of them were found to be of the "ancestral" type, while 35 were of the "modern" type, indicating that both endemic (ancestral type) and epidemic (modern type) strains cause tuberculosis in Bangladesh. Genotyping based on the spoligotype and variable-number tandem repeats (VNTR) of mycobacterial interspersed repetitive units (MIRU) was also done. A total of 34 strains (71%) were grouped by spoligotyping into nine different clusters; the largest comprised 15 isolates of the Beijing genotype, whereas the remaining eight clusters consisted of two to five isolates. MIRU-VNTR typing detected 32 different patterns among 44 tested strains, and the 15 Beijing strains were further discriminated by MIRU-VNTR typing (7 distinct patterns for the 15 isolates). These results indicate that MIRU-VNTR typing, along with spoligotyping and deletion analysis, can be used effectively for molecular epidemiological studies to determine ongoing transmission clusters; to our knowledge, this is the first report about the type of strains prevailing in Bangladesh.     

Tetanus toxin fragment C expressed in live Salmonella vaccines enhances antibody responses to its fusion partner Schistosoma haematobium glutathione S-transferase

Tetanus toxoid has been used widely as an adjuvant. The atoxic fragment C from tetanus toxin (TetC) is potently immunogenic when expressed in Salmonella vaccine strains and has been used as a fusion partner for antigens (Ag). However, there has been no formal comparison of the immunomodulatory impact of TetC on its fusion partners. In this study, we have addressed this important issue. The protective 28-kDa glutathione S-transferase (GST) from Schistosoma haematobium (Sh28GST) was expressed either as a fusion to TetC or as the full-length Sh28GST alone in a nonvirulent aroA-attenuated strain of Salmonella enterica serovar Typhimurium. The Sh28GST proteins were soluble and stably expressed in Salmonella, as evaluated by Western blotting with TetC and/or Sh28GST antisera. Mice were immunized orally with a single dose of the live recombinant Salmonella. The constructs were stable in mice but, dramatically, only the strain expressing the TetC-Sh28GST fusion elicited significant antibody (Ab) responses directed against Sh28GST as determined by enzyme-linked immunosorbent assay. An analysis of the isotype profiles showed that these mice also produced anti-Sh28GST immunoglobulin A and GST-neutralizing assays revealed high levels of neutralizing Abs in sera. These are important correlates of protection in schistosomiasis. In addition, stimulation of spleen cells from immunized mice with Sh28GST Ag showed that both strains, expressing Sh28GST alone or the TetC-Sh28GST fusion, were able to stimulate the secretion of Th1-related cytokines (gamma interferon and interleukin 2) to comparable levels. Thus, TetC has modulated the immune responses generated against its fusion partner, Sh28GST, by markedly enhancing the Ab responses elicited. These results have important implications in the rational development of live vaccines.     

Evaluation of a rapid enzyme-linked immunosorbent assay for IgG antibodies to Aspergillus fumigatus in the serological diagnosis of allergic aspergillosis

A range of 6 somatic and culture filtrate antigens of Aspergillus fumigatus were evaluated in a rapid ELISA procedure for anti-A. fumigatus IgG where the component incubation times had been reduced to 10 min. Sera from patients with allergic aspergillosis, patients with suspected allergic aspergillosis, and asthmatic patients with or without A. fumigatus precipitins were tested. For all antigens, levels of anti-A. fumigatus IgG were higher in patients with allergic aspergillosis than in the other 3 groups. Low levels of specific IgG were, however, detected in asthmatic patients who had no precipitins against A. fumigatus. None of the antigen preparations enabled all patients with proven or suspected allergic aspergillosis to be separated from the other 2 groups of asthmatic patients. Positive-negative discrimination in ELISA was achieved by the inclusion of 10 pools of precipitin test-negative sera from the 50 asthmatics without A. fumigatus precipitins. The number of sera that were classed as positive in ELISA ranged from 9 to 15 in the allergic aspergillosis group, depending on the antigen used; in the suspected aspergillosis group, the number of positive reactions ranged from 1 to 8, while in the asthmatics with precipitins, the number ranged from 0 to 2.     

Novel type of fimbriae encoded by the large plasmid of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H(-)

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) have emerged as important causes of diarrheal diseases and the hemolytic-uremic syndrome in Germany. In this study, we characterized a 32-kb fragment of the plasmid of SF EHEC O157:H(-), pSFO157, which differs markedly from plasmid pO157 of classical non-sorbitol-fermenting EHEC O157:H7. We found a cluster of six genes, termed sfpA, sfpH, sfpC, sfpD, sfpJ, and sfpG, which mediate mannose-resistant hemagglutination and the expression of fimbriae. sfp genes are similar to the pap genes, encoding P-fimbriae of uropathogenic E. coli, but the sfp cluster lacks homologues of genes encoding subunits of a tip fibrillum as well as regulatory genes. The major pilin, SfpA, despite its similarity to PapA, does not cluster together with known PapA alleles in a phylogenetic tree but is structurally related to the PmpA pilin of Proteus mirabilis. The putative adhesin gene sfpG, responsible for the hemagglutination phenotype, shows significant homology neither to papG nor to other known sequences. Sfp fimbriae are 3 to 5 nm in diameter, in contrast to P-fimbriae, which are 7 nm in diameter. PCR analyses showed that the sfp gene cluster is a characteristic of SF EHEC O157:H(-) strains and is not present in other EHEC isolates, diarrheagenic E. coli, or other Enterobacteriaceae. The sfp gene cluster is flanked by two blocks of insertion sequences and an origin of plasmid replication, indicating that horizontal gene transfer may have contributed to the presence of Sfp fimbriae in SF EHEC O157:H(-).     

Effects of fluorodeoxyuridine on the developing inner ear of the rat

The inner ear in rats develops from the surface ectoderm on day 8 of a 22-day gestational period. Labeled thymidine incorporation studies have indicated that in the developing inner ear most of the cells undergo terminal mitosis between gestational days 13 and 15. During this period the developing inner ear would be particularly vulnerable to environmental hazards. To test this hypothesis, pregnant rats were given a single intraperitoneal injection of 5-fluoro-2'-deoxyuridine (FUdR), an antimitotic substance, on gestational days 12 to 16. The rats also received one injection of 3H-thymidine 1 h prior to the removal of the fetuses. The animals were killed after various time intervals following the treatment, and the otocysts or inner ears were prepared for morphologic observations and biochemical assays. The cells in the inner ear of rats exposed to FUdR exhibited pyknotic nuclei and chromatolytic degeneration, and they eventually died. By 4 h after the administration of FUdR, pyknotic nuclei were seen in the antiluminal zone of the otic epithelium, and there was a substantial decrease in the number of the otic cells. This decline in cell number was seen until 24 h after treatment. However, the inner ears from the fetuses exposed to FUdR during gestational days 12--15 showed complete recovery from the toxic effects of the drug when examined on day 21 of gestation. The phenomenon of programmed cell death observed in the developing inner ear of the rat indicates that more cells are produced during the earlier stages of development than are required for the definitive adult structures. This phenomenon may represent an important protective feature. The redundant production of cells perhaps allows the developing otocysts to respond to an environmental stress by subtotal destruction of cells from the pool of undifferentiated cells, resulting in relatively fewer congenital anomalies of the inner ear.     

Expression of BCL-2 and p53 in oncocytic (Hürthle cell) tumors of the thyroid: An immunohistochemical study

Previous studies have established that thyroid follicular neoplasms of higher malignant potential show a high p53 and low bc1-2 expression. This however has not been well studied in Oncocytic (Hürthle cell) neoplasms, the management of which remains controversial. We therefore studied the expression of p53 and bc1-2 in 18 Hürthle cell adenomas (HCA) and 8 Hürthle cell carcinomas (HCC) and compared them with their benign and malignant counterparts, respectively, including 16 follicular adenomas (FA) and 68 papillary carcinomas (PC). All 16 FA were bc1-2 positive, 4 were 2+, and 12 were 3+. On the other hand, 14/18 (78%) HCA showed bc1-2 expression, 5 were 1+, 6 were 2+, and only 3 were 3_. Similarly, HCC showed a weaker bc1-2 staining pattern compared to PC. Only 1 FA showed grade 1, p53 staining, the remaining 15 were negative, and 15/18 HCA showed p53 expression of varying grades. This difference in p53 staining was statistically significant (p=0.005). A significant p53 overexpression was also seen in HCC compared to PC (p=0.005). In conclusion, there appears to be an inverse relationship between p53 and bc1-2 expression in thyroid follicular neoplasms. A higher expression of p53 and lower levels bc1-2 in Hürthle cell neoplasms may have biological and clinical implications. This may support a more aggressive surgical treatment for HCA compared to FA.