1887

Abstract

This study estimated the value of PCR (ISPCR) in the detection of type b (Hib) DNA in paraffin-embedded lung tissues of a murine pneumonia model. ICR mice were infected with Hib solution intranasally. In study group A (=20), physiological changes and the number of deaths were recorded for 7 consecutive days after infection. In study group B (=10), blood samples and lung tissues were obtained from the infected mice on the brink of death. In both groups, portions of the lung tissue were cultured for Hib, while other portions were submitted for histopathological studies. Conventional PCR, PCR followed by Southern blotting and ISPCR were performed to detect Hib in paraffin-embedded lung tissues. In control group A, six mice were inoculated intranasally with the same concentration of heat-inactivated Hib solution. In control group B, six healthy mice served as a blank control. Both control groups were managed using the same methods as those used in the study groups. The white blood cell count of the mice in the study group increased (F=3.295, <0.01), with a high neutrophil count (F=0.127, <0.05). In the histopathological study, various stages of pneumonia were found in the lung tissues of the infected mice examined by microscope; 80 % of the mice had moderate or severe pneumonia. Cultures of lung tissues in the study groups were all positive for Hib, while no bacteria were found in the control groups. Hib was detected in only 4 of 30 samples (13.3 %) of the study groups using conventional PCR, but in all 30 samples (100 %) using both Southern blotting and ISPCR. All three methods did not detect Hib in the control groups. Because of its sensitivity and specificity and its ability to locate the micro-organism, ISPCR can be considered suitable for the detection of Hib in paraffin-embedded lung tissues.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.2008/002204-0
2008-10-01
2024-12-12
Loading full text...

Full text loading...

/deliver/fulltext/jmm/57/10/1282.html?itemId=/content/journal/jmm/10.1099/jmm.0.2008/002204-0&mimeType=html&fmt=ahah

References

  1. Corley D. E., Kirtland S. H., Winterbauer R. H., Hammar S. P., Dail D. H., Bauermeister D. E., Bolen J. W. 1997; Reproducibility of the histologic diagnosis of pneumonia among a panel of four pathologists: analysis of a gold standard. Chest 112:458–465 [CrossRef]
    [Google Scholar]
  2. Drummond P., Clark J., Wheeler J., Galloway A., Freeman R., Cant A. 2000; Community acquired pneumonia – a prospective UK study. Arch Dis Child 83:408–412 [CrossRef]
    [Google Scholar]
  3. Hu H. L., He L. J., Dmitriev A., Hu Y. Y., Deng J. H., Zhang W. S., Yao K. H., Yang Y. H. 2008; The role of Haemophilus influenzae type b in fatal community-acquired pneumonia in Chinese children. Pediatr Infect Dis J in press
    [Google Scholar]
  4. Johansen I. S., Thomsen V. Ø., Forsgren A., Hansen B. F., Lundgren B. 2004; Detection of Mycobacterium tuberculosis complex in formalin-fixed, paraffin-embedded tissue specimens with necrotizing granulomatous inflammation by strand displacement amplification. J Mol Diagn 6:231–236 [CrossRef]
    [Google Scholar]
  5. Komminoth P., Long A. A., Ray R., Wolfe H. J. 1992; In situ polymerase chain reaction detection of viral DNA, single copy genes, and gene rearrangements in cell suspensions and cytospins. Diagn Mol Pathol 1:85–97 [CrossRef]
    [Google Scholar]
  6. Ma T. Y., Liu W. K., Chu Y. L., Jiang X. Y., An Y., Zhang M. P., Zheng J. W. 2007; Detection of human papillomavirus type 16 DNA in formalin-fixed, paraffin-embedded tissue specimens of gastric carcinoma. Eur J Gastroenterol Hepatol 19:1090–1096 [CrossRef]
    [Google Scholar]
  7. McIntosh K. 2002; Community-acquired pneumonia in children. N Engl J Med 346:429–437 [CrossRef]
    [Google Scholar]
  8. Miyazaki S., Hosoyama T., Furuya N., Ishii Y., Matsumoto T., Ohno A., Tateda K., Yamaguchi K. 2001; In vitro and in vivo antibacterial of L-084, a novel oral carbapenem, against causative organism of respiratory tract infections. Antimicrob Agents Chemother 45:203–207 [CrossRef]
    [Google Scholar]
  9. Nascimento-Carvalho C. M. 2001; Etiology of children community-acquired pneumonia and its implications for vaccination. Braz J Infect Dis 5:87–97
    [Google Scholar]
  10. Nuovo G. J., Gallery F., MacConnell P., Becker J., Bloch W. 1991; An improved technique for the in situ detection of DNA after polymerase chain reaction amplification. Am J Pathol 139:1239–1244
    [Google Scholar]
  11. Praveena P. E., Jayakumar R., Balachandran C., Thirumurugan G., Raj G. D., Manohar B. M. 2007; Detection of the rabies virus genes by in situ polymerase chain reaction. Vet Res Commun 31:775–781 [CrossRef]
    [Google Scholar]
  12. Retzel E. F., Staskus K. A., Embretson J. E., Haase A. T. 1994 The In Situ PCR: Amplification and Detection in a Cellular Context http://wheat.pw.usda.gov/∼lazo/methods/minn/chapter3.7/chapter3.7.fm.html
    [Google Scholar]
  13. Scaglione F., Mouton J. W., Mattina R., Fraschini F. 2003; Pharmacodynamics of levofloxacin and ciprofloxacin in a murine pneumonia model: peak concentration/MIC versus area under the curve/MIC ratios. Antimicrob Agents Chemother 47:2749–2755 [CrossRef]
    [Google Scholar]
  14. Tani K., Kurokawa K., Nasu M. 1998; Development of a direct in situ PCR method for detection of specific bacteria in natural environments. Appl Environ Microbiol 64:1536–1540
    [Google Scholar]
  15. Xu F., Xu Z., Zhang R., Wu Z., Lim J. H., Koga T., Li J.-D., Shen H. 2008; Nontypeable Haemophilus influenzae induces COX-2 and PGE2 expression in lung epithelial cells via activation of p38 MAPK and NF-kappa B. Respir Res 9:16 [CrossRef]
    [Google Scholar]
  16. Zhaori G. T., Shen K. L., Jiang Z. F., Li P., Liu Y. 1996; Detection of cytomegalovirus DNA in paraffin-embedded lung tissue specimens using in situ polymerase chain reaction. Chin Med J (Engl 109:361–365
    [Google Scholar]
/content/journal/jmm/10.1099/jmm.0.2008/002204-0
Loading
/content/journal/jmm/10.1099/jmm.0.2008/002204-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error