Rapid, Simultaneous Detection of Clostridium sordellii and Clostridium perfringens in Archived Tissues by a Novel PCR-Based Microsphere Assay: Diagnostic Implications for Pregnancy-Associated Toxic Shock Syndrome Cases
Clostridium sordellii and Clostridium perfringens are infrequent human pathogens; however, the case-fatality rates for the infections are very high, particularly in obstetric C. sordellii infections (>90%). Deaths from Clostridium sordellii and Clostridium perfringens toxic shock (CTS) are sudden, and diagnosis is often challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues usually are the only specimens available for sudden fatal cases, and immunohistochemistry (IHC) for Clostridia is generally performed but it cannot identify species. A clear need exists for a rapid, species-specific diagnostic assay for FFPE tissues. We developed a duplex PCR-based microsphere assay for simultaneous detection of C. sordellii and C. perfringens and evaluated DNA extracted from 42 Clostridium isolates and FFPE tissues of 28 patients with toxic shock/endometritis (20？CTS, 8？non-CTS, as confirmed by PCR and sequencing). The microsphere assay correctly identified C. sordellii and C. perfringens in all known isolates and in all CTS patients (10 C. sordellii, 8 C. perfringens, 2 both) and showed 100% concordance with PCR and sequencing results. The microsphere assay is a rapid, specific, and cost-effective method for the diagnosis of CTS and offers the advantage of simultaneous testing for C. sordellii and C. perfringens in FFPE tissues using a limited amount of DNA. 1. Introduction Clostridium species are ubiquitous Gram-positive, anaerobic, spore-forming bacteria that are generally found in soil and in the intestinal tract of humans and other animals. Clostridium sordellii has been reported to cause a variety of diseases including peritonitis, endocarditis, pneumonia, arthritis, cellulitis, and myonecrosis [1–4]. Fulminant toxic shock syndrome and sepsis among previously healthy persons have been described most often in cases associated with gynecologic infections and neonatal omphalitis [3, 5]. Clostridium perfringens is also responsible for a number of clinical conditions in humans ranging from acute food poisoning and enteritis to gas gangrene, enterotoxemia, and endometritis [6–8]. In the last couple of years, several studies reported pregnancy-associated fatal toxic shock syndrome cases due to C. sordellii and C. perfringens infections [8–10]. Among all C. sordellii infections reported in the literature, the overall case fatality ratio is 70%, while, for obstetric infections, it is more than 90% [8]. Pregnancy-associated C. perfringens fulminant septicemia also carries a very high mortality rate [8]. Deaths from C. sordellii and C. perfringens toxic shock
References
[1]
P. Barnes and J. M. Leedom, “Infective endocarditis due to Clostridium sordellii,” The American Journal of Medicine, vol. 83, no. 3, p. 605, 1987.
[2]
D. E. Soper, “Clostridial myonecrosis arising from an episiotomy,” Obstetrics and Gynecology, vol. 68, no. 3, pp. 26S–28S, 1986.
[3]
J. A. McGregor, D. E. Soper, G. Lovell, and J. K. Todd, “Maternal deaths associated with Clostridium sordellii infection,” American Journal of Obstetrics and Gynecology, vol. 161, no. 4, pp. 987–995, 1989.
[4]
M. J. Aldape, A. E. Bryant, and D. L. Stevens, “Clostridium sordellii infection: epidemiology, clinical findings, and current perspectives on diagnosis and treatment,” Clinical Infectious Diseases, vol. 43, no. 11, pp. 1436–1446, 2006.
[5]
T. V. Adamkiewicz, D. Goodman, B. Burke, D. M. Lyerly, J. Goswitz, and P. Ferrieri, “Neonatal Clostridium sordellii toxic omphalitis,” Pediatric Infectious Disease Journal, vol. 12, no. 3, pp. 253–257, 1993.
[6]
S. G. Sparks, R. J. Carman, M. R. Sarker, and B. A. McClane, “Genotyping of enterotoxigenic Clostridium perfringens fecal isolates associated with antibiotic-associated diarrhea and food poisoning in North America,” Journal of Clinical Microbiology, vol. 39, no. 3, pp. 883–888, 2001.
[7]
M. Tsokos, S. Schalinski, F. Paulsen, J. P. Sperhake, K. Puschel, and I. Sobottka, “Pathology of fatal traumatic and nontraumatic clostridial gas gangrene: a histopathological, immunohistochemical, and ultrastructural study of six autopsy cases,” International Journal of Legal Medicine, vol. 122, no. 1, pp. 35–41, 2008.
[8]
A. L. Cohen, J. Bhatnagar, S. Reagan et al., “Toxic shock associated with Clostridium sordellii and Clostridium perfringens after medical and spontaneous abortion,” Obstetrics and Gynecology, vol. 110, no. 5, pp. 1027–1033, 2007.
[9]
M. Fischer, J. Bhatnagar, J. Guarner et al., “Fatal toxic shock syndrome associated with Clostridium sordellii after medical abortion,” The New England Journal of Medicine, vol. 353, no. 22, pp. 2352–2360, 2005.
[10]
C. S. Ho, J. Bhatnagar, A. L. Cohen et al., “Undiagnosed cases of fatal Clostridium-associated toxic shock in Californian women of childbearing age,” American Journal of Obstetrics and Gynecology, vol. 201, no. 5, pp. 459.e1–459.e7, 2009.
[11]
S. Nakamura, H. Ogura, J. Tanaka et al., “Difference in susceptibility of various cell cultures to cytotoxic culture filtrates of Clostridium sordellii,” Microbiology and Immunology, vol. 28, no. 4, pp. 493–497, 1984.
[12]
S. Nakamura, K. Yamakawa, and S. Nishida, “Antibacterial susceptibility of Clostridium sordellii strains,” Zentralblatt für Bakteriologie Mikrobiologie und Hygiene, vol. 261, no. 3, pp. 345–349, 1986.
[13]
R. Eriksson, M. Jobs, C. Ekstrand et al., “Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout,” Journal of Microbiological Methods, vol. 78, no. 2, pp. 195–202, 2009.
[14]
R. Benson, M. L. Tondella, J. Bhatnagar et al., “Development and evaluation of a novel multiplex PCR technology for molecular differential detection of bacterial respiratory disease pathogens,” Journal of Clinical Microbiology, vol. 46, no. 6, pp. 2074–2077, 2008.
[15]
T. J. Dumonceaux, J. Schellenberg, V. Goleski et al., “Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres,” Journal of Clinical Microbiology, vol. 47, no. 12, pp. 4067–4077, 2009.
[16]
E. Meites, S. Zane, and C. Gould, “Fatal Clostridium sordellii infections after medical abortions,” The New England Journal of Medicine, vol. 363, no. 14, pp. 1382–1383, 2010.
[17]
P. Fach and M. R. Popoff, “Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test,” Applied and Environmental Microbiology, vol. 63, no. 11, pp. 4232–4236, 1997.
[18]
J. Guarner, J. Bhatnagar, W. J. Shieh et al., “Histopathologic, immunohistochemical, and polymerase chain reaction assays in the study of cases with fatal sporadic myocarditis,” Human Pathology, vol. 38, no. 9, pp. 1412–1419, 2007.
[19]
R. A. Tucker, E. R. Unger, B. P. Holloway, and D. C. Swan, “Real-time PCR-based fluorescent assay for quantitation of human papillomavirus types 6, 11, 16, and 18,” Molecular Diagnosis, vol. 6, no. 1, pp. 39–47, 2001.
[20]
J. Guarner, J. Bartlett, S. Reagan et al., “Immunohistochemical evidence of Clostridium sp, Staphylococcus aureus, and group A Streptococcus in severe soft tissue infections related to injection drug use,” Human Pathology, vol. 37, no. 11, pp. 1482–1488, 2006.
[21]
K. Imrit, M. Goldfischer, J. Wang et al., “Identification of bacteria in formalin-fixed, paraffin-embedded heart valve tissue via 16S rRNA gene nucleotide sequencing,” Journal of Clinical Microbiology, vol. 44, no. 7, pp. 2609–2611, 2006.
[22]
J. Guarner, J. Sumner, C. D. Paddock et al., “Diagnosis of invasive group A streptococcal infections by using immunohistochemical and molecular assays,” American Journal of Clinical Pathology, vol. 126, no. 1, pp. 148–155, 2006.
[23]
L. C. Thomas, H. F. Gidding, A. N. Ginn, T. Olma, and J. Iredell, “Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture,” Journal of Microbiological Methods, vol. 68, no. 2, pp. 296–302, 2007.
[24]
N. Lansac, F. J. Picard, C. Menard et al., “Novel genus-specific PCR-based assays for rapid identification of Neisseria species and Neisseria meningitidis,” European Journal of Clinical Microbiology and Infectious Diseases, vol. 19, no. 6, pp. 443–451, 2000.
[25]
B. Herrmann, T. Nystrom, and H. Wessel, “Detection of Neisseria gonorrhoeae from air-dried genital samples by single-tube nested PCR,” Journal of Clinical Microbiology, vol. 34, no. 10, pp. 2548–2551, 1996.
[26]
S. Das, T. M. Brown, K. L. Kellar, B. P. Holloway, and C. J. Morrison, “DNA probes for the rapid identification of medically important Candida species using a multianalyte profiling system,” FEMS Immunology and Medical Microbiology, vol. 46, no. 2, pp. 244–250, 2006.
[27]
L. S. Cowan, L. Diem, M. C. Brake, and J. T. Crawford, “Transfer of a Mycobacterium tuberculosis genotyping method, Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system,” Journal of Clinical Microbiology, vol. 42, no. 1, pp. 474–477, 2004.
[28]
S. A. Dunbar, C. A. Vander Zee, K. G. Oliver, K. L. Karem, and J. W. Jacobson, “Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system,” Journal of Microbiological Methods, vol. 53, no. 2, pp. 245–252, 2003.
[29]
J. Mahony, S. Chong, F. Merante et al., “Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay,” Journal of Clinical Microbiology, vol. 45, no. 9, pp. 2965–2970, 2007.
[30]
R. L. Sweet and W. J. Ledger, “Puerperal infectious morbidity: a two year review,” American Journal of Obstetrics and Gynecology, vol. 117, no. 8, pp. 1093–1100, 1973.
[31]
H. A. Hammill, “Normal vaginal flora in relation to vaginitis,” Obstetrics and Gynecology Clinics of North America, vol. 16, no. 2, pp. 329–336, 1989.
