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Direct Eubacterial PCR Diagnosis of Primary Sterile Body Fluids and Tissues

Broad-Range PCR Diagnosis of Pathogens

Michael Lustig, PhD,
Molecular biologist and Product Manager for molecular diagnostic products at Molzym

Culture-Independent, Broad-Range PCR Diagnosis

Culturing is the standard method for the detec­tion and identification of pathogens. However, the rate of culture-negative infections can be considerably high [17]. Well studied is infectious endocarditis for which the rate of culture-nega­tive infections can account for 12% and thus be nearly double as high as culture [11]. The high rate of false-negative results is primarily due to fastidious organisms, including species of Coxiella, Bartonella, Chlamydia, Legionella and Tropheryma some of which can be detected only by molecular means [3, 5]. In the case of sepsis and its progressive forms the cultures remain negative primarily because of the administration of antibi­otics before blood collection [4]. Examples of culture-negative infections are summarized in Table 1. Interestingly, the incidence of culture-negative infections, e. g., infectious ascites as judged by clinical parameters, can be two to three times higher than culture proven infections.

Table 1

Disease Study patients Specimens Issue Culture-positive infections (%) a Culture-negative infections (%) b Method Ref.
Acute osteoarticular infections 390 blood, aspirates, tissues MRSA incidence 50,1 49,9 clinical parameters [22]
Brain abscess 1
(case study)
tissue Fusobacterium nucleatum, Porphyromonas
endodontalis mixed infection
n.a. n.a. broad-range 16S rRNA gene PCR plus sequencing [8]
  1
(case study)
tissue Streptococcus intermedius n.a. n.a. broad-range 16S rRNA gene PCR plus sequencing [18]
Bloodstream infection 218 EDTA-blood detection of bacteremia 19,3 2,3 Gram-specific Real-Time PCR [1]
  233 blood culture rate of culture-negative infections 54,9 4,7 broad-range 16S rRNA gene PCR plus array hybridization [12]
  384 EDTA-blood MRSA incidence 1,6 2,6 target-specific Real-Time PCR [21]
Ascitic fluid infection 130 aspirates bacterial infection in hepatitis B virus cirrhotic patients 28,5 71,5 clinical parameters [9]
  187 aspirates viral causes of cirrhosis along with ascites 23,5 76,4 clinical parameters [7]
  100 tissues, aspirates 9,9 2,0 broad-range 16S rRNA gene PCR plus pyro-sequencing [2]
Infectious endocarditis 516 blood incidence of culture-negative infections 6,6 12,2 pathologically, other cultures, serology [11]
a results among all patients without assessment of clinical relevance
b clinically relevant results among all patients

Broad-range 16S rRNA gene PCR and se­quencing are acknowledged methods for the precise diagnosis of pathogenic organisms. Frequently used as an ultima ratio method, 16S rRNA gene PCR allows for the most compre­hensive detection and identification of patho­gens. It is especially useful for the etiological diagnosis of diseases which can be caused by a variety of or­ganisms from very diverse taxons. PCR methods rely on the detection of nucleic acids and, thus, do not depend on the growth of organisms. For this reason, the major strength of PCR is the proof of pathogens that do not grow in culture because of being fastidious or inhibited by antibiotics (Table 1). For molecular diagnosis, the material is extracted and the purified DNA from potentially present pathogens is analyzed using PCR or Real-Time PCR assays. Detection of DNA from pathogens by broad-range PCR or Real-Time PCR is followed by sequence analysis of amplicons which results in the identification of the species.

Direct Diagnosis of pathogens by UMD-Universal

UMD-Universal is a CE-marked test for the in-vitro diagnosis by broad-range rRNA gene PCR of bacteria and fungi. The diagnosis is performed using clinical material without cultivation. The specimens comprise EDTA, citrate or heparin-stabilized whole blood, aspirates from joints or the peritoneum, abscesses, diverse tissues(e.g., skin, organ, bone biopsies), and swabs from wounds, catheters and other material. Samples are processed following manual or automated protocols for the enrichment and purification of microbial DNA exclusively from live or intact dead cells. The DNA is analyzed by Real-Time PCR assays for bacterial and fungal sequences. In case of positive results, the amplicon is sequence-analyzed. For this purpose, a BLAST algorithm (sepsitest-blast.de) is available based on more than 7,000 quality-controlled sequence entries of 16S and 18S rRNA genes. Mixed infections of up to 6 strains can be identified by the Gram-specific primer sequencing approach combined with Ripseq® software algorithm (Isentio, Bergen, Norway).

Table 2

Primary Diagnosis Material Patients Culture-positive infections (%) a Culture-negative infections (%) b Ref.
Diverse synovial and peritoneal aspirates, tissues, CSF 84 25,0 15,5 [4]
  heart valves, synovial aspirates, CSF, blood, blood culture 66 18,2 21,2 [14]
  blood 120 13,3 18,3 [6]
Infectious endocarditis blood and heart valves 29 31,0 48,3 [10]
  catheter tip,Coryne-bacterium striatum 1 - c + [16]
Meningitis CSF 12 - c 50,0 [13]
Sepsis blood 187 18,2 13,4 [20]
  blood 26 30,8 7,7 [15]
Tick-borne disease blood,Neoehrlichia mikurensis 2 - c + [19]
a cultivation of samples in blood culture bottles; results among all patients clinically not assessed
b PCR-positive, culture-negative, clinically relevant results among all patients
c culture-negative patients

The diagnostic performance of UMD-Universal has been evaluated employing a variety of samples from more than 500 patients. An international multicenter study on sepsis diagnostics by UMD-Universal has now been completed. The diagnostic values with blood culture as standard were comparable (e.g., 80-88.5% sensitivity [4, 6, 10, 20]). Besides the rapidity of diagnosis, UMD-Universal uncovered its benefit in the identification of aetiologies in substantially less time fraction of suspected culture-negative infections (Table 2). In fact, UMD-Universal-approved infections accounted for rates of detection comparable to or even higher than those of culture. Hence, UMD-Universal constitutes a valuable tool complementing culture diagnosis.

Laboratory Service

Molzym offers an identification service for the culture-inde­pend­ent molecular identification of pathogens from a variety of specimens. After receipt of samples, reports are usually provided within 1-3 weeks, including the identity of organisms in positive samples. Please call for further information.

 

References

[1]  Chan KYY, Lam HS, Cheung HM et al. (2009) Crit Care Med 37: 2441-2447.

[2]  Gadsby NJ, Onen A, Phillips SA et al. (2011) Open J Med Microbiol 1 (doi:10.4236/ojmm.2011.11001).

[3] Geißdörfer W, Moos V, Moter A et al. (2012) J Clin Microbiol 50: 216–222.

[4]  Grif K, Heller I, Prodinger WM et al. (2012) J Clin Microbiol 50: 2250-2254.

[5]  Houpikian P, Raoult D (2005) Medicine 84: 162-173.

[6]  Irwin AD, Carrol ED, Barton T et al. (2012) Poster P1797, 22nd ECCMID, London.

[7]  Kamani L, Mumtaz K,  Ahmed US et al. (2008) BMC Gastroenterology 8:59 doi:10.1186/1471-230X-8-59.

[8]  Keller PM, Rampini SK, Bloemberg GV (2010) J Clin MIcrobiol 48: 2250–2252.

[9]  Kim SU, Kim DY, Lee CK et al. (2009) J Gastroent  Hepatol 25: 122–128.

[10]Kühn C, Disqué C, Mühl H et al. (2011) J Clin Microbiol 49: 2919-2923.

[11]Lamas CC, Eykyn SJ (2003) Heart 89: 258-262.

[12]Matsuda K, Iwaki KK, Garcia-Gomez J et al. (2011) J Clin Microbiol 49: 2031–2034.

[13]Meyer T, Polywka SKA (2011) Poster 1103, 111th ASM Meeting, New Orleans.

[14]Nolte O, Locher F, Haag H (2011) Poster P1735. 21st ECCMID, Milan.

[15]Obaro S, personal communication.

[16]Oliva A, Belvisi V, Iannetta M et al. (2010) J Clin Microbiol 48: 4669-4671.

[17]Rangel-Frausto MS, Pittet D, Costigan M et al. (1995) J Am Med Assoc 273: 117-123.

[18]Saito N, Hida A, Koide Y et al. (2012) Inter Med 51: 211-216.

[19]von Loewenich FD, Geiß-dörfer W, Disqué C et al. (2010) J Clin Microbiol 48: 2630-2635.

[20]Wellinghausen N, Kochem AJ, Disqué C et al. (2009) J Clin Microbiol 47: 2759-2765.

[21]Wellinghausen N, Siegel D, Gebert S, Winter J (2009) Eur J Clin Microbiol Infect Dis 28: 1001-1005.

[22]Williams DJ, Deis JN, Tardy J et al. (2010) Ped Infect Dis J 30: 523-525.

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