European Review for Medical and Pharmacological Sciences
2011; 15: 313-317
Rapid detection of Streptococcus pyogenes in throat swab specimens by fluorescent in situ hybridization S. TAJBAKHSH1, S. GHARIBI2, K. ZANDI1, R. YAGHOBI3, G. ASAYESH1 1
Department of Microbiology and Parasitology, Faculty of Medicine, The Persian Gulf Tropical and Infectious Diseases Research Center, Bushehr University of Medical Sciences, Bushehr (Iran) 2 Department of Microbiology, Islamic Azad University, Jahrom Branch, Jahrom (Iran) 3 Shiraz Transplant Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz (Iran)
Abstract. – Background and Objectives: Streptococcus pyogenes (S. pyogenes) is an important cause of pharyngitis. Rapid detection of this microorganism in throat specimens is essential to promptly start antibiotic therapy which could be lead to prevent complications and stop transmission of infection to other individuals. In the present study, fluorescent in situ hybridization (FISH) was compared with culture method for the detection of S. pyogenes in throat swab specimens. Materials and Methods: One hundred eleven patients with pharyngitis were included in this study. The throat swab specimens of these patients were investigated by both conventional culturing and FISH. Results: Based on the results of this investigation, the sensitivity and specificity of FISH were 88.9% and 97.8%, respectively. Strikingly, in the specimen of one patient who had received antibiotic previous to clinical sampling, S. pyogenes was detected by means of FISH, whereas the culture method could not detect this bacterium. Conclusions: It seems that FISH is a suitable method for quick identification of S. pyogenes in throat swab specimens. When FISH is positive, culturing is not necessary. But because of the limited sensitivity of FISH for detection of S. pyogenes in throat swab specimens, culturing shoud be performed if FISH was negative. Key Words: Streptococcus pyogenes, Fluorescent in situ hybridization, FISH, Pharyngitis.
Introduction Streptococcus pyogenes (S. pyogenes) is an important pathogen which causes both local and systemic infections. The most common infection
due to this organism is streptococcal sore throat or pharyngitis1. Suppurative sequelae of pharyngitis may ensue from spread of infectious agent to adjacent tissue or by bacteremia2. Also, the poststreptococcal nonsuppurative disorder, rheumatic fever, sometimes developes after pharyngitis due to cross- reaction of streptococcal antigens with human heart tissue antigens. Therefore, rapid and reliable detection of S. pyogenes is essential for initiation of appropriate therapy, rapid eradication of the agent, and prevention of poststreptococcal disorders1. Conventional bacteriological methods are relatively time – consuming so that require about 48 hours for isolation and identification of S. pyogenes 2. Moreover, antibiotic therapy before obtaining the specimens, may impede the isolation of the bacterium in culture media3. Fluorescent in situ hybridization (FISH) using fluorescently-labeled oligonucleotide probes that specifically target to ribosomal RNA (rRNA) is considered in microbiology as a molecular method for the rapid identification of bacteria4-6. FISH can be used for the direct detection and visualization of microorganisms in clinical samples without previous amplification or cultivation steps and this would lead to a considerable reduced time for specific detection of infectious agents4,5. There are many reports concerning the application of FISH for the detection of bacteria in clinical samples. For example, FISH has been used for diagnosis of Helicobacter pylori in gastric biopsy7-10, Enterococcus faecalis and Enterococcus faecium in faeces5, Staphylococcus aureus in sputum 11, oral streptococci in dental plaque12, and several bacteria in cerebrospinal fluid (CSF) samples13. Furthermore, S. pyogenes
Corresponding Author: Saeed Tajbakhsh, Ph.D; e-mail:
[email protected] and
[email protected]
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has been detected in samples such as maxillary sinus or fascial tissue by FISH4. The objective of this study was to compare FISH with the conventional culture method for the detection of S. pyogenes in throat swab specimens of patients suffering from pharyngitis.
Materials and Methods Collection and Cultivation of Throat Swab Specimens This study was approved by Ethical Committee of Bushehr University of Medical Sciences. Throat swab specimens were taken from 111 patients with pharyngitis. Two throat swabs were obtained from each patient. One swab was used for culture method and the other one for the FISH procedure. The specimens were cultured on blood agar (Merck, Germany) so that after streaking the medium, stabbing the inoculating loop into the agar was performed in order to allow subsurface growth and occur the most reliable hemolytic reactions2. The blood agar plates were then incubated at 35ºC. After 24 hours, the plates were observed to check the grown colonies. Negative cultures were reincubated for an additional 24 hours2,14. The suspicious colonies were subcultured and then the identification of S. pyogenes was carried out by means of conventional laboratory methods. Fixation of Samples for FISH The swabs for FISH were put into phosphate buffered saline (PBS) and rotated adequately to suspend the specimens in PBS. The specimens in these suspensions were then fixed by the adding an equal volume of absolute ethanol (Merck, Germany)4,11. Fixation of Reference Strains In the FISH procedure, S. pyogenes (ATCC 19615) and Enterococcus faecalis (ATCC 29212) were used as positive control and negative control, respectively. The strains were grown in Luria-Bertani (LB) broth3,15. Then, the bacterial cultures were centrifuged at 8000 rpm for 5 minutes at 4ºC. The bacterial pellets were resuspended in PBS and recentrifuged (PBS washing). The pellets were suspended again in 500 µl PBS and fixed by the addition of the equal volume of absolute ethanol4,11. Fixed bacterial strains were stored at –20ºC11,15,16. 314
FISH Two oligonucleotide probes, Strpyo and EUB338, that synthesized and 5’-labeled by Metabion (Martinsried, Germany), were used for our study. Probe Strpyo (5’-CTA ACA TGC GTT AGT CTC TC- 3’) that specifically binds and hybridizes to a 16S rRNA position of S. pyogenes, was applied for specific identification of this microorganism3,4. The 5’ end of Strpyo was labeled with fluorescent dye Cy3 which exhibits red signal. Probe EUB338 with the sequence 5’-GCT GCC TCC CGT AGG AGT- 3’ that targets and hybridizes 16S rRNA of nearly all bacteria, was labeled with Fluo (green signal) at the 5’ end17. The FISH procedure was carried out by spotting 10 µl of each fixed throat specimen or fixed control reference strain on glass slides. After air drying, the slides were put in ethanol series (50%, 80%, and absolute ethanol; 3 minutes each) for dehydration. Enzymatic treatment was done with 1 mg/ml lysozyme (Sigma, Steinheim, Germany) for 15 minutes4. In the hybridization step, each specimen or bacterial control was covered with 10 µl of hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 8], 0.01% SDS, 20% formamide)4,15 containing 5 ng/µl of the each probe EUB338-Fluo and Strpyo-Cy3 and then the slides were incubated at 46ºC for 90 minutes in humid chambers4,10. For stringent washing, the slides were incubated in washing buffer (20 mM Tris-HCl [pH 8], 0.01%SDS, 225 mM NaCl) for 15 minutes at 48ºC4. Afterwards, DAPI (4’,6-diamidine-2’-phenylindole dihydrochloride) (Roche, Mannheim, Germany) with the concentration 1 µg/ml for 5 minutes was applied for nonspecifically staining the DNA4,15,18. Subsequently, the slides were rinsed with PBS, left to air-dry, and mounted with fluorescent mounting medium (DAKO, Glostrup, Denmark) 10. The slides were observed and analysed with a Nikon 80i epi-fluorescence microscope (Tokyo, Japan) equipped with different filters and a digital camera system for taking the pictures. Microscopy was performed in a blinded manner by two persons. The examinations were performed twice. The time needed for FISH procedure was about 3 hours. Analysis of Test The sensitivity and specificity of FISH were calculated with the formulas (a/(a + c)) × 100 and (d/(b + d)) × 100, respectively, where a = true positive, b = false positive, c = false negative, and d = true negative.
Detection of S. pyogenes in throat specimens by FISH
Results Table I shows the results of the examination of 110 throat swab specimens for the detection of S. pyogenes using conventional culturing and FISH. Sixteen of the specimens were S. pyogenes positive by conventional culture and FISH. S. pyogenes was identified by FISH due to its specific red signal under the relevant filter of microscope (Figure 1). In 90 throat samples, S. pyogenes was not detected according to the both methods. S. pyogenes was detected in two specimens by means of culturing, while these specimens were FISH negative for S. pyogenes. Also, two throat specimens were culture negative but FISH positive for this bacterium. When we repeated the FISH tests, the results were same. Analysis of test in this study showed that the sensitivity and specificity of FISH for the detection of S. pyogenes in throat swab specimens were 88.9% and 97.8%, respectively. Strikingly, in the throat specimen of one patient who had received antibiotic before clinical sampling, S. pyogenes was detected by FISH, whereas the culture was negative for this microorganism. We did not include this specimen in total number of samples in calculation of sensitivity and specificity.
Discussion Pharyngitis is a common disease of S. pyogenes which has both suppurative (e.g. septicemia) and nonsuppurative (e.g. acute rheumatic fever) complications 1,2. On the other hand, some viral diseases such as infectious mononucleosis and adenovirus infection can exhibit clinical manifestations similar to S. pyogenes pharyngitis1. For these reasons, every effort must be made to rapidly identify the causative agent, correct treatment of patients and thus prevention
Figure 1. Detection of S. pyogenes within a throat sample of a patient with pharyngitis. Red fluorescence of the bacterium indicates the hybridization with probe StrpyoCy3.
of life-threatening complications. Isolation and identification of S. pyogenes by conventional culturing is not fast. Application of FISH as a rapid molecular technique to detect various bacteria within the clinical specimens has already been reported in many studies3,8,10,11,13,19. Therefore, we designed the present study to evaluate FISH for detection of S. pyogenes in throat swab specimens. Eighteen specimens were culture positive, of which 16 specimens were FISH positive, but 2 specimens were FISH negative (Table I). The sensitivity of FISH for detection of S. pyogenes was 88.9%. It is probable that the low number of S. pyogenes cells within 2 aforementioned specimens, and thus, microscopic detection limit of FISH could had been the reason of false negative results. Low number of bacterial cells in some specimens is a limitation factor for FISH which was also mentioned in other studies for samples such as sputum3,11 or gastric biopsy8. By conventional culture, 92 specimens were culture negative for S. pyogenes, of which 90
Table I. Results of the examination of 110 throat swab specimens by conventional culturing and FISH for diagnosis of Streptococcus pyogenes. Number of throat specimens
Culture results
FISH results
16 90 2 2
Positive Negative Positive Negative
Positive Negative Negative Positive
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specimens were also FISH negative, whereas 2 specimens were FISH positive. The specificity of FISH was 97.8%. Although we have supposed the FISH results of these 2 specimens as false positive, but following explanations indicate that they might be the false negative results of culture. As first explanation, it has been shown that S. pyogenes has the capability to enter a quiescent state and formation of viable but non-culturable bacterial cells20. On the other hand, during starvation the number of culturable S. pyogenes reduced quickly. However, bacterial number observed by FISH does not significantly change4. Thus, perhaps the 2 mentioned throat samples in our study contained non-culturable S. pyogenes which did not grow in culture medium, but was viable and could be detected by FISH. A similar condition has been found in H. pylori that develops from vegetative form into non-cultivable form7,21. The non-cultivable H. pylori possess an enough quantity of rRNA, so could be detected by means of FISH7. The second explanation is that the normal bacterial flora of the mouth may obscure or suppress the growth of S. pyogenes in throat specimens22. The third explanation is related to probe Strpyo. In the study performed by Trebesius et al4, probe Strpyo was examined by hybridizing several different target and non-target species. The results of mentioned study have been shown that Strpyo is highly specific for S. pyogenes. Also, during another study23, evaluation of the probe Strpyo was performed on pure cultures of clinical isolates as well as reference strains that Strpyo proved to be 100% sensitive and specific. Even if the FISH results of the 2 mentioned specimens in our investigation considered as false positive, the specificity of FISH is still high. Since, the specificity of FISH for detection of S. pyogenes in throat was found to be higher than its sensitivity, in practice two throat swabs from each patient can be used. First, the FISH procedure can be performed for one throat swab specimen and if it was FISH-positive, the follow up of the other swab by culturing is not necessary. In one patient who had received antibiotic previous to sampling, the FISH result was positive, but culture result was negative. Utilization of antibiotic before obtaining the specimen, can be reason of this discrepancy. It has been shown that FISH allows diagnosis of antibiotic exposed S. pyogenes, while its cultivation is negative4. Detection of infectious agents in pre-sampling antibiotic-treated patients is an advantage of FISH3,4. 316
In the study which was carried out by Trebesius et al4, only one throat swab specimen was examined by FISH. This specimen was FISHnegative for S. pyogenes. Hogardt et al3 applied culture and FISH techniques for 10 throat swab specimens from cystic fibrosis patients and Pseudomonas aeruginosa or S. aureus were detected by both methods. In the recent study conducted by Poppert et al23, FISH was applied for identification of beta-hemolytic streptococci. For this purpose, these Authors used clinical isolates and blood cultures, and finally, they recommended further investigations using respiratory samples. Therefore, our investigation can be a suitable study to evaluate FISH for detection of S. pyogenes in throat swab specimens. In conclusion, it seems that FISH is relatively an appropriate technique for rapid detection of S. pyogenes in throat swab specimens. Because of the limited sensitivity of FISH, we should state that it only partially can be used instead of culture method. However, since a high specificity was achieved for FISH, positive results are reliable, so that antibiotic therapy can start without delay. –––––––––––––––––––– Acknowledgements
We thank the Vice-Chancellor of Research of Bushehr University of Medical Sciences for financial support.
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