In a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection

Kooistra-Smid, Anna M. D.

Ott, Alewijn

van Zanten, Evert

Vierhout, Bastiaan

Wisselink , Guido

Dissertations of the University of Groningen

   University of GroningenTracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesVierhout, Bastiaan; van Zanten, Evert; Wisselink , Guido ; Kooistra-Smid, Anna M. D.; Ott,AlewijnPublished in:Clinics in SurgeryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vierhout, B., van Zanten, E., Wisselink , G., Kooistra-Smid, A. M. D., & Ott, A. (2018). Tracking Down theCause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clinics in Surgery, 3, [2011].http://www.clinicsinsurgery.com/full-text/cis-v3-id2011.phpCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022Remedy Publications LLC., | http://clinicsinsurgery.com/Clinics in Surgery2018 | Volume 3 | Article 20111Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesOPEN ACCESS *Correspondence:Alewijn Ott, Department of Medical Microbiology, Certe, Groningen, Netherlands,E-mail: a.ott@certe.nlReceived Date: 25 Jun 2018Accepted Date: 09 Jul 2018Published Date: 12 Jul 2018Citation: Vierhout BP, van Zanten E, Wisselink G, Kooistra-Smid MAMD, Ott A. Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clin Surg. 2018; 3: 2011.Copyright © 2018 Alewijn Ott. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Case ReportPublished: 12 Jul, 2018AbstractIn a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection.Bas P Vierhout1, Evert van Zanten2, Guido Wisselink2, Mirjam AMD Kooistra-Smid2,3 and Alewijn Ott2*1Department of Surgery, Wilhelmina Ziekenhuis, Assen, Netherlands2Department of Medical Microbiology, Certe, Groningen, Netherlands3Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsBackgroundNecrotizing Fasciitis (NF) is a severe disease of sudden onset that spreads rapidly [1]. Symptoms include red skin, severe pain and fever. Risk factors include poor immune function and a break in the skin, for instance a wound. But also healthy persons may suffer from NF, without a visible entrance through the skin, mostly in the perineal region or the limbs.The disease is feared because of its high mortality and subsequent morbidity. The infection spreads rapidly over the fascia of the muscles and open surgical drainage is the only treatment option: Infected tissue must be removed without delay, in combination with intravenous antibiotics.Most often, the causative agent is a Streptococcus from Lancefield group A (S. pyogenes). If S. pyogenes is involved antibiotic treatment with a high dose of penicillin suffices. But combinations of antibiotics with a broader spectrum are required in infections in the perineal area, because these may be polymicrobial.Case PresentationA 68-year-old female patient complained of severe pain and localized redness since a few hours, spreading over the lower abdomen. Four days before, she had some localized pain on the right side of the abdomen, which gradually shifted to the left. Mobilizing aggravated the pain and made her nauseous, and as a result, she ate and drank less. She had no abdominal surgery or relevant infectious medical history in the past. Only bad teeth had been removed 6 years earlier, to replace them by prosthesis.At the emergency room she had a fever of 39.2°C, with a pulse of 107 per minute and a tension of 111/63 mmHg. The abdominal redness was moderately delimited and there was no swelling (Figure 1). Some bowel sounds were heard and the redness felt firmer than the surrounding tissue.Laboratory tests showed a leukocytosis of 44,000*106 per liter, with a left upper shift. A hyponatremia was accompanied by elevated urea and creatinine level of 23.3 mmol/l and 272 µmol/l, respectively. C-reactive protein was 639 mg/l. An emergency CT-scan did not show intra-abdominal disorder: no abnormal air or fluid collection in the abdominal cavity and normal intra abdominal organs. But the subcutaneous fat of the anterior abdominal wall showed signs of inflammation without gas.The patient was started on piperacillin with tazobactam in combination with clindamycin and surgical exploration was performed on suspicion of NF. During the operation the fascia seemed intact, but the subcutaneous tissue, adjacent to the fascia had a discolored, greyish, avital aspect. Biopsies for cultures and histology of the tissue were taken and the wound was left open.Alewijn Ott, et al., Clinics in Surgery - Vascular SurgeryRemedy Publications LLC., | http://clinicsinsurgery.com/ 2018 | Volume 3 | Article 20112MicrobiologyBlood cultures taken on admission and culture of the tissue taken during surgery unfortunately remained negative. No bacteria were seen in the Gram stain of the tissue. Because of these negative findings and uncertainty about the best choice of antibiotics to continue the treatment we additionally examined the tissue with 16S-23S rDNANext Generation Sequencing [2]. This method is based on a PCR amplification of the 16S-23S rRNA region followed by amplicon sequencing on the MiSeq (Illumina, Inc., San Diego, CA). The resulting reads are denovo assembled into contigs. Species identification is based on an alignment of the contig sequences with the sequences deposited in the reference databases. Th is resulted in identification of Streptococcus pyogenes only, with a Relative Abundance (RA) of 99.7%. The RA indicates the relative percentage of reads that represents the same species in a sample.Figure 1: Redness of the lower abdomen of the patient before surgery. The line around the redness was drawn in the emergency department in order to indicate any increase.DiscussionThe best remedy for NF is early recognition, followed by surgery and antibiotic treatment focused upon the causative agent(s). Unfortunately, in some cases, these agents cannot be identified, as was the case in this patient. Most likely this was due to the high dose of intravenous antibiotic that was already given at the emergency department. The infection was in an early stage and therefore tissue circulation was still intact. Streptococcus pyogenes is usually highly sensitive to the penicillin class antibiotics. Knowing the causative bacteria enabled us to finish the treatment with oral amoxicillin after the patient recovered. 16S-23S rDNA NGS could exclude the alternative diagnosis in this patient of the polybacterialFournier NF, which requires a broader spectrum of antibiotics [3]. 16S-23S rDNA NGS can therefore contribute to rational antibiotic use.References1. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 2017;377:2253-65.2. Sabat AJ, van Zanten E, Akkerboom V, Wisselink G, van Slochteren K, de Boer RF, et al. Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species. Sci Rep. 2017;7(1):3434.3. Mallikarjuna MN, Vijayakumar A, Patil VS, Shivswamy BS. Fournier's Gangrene: Current Practices. ISRN Surg. 2012:942437.

English

Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures

Wisselink, Guido

NARCIS 

   University of GroningenTracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesVierhout, Bastiaan; van Zanten, Evert; Wisselink , Guido ; Kooistra-Smid, Anna M. D.; Ott,AlewijnPublished in:Clinics in SurgeryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vierhout, B., van Zanten, E., Wisselink , G., Kooistra-Smid, A. M. D., & Ott, A. (2018). Tracking Down theCause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clinics in Surgery, 3, Article 2011.http://www.clinicsinsurgery.com/full-text/cis-v3-id2011.phpCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 01-11-2023Remedy Publications LLC., | http://clinicsinsurgery.com/Clinics in Surgery2018 | Volume 3 | Article 20111Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesOPEN ACCESS *Correspondence:Alewijn Ott, Department of Medical Microbiology, Certe, Groningen, Netherlands,E-mail: a.ott@certe.nlReceived Date: 25 Jun 2018Accepted Date: 09 Jul 2018Published Date: 12 Jul 2018Citation: Vierhout BP, van Zanten E, Wisselink G, Kooistra-Smid MAMD, Ott A. Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clin Surg. 2018; 3: 2011.Copyright © 2018 Alewijn Ott. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Case ReportPublished: 12 Jul, 2018AbstractIn a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection.Bas P Vierhout1, Evert van Zanten2, Guido Wisselink2, Mirjam AMD Kooistra-Smid2,3 and Alewijn Ott2*1Department of Surgery, Wilhelmina Ziekenhuis, Assen, Netherlands2Department of Medical Microbiology, Certe, Groningen, Netherlands3Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsBackgroundNecrotizing Fasciitis (NF) is a severe disease of sudden onset that spreads rapidly [1]. Symptoms include red skin, severe pain and fever. Risk factors include poor immune function and a break in the skin, for instance a wound. But also healthy persons may suffer from NF, without a visible entrance through the skin, mostly in the perineal region or the limbs.The disease is feared because of its high mortality and subsequent morbidity. The infection spreads rapidly over the fascia of the muscles and open surgical drainage is the only treatment option: Infected tissue must be removed without delay, in combination with intravenous antibiotics.Most often, the causative agent is a Streptococcus from Lancefield group A (S. pyogenes). If S. pyogenes is involved antibiotic treatment with a high dose of penicillin suffices. But combinations of antibiotics with a broader spectrum are required in infections in the perineal area, because these may be polymicrobial.Case PresentationA 68-year-old female patient complained of severe pain and localized redness since a few hours, spreading over the lower abdomen. Four days before, she had some localized pain on the right side of the abdomen, which gradually shifted to the left. Mobilizing aggravated the pain and made her nauseous, and as a result, she ate and drank less. She had no abdominal surgery or relevant infectious medical history in the past. Only bad teeth had been removed 6 years earlier, to replace them by prosthesis.At the emergency room she had a fever of 39.2°C, with a pulse of 107 per minute and a tension of 111/63 mmHg. The abdominal redness was moderately delimited and there was no swelling (Figure 1). Some bowel sounds were heard and the redness felt firmer than the surrounding tissue.Laboratory tests showed a leukocytosis of 44,000*106 per liter, with a left upper shift. A hyponatremia was accompanied by elevated urea and creatinine level of 23.3 mmol/l and 272 µmol/l, respectively. C-reactive protein was 639 mg/l. An emergency CT-scan did not show intra-abdominal disorder: no abnormal air or fluid collection in the abdominal cavity and normal intra abdominal organs. But the subcutaneous fat of the anterior abdominal wall showed signs of inflammation without gas.The patient was started on piperacillin with tazobactam in combination with clindamycin and surgical exploration was performed on suspicion of NF. During the operation the fascia seemed intact, but the subcutaneous tissue, adjacent to the fascia had a discolored, greyish, avital aspect. Biopsies for cultures and histology of the tissue were taken and the wound was left open.Alewijn Ott, et al., Clinics in Surgery - Vascular SurgeryRemedy Publications LLC., | http://clinicsinsurgery.com/ 2018 | Volume 3 | Article 20112MicrobiologyBlood cultures taken on admission and culture of the tissue taken during surgery unfortunately remained negative. No bacteria were seen in the Gram stain of the tissue. Because of these negative findings and uncertainty about the best choice of antibiotics to continue the treatment we additionally examined the tissue with 16S-23S rDNANext Generation Sequencing [2]. This method is based on a PCR amplification of the 16S-23S rRNA region followed by amplicon sequencing on the MiSeq (Illumina, Inc., San Diego, CA). The resulting reads are denovo assembled into contigs. Species identification is based on an alignment of the contig sequences with the sequences deposited in the reference databases. Th is resulted in identification of Streptococcus pyogenes only, with a Relative Abundance (RA) of 99.7%. The RA indicates the relative percentage of reads that represents the same species in a sample.Figure 1: Redness of the lower abdomen of the patient before surgery. The line around the redness was drawn in the emergency department in order to indicate any increase.DiscussionThe best remedy for NF is early recognition, followed by surgery and antibiotic treatment focused upon the causative agent(s). Unfortunately, in some cases, these agents cannot be identified, as was the case in this patient. Most likely this was due to the high dose of intravenous antibiotic that was already given at the emergency department. The infection was in an early stage and therefore tissue circulation was still intact. Streptococcus pyogenes is usually highly sensitive to the penicillin class antibiotics. Knowing the causative bacteria enabled us to finish the treatment with oral amoxicillin after the patient recovered. 16S-23S rDNA NGS could exclude the alternative diagnosis in this patient of the polybacterialFournier NF, which requires a broader spectrum of antibiotics [3]. 16S-23S rDNA NGS can therefore contribute to rational antibiotic use.References1. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 2017;377:2253-65.2. Sabat AJ, van Zanten E, Akkerboom V, Wisselink G, van Slochteren K, de Boer RF, et al. Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species. Sci Rep. 2017;7(1):3434.3. Mallikarjuna MN, Vijayakumar A, Patil VS, Shivswamy BS. Fournier's Gangrene: Current Practices. ISRN Surg. 2012:942437.

University of Groningen Research Database

  
 University of Groningen
Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures
Vierhout, Bastiaan; van Zanten, Evert; Wisselink , Guido ; Kooistra-Smid, Anna M. D.; Ott,
Alewijn
Published in:
Clinics in Surgery
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2018
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Vierhout, B., van Zanten, E., Wisselink , G., Kooistra-Smid, A. M. D., & Ott, A. (2018). Tracking Down the
Cause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clinics in Surgery, 3, [2011].
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 29-04-2019
Remedy Publications LLC., | http://clinicsinsurgery.com/
Clinics in Surgery
2018 | Volume 3 | Article 20111
Tracking Down the Cause of Necrotizing Fasciitis in a 
Patient with Negative Cultures
OPEN ACCESS
 *Correspondence:
Alewijn Ott, Department of Medical 
Microbiology, Certe, Groningen, 
Netherlands,
E-mail: a.ott@certe.nl
Received Date: 25 Jun 2018
Accepted Date: 09 Jul 2018
Published Date: 12 Jul 2018
Citation: 
Vierhout BP, van Zanten E, Wisselink G, 
Kooistra-Smid MAMD, Ott A. Tracking 
Down the Cause of Necrotizing Fasciitis 
in a Patient with Negative Cultures. Clin 
Surg. 2018; 3: 2011.
Copyright © 2018 Alewijn Ott. This is 
an open access article distributed under 
the Creative Commons Attribution 
License, which permits unrestricted 
use, distribution, and reproduction in 
any medium, provided the original work 
is properly cited.
Case Report
Published: 12 Jul, 2018
Abstract
In a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, 
possibly because broad spectrum antibiotics had already been given before tissue for culture was 
obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of 
Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in 
polymicrobial infection.
Bas P Vierhout1, Evert van Zanten2, Guido Wisselink2, Mirjam AMD Kooistra-Smid2,3 and Alewijn 
Ott2*
1Department of Surgery, Wilhelmina Ziekenhuis, Assen, Netherlands
2Department of Medical Microbiology, Certe, Groningen, Netherlands
3Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, 
Netherlands
Background
Necrotizing Fasciitis (NF) is a severe disease of sudden onset that spreads rapidly [1]. Symptoms 
include red skin, severe pain and fever. Risk factors include poor immune function and a break 
in the skin, for instance a wound. But also healthy persons may suffer from NF, without a visible 
entrance through the skin, mostly in the perineal region or the limbs.
The disease is feared because of its high mortality and subsequent morbidity. The infection 
spreads rapidly over the fascia of the muscles and open surgical drainage is the only treatment 
option: Infected tissue must be removed without delay, in combination with intravenous antibiotics.
Most often, the causative agent is a Streptococcus from Lancefield group A (S. pyogenes). If S. 
pyogenes is involved antibiotic treatment with a high dose of penicillin suffices. But combinations 
of antibiotics with a broader spectrum are required in infections in the perineal area, because these 
may be polymicrobial.
Case Presentation
A 68-year-old female patient complained of severe pain and localized redness since a few hours, 
spreading over the lower abdomen. Four days before, she had some localized pain on the right 
side of the abdomen, which gradually shifted to the left. Mobilizing aggravated the pain and made 
her nauseous, and as a result, she ate and drank less. She had no abdominal surgery or relevant 
infectious medical history in the past. Only bad teeth had been removed 6 years earlier, to replace 
them by prosthesis.
At the emergency room she had a fever of 39.2°C, with a pulse of 107 per minute and a tension of 
111/63 mmHg. The abdominal redness was moderately delimited and there was no swelling (Figure 
1). Some bowel sounds were heard and the redness felt firmer than the surrounding tissue.
Laboratory tests showed a leukocytosis of 44,000*106 per liter, with a left upper shift. A 
hyponatremia was accompanied by elevated urea and creatinine level of 23.3 mmol/l and 272 
µmol/l, respectively. C-reactive protein was 639 mg/l. An emergency CT-scan did not show intra-
abdominal disorder: no abnormal air or fluid collection in the abdominal cavity and normal 
intra abdominal organs. But the subcutaneous fat of the anterior abdominal wall showed signs of 
inflammation without gas.
The patient was started on piperacillin with tazobactam in combination with clindamycin and 
surgical exploration was performed on suspicion of NF. During the operation the fascia seemed 
intact, but the subcutaneous tissue, adjacent to the fascia had a discolored, greyish, avital aspect. 
Biopsies for cultures and histology of the tissue were taken and the wound was left open.
Alewijn Ott, et al., Clinics in Surgery - Vascular Surgery
Remedy Publications LLC., | http://clinicsinsurgery.com/ 2018 | Volume 3 | Article 20112
Microbiology
Blood cultures taken on admission and culture of the tissue 
taken during surgery unfortunately remained negative. No bacteria 
were seen in the Gram stain of the tissue. Because of these negative 
findings and uncertainty about the best choice of antibiotics to 
continue the treatment we additionally examined the tissue with 
16S-23S rDNANext Generation Sequencing [2]. This method is 
based on a PCR amplification of the 16S-23S rRNA region followed 
by amplicon sequencing on the MiSeq (Illumina, Inc., San Diego, 
CA). The resulting reads are denovo assembled into contigs. Species 
identification is based on an alignment of the contig sequences with 
the sequences deposited in the reference databases. Th is resulted 
in identification of Streptococcus pyogenes only, with a Relative 
Abundance (RA) of 99.7%. The RA indicates the relative percentage 
of reads that represents the same species in a sample.
Figure 1: Redness of the lower abdomen of the patient before surgery. The 
line around the redness was drawn in the emergency department in order to 
indicate any increase.
Discussion
The best remedy for NF is early recognition, followed by surgery 
and antibiotic treatment focused upon the causative agent(s). 
Unfortunately, in some cases, these agents cannot be identified, as 
was the case in this patient. Most likely this was due to the high dose 
of intravenous antibiotic that was already given at the emergency 
department. The infection was in an early stage and therefore tissue 
circulation was still intact. Streptococcus pyogenes is usually highly 
sensitive to the penicillin class antibiotics. 
Knowing the causative bacteria enabled us to finish the treatment 
with oral amoxicillin after the patient recovered. 16S-23S rDNA 
NGS could exclude the alternative diagnosis in this patient of the 
polybacterialFournier NF, which requires a broader spectrum of 
antibiotics [3]. 16S-23S rDNA NGS can therefore contribute to 
rational antibiotic use.
References
1. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 
2017;377:2253-65.
2. Sabat AJ, van Zanten E, Akkerboom V, Wisselink G, van Slochteren K, 
de Boer RF, et al. Targeted next-generation sequencing of the 16S-23S 
rRNA region for culture-independent bacterial identification - increased 
discrimination of closely related species. Sci Rep. 2017;7(1):3434.
3. Mallikarjuna MN, Vijayakumar A, Patil VS, Shivswamy BS. Fournier's 
Gangrene: Current Practices. ISRN Surg. 2012:942437.


Proceedings - University of Groningen

   University of GroningenTracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesVierhout, Bastiaan; van Zanten, Evert; Wisselink , Guido ; Kooistra-Smid, Anna M. D.; Ott,AlewijnPublished in:Clinics in SurgeryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vierhout, B., van Zanten, E., Wisselink , G., Kooistra-Smid, A. M. D., & Ott, A. (2018). Tracking Down theCause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clinics in Surgery, 3, [2011].http://www.clinicsinsurgery.com/full-text/cis-v3-id2011.phpCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 03-06-2022Remedy Publications LLC., | http://clinicsinsurgery.com/Clinics in Surgery2018 | Volume 3 | Article 20111Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesOPEN ACCESS *Correspondence:Alewijn Ott, Department of Medical Microbiology, Certe, Groningen, Netherlands,E-mail: a.ott@certe.nlReceived Date: 25 Jun 2018Accepted Date: 09 Jul 2018Published Date: 12 Jul 2018Citation: Vierhout BP, van Zanten E, Wisselink G, Kooistra-Smid MAMD, Ott A. Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clin Surg. 2018; 3: 2011.Copyright © 2018 Alewijn Ott. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Case ReportPublished: 12 Jul, 2018AbstractIn a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection.Bas P Vierhout1, Evert van Zanten2, Guido Wisselink2, Mirjam AMD Kooistra-Smid2,3 and Alewijn Ott2*1Department of Surgery, Wilhelmina Ziekenhuis, Assen, Netherlands2Department of Medical Microbiology, Certe, Groningen, Netherlands3Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsBackgroundNecrotizing Fasciitis (NF) is a severe disease of sudden onset that spreads rapidly [1]. Symptoms include red skin, severe pain and fever. Risk factors include poor immune function and a break in the skin, for instance a wound. But also healthy persons may suffer from NF, without a visible entrance through the skin, mostly in the perineal region or the limbs.The disease is feared because of its high mortality and subsequent morbidity. The infection spreads rapidly over the fascia of the muscles and open surgical drainage is the only treatment option: Infected tissue must be removed without delay, in combination with intravenous antibiotics.Most often, the causative agent is a Streptococcus from Lancefield group A (S. pyogenes). If S. pyogenes is involved antibiotic treatment with a high dose of penicillin suffices. But combinations of antibiotics with a broader spectrum are required in infections in the perineal area, because these may be polymicrobial.Case PresentationA 68-year-old female patient complained of severe pain and localized redness since a few hours, spreading over the lower abdomen. Four days before, she had some localized pain on the right side of the abdomen, which gradually shifted to the left. Mobilizing aggravated the pain and made her nauseous, and as a result, she ate and drank less. She had no abdominal surgery or relevant infectious medical history in the past. Only bad teeth had been removed 6 years earlier, to replace them by prosthesis.At the emergency room she had a fever of 39.2°C, with a pulse of 107 per minute and a tension of 111/63 mmHg. The abdominal redness was moderately delimited and there was no swelling (Figure 1). Some bowel sounds were heard and the redness felt firmer than the surrounding tissue.Laboratory tests showed a leukocytosis of 44,000*106 per liter, with a left upper shift. A hyponatremia was accompanied by elevated urea and creatinine level of 23.3 mmol/l and 272 µmol/l, respectively. C-reactive protein was 639 mg/l. An emergency CT-scan did not show intra-abdominal disorder: no abnormal air or fluid collection in the abdominal cavity and normal intra abdominal organs. But the subcutaneous fat of the anterior abdominal wall showed signs of inflammation without gas.The patient was started on piperacillin with tazobactam in combination with clindamycin and surgical exploration was performed on suspicion of NF. During the operation the fascia seemed intact, but the subcutaneous tissue, adjacent to the fascia had a discolored, greyish, avital aspect. Biopsies for cultures and histology of the tissue were taken and the wound was left open.Alewijn Ott, et al., Clinics in Surgery - Vascular SurgeryRemedy Publications LLC., | http://clinicsinsurgery.com/ 2018 | Volume 3 | Article 20112MicrobiologyBlood cultures taken on admission and culture of the tissue taken during surgery unfortunately remained negative. No bacteria were seen in the Gram stain of the tissue. Because of these negative findings and uncertainty about the best choice of antibiotics to continue the treatment we additionally examined the tissue with 16S-23S rDNANext Generation Sequencing [2]. This method is based on a PCR amplification of the 16S-23S rRNA region followed by amplicon sequencing on the MiSeq (Illumina, Inc., San Diego, CA). The resulting reads are denovo assembled into contigs. Species identification is based on an alignment of the contig sequences with the sequences deposited in the reference databases. Th is resulted in identification of Streptococcus pyogenes only, with a Relative Abundance (RA) of 99.7%. The RA indicates the relative percentage of reads that represents the same species in a sample.Figure 1: Redness of the lower abdomen of the patient before surgery. The line around the redness was drawn in the emergency department in order to indicate any increase.DiscussionThe best remedy for NF is early recognition, followed by surgery and antibiotic treatment focused upon the causative agent(s). Unfortunately, in some cases, these agents cannot be identified, as was the case in this patient. Most likely this was due to the high dose of intravenous antibiotic that was already given at the emergency department. The infection was in an early stage and therefore tissue circulation was still intact. Streptococcus pyogenes is usually highly sensitive to the penicillin class antibiotics. Knowing the causative bacteria enabled us to finish the treatment with oral amoxicillin after the patient recovered. 16S-23S rDNA NGS could exclude the alternative diagnosis in this patient of the polybacterialFournier NF, which requires a broader spectrum of antibiotics [3]. 16S-23S rDNA NGS can therefore contribute to rational antibiotic use.References1. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 2017;377:2253-65.2. Sabat AJ, van Zanten E, Akkerboom V, Wisselink G, van Slochteren K, de Boer RF, et al. Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species. Sci Rep. 2017;7(1):3434.3. Mallikarjuna MN, Vijayakumar A, Patil VS, Shivswamy BS. Fournier's Gangrene: Current Practices. ISRN Surg. 2012:942437.

University of Groningen

   University of GroningenTracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesVierhout, Bastiaan; van Zanten, Evert; Wisselink , Guido ; Kooistra-Smid, Anna M. D.; Ott,AlewijnPublished in:Clinics in SurgeryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vierhout, B., van Zanten, E., Wisselink , G., Kooistra-Smid, A. M. D., & Ott, A. (2018). Tracking Down theCause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clinics in Surgery, 3, [2011].CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 13-11-2019Remedy Publications LLC., | http://clinicsinsurgery.com/Clinics in Surgery2018 | Volume 3 | Article 20111Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesOPEN ACCESS *Correspondence:Alewijn Ott, Department of Medical Microbiology, Certe, Groningen, Netherlands,E-mail: a.ott@certe.nlReceived Date: 25 Jun 2018Accepted Date: 09 Jul 2018Published Date: 12 Jul 2018Citation: Vierhout BP, van Zanten E, Wisselink G, Kooistra-Smid MAMD, Ott A. Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clin Surg. 2018; 3: 2011.Copyright © 2018 Alewijn Ott. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Case ReportPublished: 12 Jul, 2018AbstractIn a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection.Bas P Vierhout1, Evert van Zanten2, Guido Wisselink2, Mirjam AMD Kooistra-Smid2,3 and Alewijn Ott2*1Department of Surgery, Wilhelmina Ziekenhuis, Assen, Netherlands2Department of Medical Microbiology, Certe, Groningen, Netherlands3Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsBackgroundNecrotizing Fasciitis (NF) is a severe disease of sudden onset that spreads rapidly [1]. Symptoms include red skin, severe pain and fever. Risk factors include poor immune function and a break in the skin, for instance a wound. But also healthy persons may suffer from NF, without a visible entrance through the skin, mostly in the perineal region or the limbs.The disease is feared because of its high mortality and subsequent morbidity. The infection spreads rapidly over the fascia of the muscles and open surgical drainage is the only treatment option: Infected tissue must be removed without delay, in combination with intravenous antibiotics.Most often, the causative agent is a Streptococcus from Lancefield group A (S. pyogenes). If S. pyogenes is involved antibiotic treatment with a high dose of penicillin suffices. But combinations of antibiotics with a broader spectrum are required in infections in the perineal area, because these may be polymicrobial.Case PresentationA 68-year-old female patient complained of severe pain and localized redness since a few hours, spreading over the lower abdomen. Four days before, she had some localized pain on the right side of the abdomen, which gradually shifted to the left. Mobilizing aggravated the pain and made her nauseous, and as a result, she ate and drank less. She had no abdominal surgery or relevant infectious medical history in the past. Only bad teeth had been removed 6 years earlier, to replace them by prosthesis.At the emergency room she had a fever of 39.2°C, with a pulse of 107 per minute and a tension of 111/63 mmHg. The abdominal redness was moderately delimited and there was no swelling (Figure 1). Some bowel sounds were heard and the redness felt firmer than the surrounding tissue.Laboratory tests showed a leukocytosis of 44,000*106 per liter, with a left upper shift. A hyponatremia was accompanied by elevated urea and creatinine level of 23.3 mmol/l and 272 µmol/l, respectively. C-reactive protein was 639 mg/l. An emergency CT-scan did not show intra-abdominal disorder: no abnormal air or fluid collection in the abdominal cavity and normal intra abdominal organs. But the subcutaneous fat of the anterior abdominal wall showed signs of inflammation without gas.The patient was started on piperacillin with tazobactam in combination with clindamycin and surgical exploration was performed on suspicion of NF. During the operation the fascia seemed intact, but the subcutaneous tissue, adjacent to the fascia had a discolored, greyish, avital aspect. Biopsies for cultures and histology of the tissue were taken and the wound was left open.Alewijn Ott, et al., Clinics in Surgery - Vascular SurgeryRemedy Publications LLC., | http://clinicsinsurgery.com/ 2018 | Volume 3 | Article 20112MicrobiologyBlood cultures taken on admission and culture of the tissue taken during surgery unfortunately remained negative. No bacteria were seen in the Gram stain of the tissue. Because of these negative findings and uncertainty about the best choice of antibiotics to continue the treatment we additionally examined the tissue with 16S-23S rDNANext Generation Sequencing [2]. This method is based on a PCR amplification of the 16S-23S rRNA region followed by amplicon sequencing on the MiSeq (Illumina, Inc., San Diego, CA). The resulting reads are denovo assembled into contigs. Species identification is based on an alignment of the contig sequences with the sequences deposited in the reference databases. Th is resulted in identification of Streptococcus pyogenes only, with a Relative Abundance (RA) of 99.7%. The RA indicates the relative percentage of reads that represents the same species in a sample.Figure 1: Redness of the lower abdomen of the patient before surgery. The line around the redness was drawn in the emergency department in order to indicate any increase.DiscussionThe best remedy for NF is early recognition, followed by surgery and antibiotic treatment focused upon the causative agent(s). Unfortunately, in some cases, these agents cannot be identified, as was the case in this patient. Most likely this was due to the high dose of intravenous antibiotic that was already given at the emergency department. The infection was in an early stage and therefore tissue circulation was still intact. Streptococcus pyogenes is usually highly sensitive to the penicillin class antibiotics. Knowing the causative bacteria enabled us to finish the treatment with oral amoxicillin after the patient recovered. 16S-23S rDNA NGS could exclude the alternative diagnosis in this patient of the polybacterialFournier NF, which requires a broader spectrum of antibiotics [3]. 16S-23S rDNA NGS can therefore contribute to rational antibiotic use.References1. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 2017;377:2253-65.2. Sabat AJ, van Zanten E, Akkerboom V, Wisselink G, van Slochteren K, de Boer RF, et al. Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species. Sci Rep. 2017;7(1):3434.3. Mallikarjuna MN, Vijayakumar A, Patil VS, Shivswamy BS. Fournier's Gangrene: Current Practices. ISRN Surg. 2012:942437.

ARTS repository - University of Groningen

   University of GroningenTracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesVierhout, Bastiaan; van Zanten, Evert; Wisselink , Guido ; Kooistra-Smid, Anna M. D.; Ott,AlewijnPublished in:Clinics in SurgeryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vierhout, B., van Zanten, E., Wisselink , G., Kooistra-Smid, A. M. D., & Ott, A. (2018). Tracking Down theCause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clinics in Surgery, 3, Article 2011.http://www.clinicsinsurgery.com/full-text/cis-v3-id2011.phpCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 11-09-2023Remedy Publications LLC., | http://clinicsinsurgery.com/Clinics in Surgery2018 | Volume 3 | Article 20111Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesOPEN ACCESS *Correspondence:Alewijn Ott, Department of Medical Microbiology, Certe, Groningen, Netherlands,E-mail: a.ott@certe.nlReceived Date: 25 Jun 2018Accepted Date: 09 Jul 2018Published Date: 12 Jul 2018Citation: Vierhout BP, van Zanten E, Wisselink G, Kooistra-Smid MAMD, Ott A. Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clin Surg. 2018; 3: 2011.Copyright © 2018 Alewijn Ott. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Case ReportPublished: 12 Jul, 2018AbstractIn a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection.Bas P Vierhout1, Evert van Zanten2, Guido Wisselink2, Mirjam AMD Kooistra-Smid2,3 and Alewijn Ott2*1Department of Surgery, Wilhelmina Ziekenhuis, Assen, Netherlands2Department of Medical Microbiology, Certe, Groningen, Netherlands3Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsBackgroundNecrotizing Fasciitis (NF) is a severe disease of sudden onset that spreads rapidly [1]. Symptoms include red skin, severe pain and fever. Risk factors include poor immune function and a break in the skin, for instance a wound. But also healthy persons may suffer from NF, without a visible entrance through the skin, mostly in the perineal region or the limbs.The disease is feared because of its high mortality and subsequent morbidity. The infection spreads rapidly over the fascia of the muscles and open surgical drainage is the only treatment option: Infected tissue must be removed without delay, in combination with intravenous antibiotics.Most often, the causative agent is a Streptococcus from Lancefield group A (S. pyogenes). If S. pyogenes is involved antibiotic treatment with a high dose of penicillin suffices. But combinations of antibiotics with a broader spectrum are required in infections in the perineal area, because these may be polymicrobial.Case PresentationA 68-year-old female patient complained of severe pain and localized redness since a few hours, spreading over the lower abdomen. Four days before, she had some localized pain on the right side of the abdomen, which gradually shifted to the left. Mobilizing aggravated the pain and made her nauseous, and as a result, she ate and drank less. She had no abdominal surgery or relevant infectious medical history in the past. Only bad teeth had been removed 6 years earlier, to replace them by prosthesis.At the emergency room she had a fever of 39.2°C, with a pulse of 107 per minute and a tension of 111/63 mmHg. The abdominal redness was moderately delimited and there was no swelling (Figure 1). Some bowel sounds were heard and the redness felt firmer than the surrounding tissue.Laboratory tests showed a leukocytosis of 44,000*106 per liter, with a left upper shift. A hyponatremia was accompanied by elevated urea and creatinine level of 23.3 mmol/l and 272 µmol/l, respectively. C-reactive protein was 639 mg/l. An emergency CT-scan did not show intra-abdominal disorder: no abnormal air or fluid collection in the abdominal cavity and normal intra abdominal organs. But the subcutaneous fat of the anterior abdominal wall showed signs of inflammation without gas.The patient was started on piperacillin with tazobactam in combination with clindamycin and surgical exploration was performed on suspicion of NF. During the operation the fascia seemed intact, but the subcutaneous tissue, adjacent to the fascia had a discolored, greyish, avital aspect. Biopsies for cultures and histology of the tissue were taken and the wound was left open.Alewijn Ott, et al., Clinics in Surgery - Vascular SurgeryRemedy Publications LLC., | http://clinicsinsurgery.com/ 2018 | Volume 3 | Article 20112MicrobiologyBlood cultures taken on admission and culture of the tissue taken during surgery unfortunately remained negative. No bacteria were seen in the Gram stain of the tissue. Because of these negative findings and uncertainty about the best choice of antibiotics to continue the treatment we additionally examined the tissue with 16S-23S rDNANext Generation Sequencing [2]. This method is based on a PCR amplification of the 16S-23S rRNA region followed by amplicon sequencing on the MiSeq (Illumina, Inc., San Diego, CA). The resulting reads are denovo assembled into contigs. Species identification is based on an alignment of the contig sequences with the sequences deposited in the reference databases. Th is resulted in identification of Streptococcus pyogenes only, with a Relative Abundance (RA) of 99.7%. The RA indicates the relative percentage of reads that represents the same species in a sample.Figure 1: Redness of the lower abdomen of the patient before surgery. The line around the redness was drawn in the emergency department in order to indicate any increase.DiscussionThe best remedy for NF is early recognition, followed by surgery and antibiotic treatment focused upon the causative agent(s). Unfortunately, in some cases, these agents cannot be identified, as was the case in this patient. Most likely this was due to the high dose of intravenous antibiotic that was already given at the emergency department. The infection was in an early stage and therefore tissue circulation was still intact. Streptococcus pyogenes is usually highly sensitive to the penicillin class antibiotics. Knowing the causative bacteria enabled us to finish the treatment with oral amoxicillin after the patient recovered. 16S-23S rDNA NGS could exclude the alternative diagnosis in this patient of the polybacterialFournier NF, which requires a broader spectrum of antibiotics [3]. 16S-23S rDNA NGS can therefore contribute to rational antibiotic use.References1. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 2017;377:2253-65.2. Sabat AJ, van Zanten E, Akkerboom V, Wisselink G, van Slochteren K, de Boer RF, et al. Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species. Sci Rep. 2017;7(1):3434.3. Mallikarjuna MN, Vijayakumar A, Patil VS, Shivswamy BS. Fournier's Gangrene: Current Practices. ISRN Surg. 2012:942437.

   University of GroningenTracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesVierhout, Bastiaan; van Zanten, Evert; Wisselink , Guido ; Kooistra-Smid, Anna M. D.; Ott,AlewijnPublished in:Clinics in SurgeryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vierhout, B., van Zanten, E., Wisselink , G., Kooistra-Smid, A. M. D., & Ott, A. (2018). Tracking Down theCause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clinics in Surgery, 3, [2011].http://www.clinicsinsurgery.com/full-text/cis-v3-id2011.phpCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 13-02-2023Remedy Publications LLC., | http://clinicsinsurgery.com/Clinics in Surgery2018 | Volume 3 | Article 20111Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesOPEN ACCESS *Correspondence:Alewijn Ott, Department of Medical Microbiology, Certe, Groningen, Netherlands,E-mail: a.ott@certe.nlReceived Date: 25 Jun 2018Accepted Date: 09 Jul 2018Published Date: 12 Jul 2018Citation: Vierhout BP, van Zanten E, Wisselink G, Kooistra-Smid MAMD, Ott A. Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clin Surg. 2018; 3: 2011.Copyright © 2018 Alewijn Ott. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Case ReportPublished: 12 Jul, 2018AbstractIn a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection.Bas P Vierhout1, Evert van Zanten2, Guido Wisselink2, Mirjam AMD Kooistra-Smid2,3 and Alewijn Ott2*1Department of Surgery, Wilhelmina Ziekenhuis, Assen, Netherlands2Department of Medical Microbiology, Certe, Groningen, Netherlands3Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsBackgroundNecrotizing Fasciitis (NF) is a severe disease of sudden onset that spreads rapidly [1]. Symptoms include red skin, severe pain and fever. Risk factors include poor immune function and a break in the skin, for instance a wound. But also healthy persons may suffer from NF, without a visible entrance through the skin, mostly in the perineal region or the limbs.The disease is feared because of its high mortality and subsequent morbidity. The infection spreads rapidly over the fascia of the muscles and open surgical drainage is the only treatment option: Infected tissue must be removed without delay, in combination with intravenous antibiotics.Most often, the causative agent is a Streptococcus from Lancefield group A (S. pyogenes). If S. pyogenes is involved antibiotic treatment with a high dose of penicillin suffices. But combinations of antibiotics with a broader spectrum are required in infections in the perineal area, because these may be polymicrobial.Case PresentationA 68-year-old female patient complained of severe pain and localized redness since a few hours, spreading over the lower abdomen. Four days before, she had some localized pain on the right side of the abdomen, which gradually shifted to the left. Mobilizing aggravated the pain and made her nauseous, and as a result, she ate and drank less. She had no abdominal surgery or relevant infectious medical history in the past. Only bad teeth had been removed 6 years earlier, to replace them by prosthesis.At the emergency room she had a fever of 39.2°C, with a pulse of 107 per minute and a tension of 111/63 mmHg. The abdominal redness was moderately delimited and there was no swelling (Figure 1). Some bowel sounds were heard and the redness felt firmer than the surrounding tissue.Laboratory tests showed a leukocytosis of 44,000*106 per liter, with a left upper shift. A hyponatremia was accompanied by elevated urea and creatinine level of 23.3 mmol/l and 272 µmol/l, respectively. C-reactive protein was 639 mg/l. An emergency CT-scan did not show intra-abdominal disorder: no abnormal air or fluid collection in the abdominal cavity and normal intra abdominal organs. But the subcutaneous fat of the anterior abdominal wall showed signs of inflammation without gas.The patient was started on piperacillin with tazobactam in combination with clindamycin and surgical exploration was performed on suspicion of NF. During the operation the fascia seemed intact, but the subcutaneous tissue, adjacent to the fascia had a discolored, greyish, avital aspect. Biopsies for cultures and histology of the tissue were taken and the wound was left open.Alewijn Ott, et al., Clinics in Surgery - Vascular SurgeryRemedy Publications LLC., | http://clinicsinsurgery.com/ 2018 | Volume 3 | Article 20112MicrobiologyBlood cultures taken on admission and culture of the tissue taken during surgery unfortunately remained negative. No bacteria were seen in the Gram stain of the tissue. Because of these negative findings and uncertainty about the best choice of antibiotics to continue the treatment we additionally examined the tissue with 16S-23S rDNANext Generation Sequencing [2]. This method is based on a PCR amplification of the 16S-23S rRNA region followed by amplicon sequencing on the MiSeq (Illumina, Inc., San Diego, CA). The resulting reads are denovo assembled into contigs. Species identification is based on an alignment of the contig sequences with the sequences deposited in the reference databases. Th is resulted in identification of Streptococcus pyogenes only, with a Relative Abundance (RA) of 99.7%. The RA indicates the relative percentage of reads that represents the same species in a sample.Figure 1: Redness of the lower abdomen of the patient before surgery. The line around the redness was drawn in the emergency department in order to indicate any increase.DiscussionThe best remedy for NF is early recognition, followed by surgery and antibiotic treatment focused upon the causative agent(s). Unfortunately, in some cases, these agents cannot be identified, as was the case in this patient. Most likely this was due to the high dose of intravenous antibiotic that was already given at the emergency department. The infection was in an early stage and therefore tissue circulation was still intact. Streptococcus pyogenes is usually highly sensitive to the penicillin class antibiotics. Knowing the causative bacteria enabled us to finish the treatment with oral amoxicillin after the patient recovered. 16S-23S rDNA NGS could exclude the alternative diagnosis in this patient of the polybacterialFournier NF, which requires a broader spectrum of antibiotics [3]. 16S-23S rDNA NGS can therefore contribute to rational antibiotic use.References1. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 2017;377:2253-65.2. Sabat AJ, van Zanten E, Akkerboom V, Wisselink G, van Slochteren K, de Boer RF, et al. Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species. Sci Rep. 2017;7(1):3434.3. Mallikarjuna MN, Vijayakumar A, Patil VS, Shivswamy BS. Fournier's Gangrene: Current Practices. ISRN Surg. 2012:942437.

   University of GroningenTracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesVierhout, Bastiaan; van Zanten, Evert; Wisselink , Guido ; Kooistra-Smid, Anna M. D.; Ott,AlewijnPublished in:Clinics in SurgeryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vierhout, B., van Zanten, E., Wisselink , G., Kooistra-Smid, A. M. D., & Ott, A. (2018). Tracking Down theCause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clinics in Surgery, 3, Article 2011.http://www.clinicsinsurgery.com/full-text/cis-v3-id2011.phpCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 31-10-2023Remedy Publications LLC., | http://clinicsinsurgery.com/Clinics in Surgery2018 | Volume 3 | Article 20111Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative CulturesOPEN ACCESS *Correspondence:Alewijn Ott, Department of Medical Microbiology, Certe, Groningen, Netherlands,E-mail: a.ott@certe.nlReceived Date: 25 Jun 2018Accepted Date: 09 Jul 2018Published Date: 12 Jul 2018Citation: Vierhout BP, van Zanten E, Wisselink G, Kooistra-Smid MAMD, Ott A. Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures. Clin Surg. 2018; 3: 2011.Copyright © 2018 Alewijn Ott. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Case ReportPublished: 12 Jul, 2018AbstractIn a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection.Bas P Vierhout1, Evert van Zanten2, Guido Wisselink2, Mirjam AMD Kooistra-Smid2,3 and Alewijn Ott2*1Department of Surgery, Wilhelmina Ziekenhuis, Assen, Netherlands2Department of Medical Microbiology, Certe, Groningen, Netherlands3Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsBackgroundNecrotizing Fasciitis (NF) is a severe disease of sudden onset that spreads rapidly [1]. Symptoms include red skin, severe pain and fever. Risk factors include poor immune function and a break in the skin, for instance a wound. But also healthy persons may suffer from NF, without a visible entrance through the skin, mostly in the perineal region or the limbs.The disease is feared because of its high mortality and subsequent morbidity. The infection spreads rapidly over the fascia of the muscles and open surgical drainage is the only treatment option: Infected tissue must be removed without delay, in combination with intravenous antibiotics.Most often, the causative agent is a Streptococcus from Lancefield group A (S. pyogenes). If S. pyogenes is involved antibiotic treatment with a high dose of penicillin suffices. But combinations of antibiotics with a broader spectrum are required in infections in the perineal area, because these may be polymicrobial.Case PresentationA 68-year-old female patient complained of severe pain and localized redness since a few hours, spreading over the lower abdomen. Four days before, she had some localized pain on the right side of the abdomen, which gradually shifted to the left. Mobilizing aggravated the pain and made her nauseous, and as a result, she ate and drank less. She had no abdominal surgery or relevant infectious medical history in the past. Only bad teeth had been removed 6 years earlier, to replace them by prosthesis.At the emergency room she had a fever of 39.2°C, with a pulse of 107 per minute and a tension of 111/63 mmHg. The abdominal redness was moderately delimited and there was no swelling (Figure 1). Some bowel sounds were heard and the redness felt firmer than the surrounding tissue.Laboratory tests showed a leukocytosis of 44,000*106 per liter, with a left upper shift. A hyponatremia was accompanied by elevated urea and creatinine level of 23.3 mmol/l and 272 µmol/l, respectively. C-reactive protein was 639 mg/l. An emergency CT-scan did not show intra-abdominal disorder: no abnormal air or fluid collection in the abdominal cavity and normal intra abdominal organs. But the subcutaneous fat of the anterior abdominal wall showed signs of inflammation without gas.The patient was started on piperacillin with tazobactam in combination with clindamycin and surgical exploration was performed on suspicion of NF. During the operation the fascia seemed intact, but the subcutaneous tissue, adjacent to the fascia had a discolored, greyish, avital aspect. Biopsies for cultures and histology of the tissue were taken and the wound was left open.Alewijn Ott, et al., Clinics in Surgery - Vascular SurgeryRemedy Publications LLC., | http://clinicsinsurgery.com/ 2018 | Volume 3 | Article 20112MicrobiologyBlood cultures taken on admission and culture of the tissue taken during surgery unfortunately remained negative. No bacteria were seen in the Gram stain of the tissue. Because of these negative findings and uncertainty about the best choice of antibiotics to continue the treatment we additionally examined the tissue with 16S-23S rDNANext Generation Sequencing [2]. This method is based on a PCR amplification of the 16S-23S rRNA region followed by amplicon sequencing on the MiSeq (Illumina, Inc., San Diego, CA). The resulting reads are denovo assembled into contigs. Species identification is based on an alignment of the contig sequences with the sequences deposited in the reference databases. Th is resulted in identification of Streptococcus pyogenes only, with a Relative Abundance (RA) of 99.7%. The RA indicates the relative percentage of reads that represents the same species in a sample.Figure 1: Redness of the lower abdomen of the patient before surgery. The line around the redness was drawn in the emergency department in order to indicate any increase.DiscussionThe best remedy for NF is early recognition, followed by surgery and antibiotic treatment focused upon the causative agent(s). Unfortunately, in some cases, these agents cannot be identified, as was the case in this patient. Most likely this was due to the high dose of intravenous antibiotic that was already given at the emergency department. The infection was in an early stage and therefore tissue circulation was still intact. Streptococcus pyogenes is usually highly sensitive to the penicillin class antibiotics. Knowing the causative bacteria enabled us to finish the treatment with oral amoxicillin after the patient recovered. 16S-23S rDNA NGS could exclude the alternative diagnosis in this patient of the polybacterialFournier NF, which requires a broader spectrum of antibiotics [3]. 16S-23S rDNA NGS can therefore contribute to rational antibiotic use.References1. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 2017;377:2253-65.2. Sabat AJ, van Zanten E, Akkerboom V, Wisselink G, van Slochteren K, de Boer RF, et al. Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species. Sci Rep. 2017;7(1):3434.3. Mallikarjuna MN, Vijayakumar A, Patil VS, Shivswamy BS. Fournier's Gangrene: Current Practices. ISRN Surg. 2012:942437.

Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures

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