A man with culture sterile cavitating lung lesions: molecular techniques can provide the key to diagnosis

A 20-year-old male presented to the Emergency Department with pyrexia, dyspnoea, chest pain and haemoptysis. Cavitating lung lesions were noted on chest X-ray and the patient was admitted to the intensive care unit where he was intubated and ventilated. Routine investigations including serial cultures did not provide an aetiological diagnosis. As such, a CT-guided lung biopsy was carried out and 16S rDNA PCR was undertaken on the sample. This identified Fusobacterium necrophorum as the causative organism. The patient was treated for Lemierre’s syndrome and successfully discharged from hospital. This case highlights how DNA tissue typing on a lung biopsy sample can be the key to successful diagnosis in an atypical pneumonia and raises the question as to whether this infrequently used approach should be added to forthcoming community acquired pneumonia guidelines.

Arterial blood analysis revealed type one respiratory failure (pH 7.41, PaO 2 11, PaCO 2 4.8).He was admitted into the intensive care unit (ICU) due to the high oxygen requirements and he was commenced on oxygen supplementation with high flow nasal oxygen, requiring a FiO 2 of 70 % and flow rate of 50 Lpm in order to achieve adequate arterial oxygen saturation.Empirical antibiotics for presumed lower respiratory tract infection were commenced with a combination of piperacillin-tazobactam and metronidazole for broad spectrum coverage.
His chest X-ray showed multiple bilateral cavitary lesions, Fig. 1a.The differential diagnoses at this point were a lower respiratory tract infection possibly caused by Staphylococcus aureus or atypical organisms, pulmonary tuberculosis, or a non-infectious process such as metastatic pulmonary lesions, or a vasculitis.Testicular examination, to exclude a primary testicular tumour, was normal.No other features suggestive of a malignancy were noted on history or examination.
A CT scan of his thorax, abdomen and pelvis confirmed the presence of multiple lung lesions with early cavitation, Fig. 1b,  c.Bi-basal consolidation and collapse, bilateral pleural effusions, and hepatosplenomegaly were also noted.There was no mediastinal lymphadenopathy.A conclusive radiological diagnosis was not achieved thus a broad range of differentials were proffered; infective pathologies such as multifocal bacterial pneumonia, fungal pneumonia, and atypical tuberculosis, and autoimmune diseases such as autoimmune granulomatosis with polyangiitis.
A vasculitis screen and autoimmune serology were requested and returned as negative.HIV, acid fast bacilli smears, cultures, and viral hepatitis serology were also negative.Pneumococcal and Legionella urine antigens were negative.Serial blood culture samples returned as sterile.We carried out a bronchoalveolar lavage and sent the fluid sample for culture.This also returned sterile.At this point we considered infective endocarditis as one of the possible diagnoses, however no vegetations were noted on transthoracic echocardiogram.

DR RANVIR CHEEMA, ICU SHO
Despite treatment with antibiotics and oxygen supplementation, the patient's oxygen requirement and work of breathing continued to deteriorate.He was intubated on the second day of admission.His antimicrobial therapy was changed to piperacillin-tazobactam and linezolid, to cover the possibility of Panton-Valentine Leucocidin-producing Staphylococcus aureus.

DR PRABHJOYT KLER, ICU REGISTRAR
Intercostal drains for bilateral pleural effusions were inserted on his third day of admission.One litre of blood-stained fluid was drained from each side and pleural fluid analysis was suggestive of a transudate; cytology was negative for malignant cells and indicative of a parapneumonic effusion.
By the eighth day of admission, we still did not have a definitive diagnosis despite extensive investigations.We repeated a CT scan of the thorax and on the advice of the microbiologists, conducted a CT-guided lung biopsy.The CT scan showed progression of the lung lesions with further cavitations and persistent bilateral pleural effusions.Histology from the biopsy confirmed an acute inflammatory process in keeping with an abscess.The tissue culture also returned sterile.
With no clear diagnosis, a broad range 16S rDNA was requested on the pleural fluid; this was negative.

DR RANVIR CHEEMA, ICU SHO
Following improvements in respiratory parameters, he was successfully extubated on the eleventh day of admission and intercostal drains were removed.Inflammatory markers had not significantly improved (CRP 245 mg l −1 ) and he remained pyrexial, thus antibiotic therapy was escalated to meropenem, doxycycline and metronidazole.

DR PRABHJOYT KLER, ICU REGISTRAR
By the eighteenth day and under the guidance of the microbiologists, a 16S rDNA PCR was requested on the lung biopsy sample and this identified Fusobacterium necrophorum pointing towards a diagnosis of Lemierre's syndrome (LS).A subsequent bedside ultrasound scan of the neck showed a thrombus in the right internal jugular vein (IJV), consolidating the diagnosis.

DR JOANNA MACVE, MICROBIOLOGY CONSULTANT AND DR RANVIR CHEEMA, ICU SHO
Broad range 16S rDNA sequencing has become more available in the last two decades, with the widespread use of PCR and DNA sequencing.In the UK it is not available in most hospital microbiology laboratories, but samples can be sent to the reference laboratories for testing.It has particularly helped in rapidly and accurately identifying bacteria with unusual phenotypic profiles, that are rare, slow-growing or non-viable, causing culture-negative infections [1].The 16S ribosomal subunit is present in all bacteria and is encoded for by the 16S rDNA gene.It contains highly conserved regions, separated by variable sequences.Primers targeted at the conserved regions are used to amplify the bacterial genome, which then allows sequencing of the variable regions to be carried out.This method has a significant advantage over routine cultures, in that it can detect non-viable DNA, thus can be effectively used in patients who have commenced a prolonged course of antimicrobial therapy.It is being increasingly used for aetiological diagnosis in culture-negative infective endocarditis; Patel [2] found that this method can prevent delayed diagnosis of LS.

DR ALPHA MADU, ICU SHO, CT2 ACCS ACUTE MEDICINE
LS is a septic thromboembolic complication of bacterial pharyngotonsillitis that can be fatal if not treated early.It is classically defined as a triad of a deep-seated neck infection and septicaemia, thrombophlebitis of the IJV, and metastatic infections from septic emboli.It usually presents with sore throat and subsequent involvement of the IJV causing thrombophlebitis, culminating in a fulminant multi-systemic organ failure.Multi-organ involvement is usually present by the time of diagnosis [3].Joint involvement is reported to occur in 16-26 % of cases.
About two-thirds of LS cases are caused by Fusobacterium necrophorum, a non-spore forming obligate anaerobic Gram-negative bacilli that forms part of the normal flora of the upper respiratory tract in humans and animals.It invades the connective tissue of the pharynx and causes a localised septic thrombophlebitis in the tonsillar veins and IJV.Septic embolization from the IJV thrombophlebitis is responsible for the systemic manifestations of the disease and explains the joint swelling noted on admission.
Pulmonary involvement is the commonest systemic manifestation of the disease and may present as pleuritic chest pain, cough, dyspnoea, or haemoptysis, all of which were present in our patient.Around 10-15 % of patients develop an empyema [3].A striking feature of LS is the rapid progression of the lung lesions and pleural effusion despite antibiotic therapy as seen in the index case [3].Some patients develop acute respiratory distress syndrome (ARDS) with less than 10 % requiring mechanical ventilation [3].
LS is rare (one case per one million people per year) [3] hence its diagnosis is challenging.A positive culture helps confirm the diagnosis.Our case was particularly challenging as serial blood cultures as well as tissue and bronchoalveolar lavage (BAL) cultures were all negative.There have been multiple recent case reports of LS thought to be precipitated by COVID-19 infection [4][5][6]; it is possible that the COVID-19 infection may have provoked LS in this case.
The treatment of LS is with an extended course of antibiotics (minimum of 4 weeks), usually metronidazole and β-lactam antibiotics [3].Complications of LS occur as a result of the metastatic septic emboli.Mortality rate is as high as 90 % without antibiotic treatment and less than 20 % with early antibiotic therapy.Anticoagulation has not been demonstrated to reduce mortality or increase vessel recanalization [7], however in this case, due to the presence of the IJV thrombus, we chose to anticoagulate the patient with treatment dose low molecular weight heparin whilst he remained an inpatient and this was then converted to a direct oral anticoagulant on discharge.

DR JOANNA MACVE, CONSULTANT IN MICROBIOLOGY
This case demonstrates the utility of 16S rDNA PCR of tissue samples in aiding diagnosis of infections caused by bacteria that are fastidious or cannot be cultured, or where prior antibiotic use affects organism recovery.Currently the cost and availability mean that it can only be used selectively, but it is vital to keep it in mind when cultures are negative, but infection is strongly suspected.

DR MACDUFF, CONSULTANT IN INTENSIVE CARE AND RESPIRATORY MEDICINE
This was an interesting case of a young man with a multi-system illness presenting with acute respiratory failure.Initial treatment clearly focused on a possible infectious aetiology with broad spectrum antibiotics in line with national guidelines.National (BTS) [8] and international (ATS) [9] guidelines recommend identifying the causative organism if possible.However, here all standard microbiology investigations were unhelpful.Given the diagnostic uncertainty with the possibility of exotic infectious agents or of non-infectious processes (vasculitides, neoplasia) being missed, we proceeded to undertake a lung biopsy, an uncommon procedure in intensive care.
Histopathology was unhelpful and tissue culture was sterile.A definitive diagnosis was only possible by the astute request of 16S rDNA PCR on the sample by our microbiology colleagues.This shows the importance of a strong working relationship between critical care and our microbiology colleagues.We are fortunate to have managed to continue daily microbiology ward rounds during the COVID-19 pandemic and subsequently.
After discharge from the ICU to a respiratory ward, the patient received intravenous antibiotics for a total of 25 days and was discharged home after a 27 day hospital stay on a 3 month course of oral co-amoxiclav.

Reviewers' comments and responses to custom questions:
Comments to Author: Dear author, after reviewing your manuscript, I found some notes that needed revision.First, you must write the complete term for the first time mentioned (lines 21, 125).Second, the scientific name should be italicized (line 51).You didn't indicate figure 1  This case describes a gentleman requiring invasive ventilation for community acquired pneumonia with cavitation, in which 16S rDNA sequencing assisted in identification of Fusobacaterium necrophorum and subsequent diagnosis of Lemierre's syndrome.It is a short article that reads well as an evolving clinical case with discussion as it happens.It raises the important and interesting consideration of molecular testing in critical care respiratory infection, where there has been much progress but little practical bedside utility.

History
I was interested to read of the gentleman's Iranian origin (particularly that he was Kurdish), and his recent infection with COVID-19.However, there is no description of the time since COVID-19 infection or of its confirmation (e.g.PCR positivity) or clinical course (assuming he was not admitted) or overlap with symptoms.Please also ensure including whether any sore throat or neck swelling were present at onset.The nature of his chest pain (type, location, severity) and haemoptysis (description, frequency, volume) would be important to describe.Thank you for your comments.Unfortunately, due to the patient presenting to hospital in extremis, we were unable to ascertain all this information and this information has not been recorded in the case notes.The history of COVID-19 infection was reported by the patient and confirmed in collateral history from his friend, but it is unclear how the infection was confirmed, as such unfortunately we have had to leave this detail out.
I was surprised that there was no brief or speculation over the possibility that COVID-19 infection may have predisposed this gentleman to his infection.Some may even have included the post-COVID-19 status with prominence, even in the title -I do not recommend necessarily doing this but recommend at least describing the time lapse between infections and the absence of any PCR positivity for SARS-CoV-2.
Thank you, we have added this, of note there have been a few recent case reports that have suggested a link between the two.

Examination
The clinical assessment is concise.as well as (i) the oxygen delivery method and flow rate at each point (line 37 and line 42) (ii) presence of sinus rhythm (iii) presence of any confusion (iv) abdominal examination (v) presence of any rash, particularly since the possibility of vasculitis is raised (53) (vi) leg swelling, if known.The gentleman's sore finger is mentioned, but not elaborated on later (was this thought to represent a septic embolus?Did this resolve?).Please state the PaO2 and PaCO2 and pH at which this gentleman was diagnosed as being in respiratory failure (line 40).

Investigations
The differential diagnosis is listed similarly around line 50 and 59.Please consider concatenation.The notion of "ruling out" vegetations by TTE is technically incorrect (line 66), please consider rephrasing to say only that no vegetations were seen, since TOE would be required to effectively rule out from a clinical perspective.

Thank you, this change has now been made.
The methodology of the 16S rDNA PCR would be useful to know to ensure this paper's findings can be replicated and acted on.A "broad range" 16S rDNA was requested (line 81), but with what kit etc? How broad was the testing used (how many species)?Were any other organisms identified?Where there any other notable negatives or false positives?The 16S PCR was not done on site but instead it was sent away to the reference laboratory (we don't have 16S PCR on site).The reference lab was Bacterial Identification Services Laboratory (BIDS), UKHSA Colindale, London.On the report they don't specify what test they use.No other organisms were identified.

Management
I would be interested to know why was the left-sided effusion drained (line 73), given that the CT scan in particular only seems to show the right-sided element?Was the right-sided effusion larger?Was the left-sided effusion easier to drain?How much fluid was drained?How many days did the chest drain remain in for?
The effusions were drained to improve ventilation and in preparation for the CT guided lung biopsy.The case report has been updated with details of when the drains were removed (day 11) What was the reason for escalation of antibiotics on day eleven?(line 85).The article states that a minimum of 4 weeks antibiotics are essential (line 126), but that this patient only received a total of 25 days (149).Additionally, why were antibiotics IV for so long?The case report has been updated to include these details.

Diagnosis
The statement "enabling us to clinch the diagnosis of " sounds a little triumphant.Perhaps more appropriate to say "pointing towards the diagnosis of ".The authors raise the possibility of F. necrophorum being part of normal flora in humans and animals (cockatoos?There is no literature, apparently).There is no comment on the possibility that this is a false positive or potentially a representation of normal flora.There is no statement on the percentage of individuals that have identifiable 16S rDNA PCR in healthy/unwell patients, or those with cavitatory pneumonia.Please consider stating a specificity and sensitivity for the test, or whether these data are available, particularly in lining up the syndrome with the molecular identification.Metagenomic testing would be the next question.Would the authors see an advantage to having performed the test earlier, or to having a broader or more accurate set of tests?The argument made by the authors is that use of molecular testing "can prevent delayed diagnosis"; at what point would you recommend testing, then?Many advocate for PCR panels in diagnosis of respiratory infection (and commercial companies produce such tests).What would your recommendation to NICE be?In this case, there was no delayed management.
Thank you, the case report has been updated to address these points.
You recommend consideration of the 16S rRNA approach in "forthcoming BTS community acquired pneumonia guidelines" (line 20).However, my understanding is that BTS are not due to be improved (https://www.brit-thoracic.org.uk/quality-improvement/guidelines/) since NICE are in the process of development of guideline GID-NG10357 on Pneumonia in adults (https://www.nice.org.uk/guidance/indevelopment/gid-ng10357) and is due in 2024.You could mention that 16S rRNA is not mentioned in the guideline from 2014 (CG191), though this guideline is only 22 pages, and does not mention PCR or any other molecular techniques.However, the UK Standards for Microbiology Investigations (SMI) guideline B 57 does mention the use of PCR, especially for time sensitive investigations (https://www.gov.uk/government/publications/smi-b-57-investigation-of-bronchoalveolarlavage-sputum-and-associated-specimens).I recommend that the authors consider emailing the NICE guideline development team ( pneumoniaupdate@ nice.org.uk) to open discussion.Thank you for this valuable advice, we will get in touch with the NICE guideline development team, as per your recommendation.
Perhaps the identification of F. necrophorum led to the return to the bedside and repeat assessment and identification of the IJV thrombus, and this was the key step in improving management.It is notable that the original therapy included pip-taz.and metronidazole.This case demonstrates the power of anaerobic cover in treating an anaerobic organism at the outset; I wonder if this point should be emphasised, as this is the ultimate treatment decision that covered the infection.Thank you, this has now been included.
The presence of an IJV thrombus is arguably what "clinches" the diagnosis of the syndrome in question.So, why was an IJV thrombus not identified earlier?One would presume this gentleman had received more than one central venous catheter during his stay, for which the most likely site would have been the right IJV, where the thrombus was eventually identified (line 91).Perhaps the vascular ultrasound operator did not identify this, perhaps it was not present, perhaps there is a need for further training in venous thrombosis assessment?Please consider noting whether or this was detected earlier or not, with any additional opinions if necessary.
Yes, the patient had had multiple CVCs during his ITU stay but unfortunately an IJV thrombus had not been noted.Furthermore, I could not see a discussion or mention of anticoagulation for this patient.Did the authors anticoagulate?Was prophylactic low molecular weight heparin (as I assume he was on) enough in their opinion?Is there a lack of consensus on this point?Thank you for highlighting this important point.We have added a section on anticoagulation.
Finally, please consider whether the patient might be willing to give an opinion on their case and management, as per CARE case report guidelines (https://www.carestatement.org/checklist).Other suggestions from the CARE checklist are (i) fewer key words (I suggest abandoning Lemierre's syndrome, it gives the game away under the title) (ii) inclusion of "case report" in the key words (iii) removal of "BTS pneumonia guidelines" (iv) include dose and strength of antimicrobials (v) any long term post-ICU follow-up.Unfortunately the patient has not provided a personal report.
Please would you additionally address stylistic comments relating to the following lines: 1: "Dilemma" -Cambridge online dictionary defines a dilemma as a "a situation in which a difficult choice has to be made between two different things you could do."What dilemma are you referring to?I recommend considering the use of another title that indicates the case's main story: the major challenge of diagnosis without use of a molecular technique.16: Please consider use of "CT-guided lung biopsy" for clarity in the abstract to specify the methodology.28, 41, 45, 67, 72, 83, 87, 105, 135: Please consider standardising the term "ICU" or "ITU."I recommend using the term "ICU" exclusively and abandoning "ITU."I would also be interested to know whether the ICU SHO and registrar are core trainees in e.g.Internal Medicine, Anaesthesia, or whether they are a Specialty doctor or fellow, et cetera.Or perhaps a single CCT trainee in Intensive Care Medicine, et cetera.32: "pack year history": over what time period?33: "but denied any recent": please consider rephrasing e.g."and denied any other recent."38, 47 "54mmHg": please have a space between numbers and units.50 "multiple, bilateral": please remove comma.
71 "Panton Valentine Leucocidin Staphylococcus aureus": please phrase as in other literature as "Panton-Valentine Leucocidinproducing Staphylococcus aureus".Thank you, these changes have been made.

Reviewer 3
Comments to Author 1.
Description of the case(s): The authors described an interesting case of infection with Fusobacterium necrophorus but serial culture remained negative with multiple tissue samples (blood, bronchoalveolar lavage, lung biopsy) for at least 18 days amer admission in a urban hospital in the UK.The final diagnosis of Lemierre's syndrome was made amer the bacterium was identified by 16S rRNA gene sequencing before the patient was treated with appropriate antibiotics successfully and was discharged home amer a 27-day hospital stay.Thank you for your kind comments.

2.
Presentation of results: The detail and clear description of the case was presented by the corresponding physicians and the microbiologist.The X-ray and CT scan slides in Fig 1 demonstrated cavitary lesions in the lungs.The result of 16S-rRNA gene sequencing was presented.
Thank you.

3.
How the style and organization of the paper: communicates and represents key findings The manuscript was descriptive in discussion style, and is easy to be comprehensive to clinical community.

4.
Literature analysis or discussion: Literature analysis and discussion were complete and appropriate.

5.
Any other relevant comments: It might be more meaningful to include the discussion of how to make timely diagnosis in the future for such rare infections.Thank you, we have tried to elaborate on this in the revised manuscript.The authors described an interesting case of infection with Fusobacterium necrophorus but serial culture remained negative with multiple tissue samples (blood, bronchoalveolar lavage, lung biopsy) for at least 18 days after admission in a urband hospital in the UK.The final diagnosis of Lemierre's syndrome was made after the bacterium was identified by 16S rRNA gene sequencing before the patient was treated with appropriate antibiotics successfully and was discharged home after a 27-day hospital stay.2. Presentation of results The detail and clear description of the case was presented by the corresponding physicians and the microbiologist.The X-ray and CT scan slides in Fig 1 demonstrated cavitary lesions in the lungs.The result of 16S-rRNA gene sequencing was presented.3. How the style and organization of the paper communicates and represents key findings The manuscript was descriptive in discussion style, and is easy to be conprehensive to clinical community.4. Literature analysis or discussion Literature analysis and discussion were complete and appropriate.5. Any other relevant comments It might be more meaningful to include the discussion of how to make timely diagnosis in the future for such rare infections.

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Yes
Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000510.v1.3 © 2022 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Anonymous.
Date report received: 15 December 2022 Recommendation: Minor Amendment Comments: Broad Summary This case describes a gentleman requiring invasive ventilation for community acquired pneumonia with cavitation, in which 16S rDNA sequencing assisted in identification of Fusobacaterium necrophorum and subsequent diagnosis of Lemierre's syndrome.It is a short article that reads well as an evolving clinical case with discussion as it happens.It raises the important and interesting consideration of molecular testing in critical care respiratory infection, where there has been much progress but little practical bedside utility.History I was interested to read of the gentleman's Iranian origin (particularly that he was Kurdish), and his recent infection with COVID-19.However, there is no description of the time since COVID-19 infection or of its confirmation (e.g.PCR positivity) or clinical course (assuming he was not admitted) or overlap with symptoms.Please also ensure including whether any sore throat or neck swelling were present at onset.The nature of his chest pain (type, location, severity) and haemoptysis (description, frequency, volume) would be important to describe.I was surprised that there was no brief or speculation over the possibility that COVID-19 infection may have predisposed this gentleman to his infection.Some may even have included the post-COVID-19 status with prominence, even in the title -I do not recommend necessarily doing this but recommend at least describing the time lapse between infections and the absence of any PCR positivity for SARS-CoV-2.Examination The clinical assessment is concise.as well as (i) the oxygen delivery method and flow rate at each point (line 37 and line 42) (ii) presence of sinus rhythm (iii) presence of any confusion (iv) abdominal examination (v) presence of any rash, particularly since the possibility of vasculitis is raised (53) (vi) leg swelling, if known.The gentleman's sore finger is mentioned, but not elaborated on later (was this thought to represent a septic embolus?Did this resolve?).Please state the PaO2 and PaCO2 and pH at which this gentleman was diagnosed as being in respiratory failure (line 40).Investigations The differential diagnosis is listed similarly around line 50 and 59.Please consider concatenation.The notion of "ruling out" vegetations by TTE is technically (line 66), please consider rephrasing to say only that no vegetations were seen, since TOE would be required to effectively rule out from a clinical perspective.The methodology of the 16S rDNA PCR would be useful to know to ensure this paper's findings can be replicated and acted on.A "broad range" 16S rDNA was requested (line 81), but with what kit etc? How broad was the testing used (how many species)?Were any other organisms identified?Where there any other notable negatives or false positives?Management I would be interested to know why was the left-sided effusion drained (line 73), given that the CT scan in particular only seems to show the right-sided element?Was the right-sided effusion larger?Was the left-sided effusion easier to drain?How much fluid was drained?How many days did the chest drain remain in for?What was the reason for escalation of antibiotics on day eleven?(line 85).The article states that a minimum of 4 weeks antibiotics are essential (line 126), but that this patient only received a total of 25 days (149).Additionally, why were antibiotics IV for so long?Diagnosis The statement "enabling us to clinch the diagnosis of " sounds a little triumphant.Perhaps more appropriate to say "pointing towards the diagnosis of ".The authors raise the possibility of F. necrophorum being part of normal flora in humans and animals (cockatoos?There is no literature, apparently).There is no comment on the possibility that this is a false positive or potentially a representation of normal flora.There is no statement on the percentage of individuals that have identifiable 16S rDNA PCR in healthy/unwell patients, or those with cavitatory pneumonia.Please consider stating a specificity and sensitivity for the test, or whether these data are available, particularly in lining up the syndrome with the molecular identification.Metagenomic testing would be the next question.Would the authors see an advantage to having performed the test earlier, or to having a broader or more accurate set of tests?The argument made by the authors is that use of molecular testing "can prevent delayed diagnosis"; at what point would you recommend testing, then?Many advocate for PCR panels in diagnosis of respiratory infection (and commercial companies produce such tests).What would your recommendation to NICE be?In this case, there was no delayed management.You recommend consideration of the 16S rRNA approach in "forthcoming BTS community acquired pneumonia guidelines" (line 20).However, my understanding is that BTS are not due to be improved (https://www.brit-thoracic.org.uk/ quality-improvement/ guidelines/) since NICE are in the process of development of guideline GID-NG10357 on Pneumonia in adults (https:// www.nice.org.uk/ guidance/ indevelopment/ gid-ng10357) and is due in 2024.You could mention that 16S rRNA is not mentioned in the guideline from 2014 (CG191), though this guideline is only 22 pages, and does not mention PCR or any other molecular techniques.However, the UK Standards for Microbiology Investigations (SMI) guideline B 57 does mention the use of PCR, especially for time sensitive investigations (https://www.gov.uk/ government/ publications/ smi-b-57-investigation-of-bronchoalveolar-lavage-sputum-and-associated-specimens).I recommend that the authors consider emailing the NICE guideline development team ( pneumoniaupdate@ nice.org.uk) to open discussion.Perhaps the identification of F. necrophorum led to the return to the bedside and repeat assessment and identification of the IJV thrombus, and this was the key step in improving management.It is notable that the original therapy included pip-taz.and metronidazole.This case demonstrates the power of anaerobic cover in treating an anaerobic organism at the outset; I wonder if this point should be emphasised, as this is the ultimate treatment decision that covered the infection.The presence of an IJV thrombus is arguably what "clinches" the diagnosis of the syndrome in question.So, why was an IJV thrombus not identified earlier?One would presume this gentleman had received more than one central venous catheter during his stay, for which the most likely site would have been the right IJV, where the thrombus was eventually identified (line 91).Perhaps the vascular ultrasound operator did not identify this, perhaps it was not present, perhaps there is a need for further training in venous thrombosis assessment?Please consider noting whether or this was detected earlier or not, with any additional opinions if necessary.Furthermore, I could not see a discussion or mention of anticoagulation for this patient.Did the authors anticoagulate?Was prophylactic low molecular weight heparin (as I assume he was on) enough in their opinion?Is there a lack of consensus on this point?Finally, please consider whether the patient might be willing to give an opinion on their case and management, as per CARE case report guidelines (https://www.care-statement.org/ checklist).Other suggestions from the CARE checklist are (i) fewer key words (I suggest abandoning Lemierre's syndrome, it gives the game away under the title) (ii) inclusion of "case report" in the key words (iii) removal of "BTS pneumonia guidelines" (iv) include dose and strength of antimicrobials (v) any long term post-ICU follow-up.Please would you additionally address stylistic comments relating to the following lines: 1: "Dilemma" -Cambridge online dictionary defines a dilemma as a "a situation in which a difficult choice has to be made between two different things you could do."What dilemma are you referring to?I recommend considering the use of another title that indicates the case's main story: the major challenge of diagnosis without use of a molecular technique.16: Please consider use of "CT-guided lung biopsy" for clarity in the abstract to specify the methodology.28, 41, 45, 67, 72, 83, 87, 105, 135: Please consider standardising the term "ICU" or "ITU."I recommend using the term "ICU" exclusively and abandoning "ITU."I would also be interested to know whether the ICU SHO and registrar are core trainees in e.g.Internal Medicine, Anaesthesia, or whether they are a Specialty doctor or fellow, et cetera.Or perhaps a single CCT trainee in Intensive Care Medicine, et cetera.32: "pack year history": over what time period?33: "but denied any recent": please consider rephrasing e.g."and denied any other recent."38, 47 "54mmHg": please have a space between numbers and units.50 "multiple, bilateral": please remove comma.51 "Staphylococcus aureus": please italicise.71 "Panton Valentine Leucocidin Staphylococcus aureus": please phrase as in other literature as "Panton-Valentine Leucocidin-producing Staphylococcus aureus".
Please rate the quality of the presentation and structure of the manuscript Good

Fig. 1 .
Fig. 1.Chest X-ray (a) and axial CT slices (b and c) demonstrating multiple cavitary lesions present in both lungs.
in the manuscript text; it should display in the correct position.Thank you, these changes have been made.Please rate the quality of the presentation and structure of the manuscript Reviewer 2: Good To what extent are the conclusions supported by the data?Reviewer 2: Partially support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?Reviewer 2: No: If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Reviewer 2: Yes: Reviewer 2 Comments to Author: Broad Summary Please rate the quality of the presentation and structure of the manuscript Good To what extent are the conclusions supported by the data?Strongly support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?No Is there a potential financial or other conflict of interest between yourself and the author(s)?No If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Thank you for your peer review.Jason Chen; Columbia University, Pathology & Cell Biology, Columbia University, 650 West 168th Street, P&S 12-403, New York, UNITED STATES https://orcid.org/0000-0001-7535-6341Date report received: 31 March 2023 Recommendation: Minor Amendment Comments: 1. Description of the case(s)

Reviewer 2
recommendation and comments https://doi.org/10.1099/acmi.0.000510.v1.4 2023 Abbas A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Ali Abbas; University of Baghdad Al-Jaderyia Campus College of Science, Tropical-Biological Research Unit, Jadiryia St., Baghdad, IRAQ https://orcid.org/0000-0001-5314-3193Date report received: 17 February 2023 Recommendation: Accept Comments: Dear author, after reviewing your manuscript, I found some notes that needed revision.First, you must write the complete term for the first time mentioned (lines 21, 125).Second, the scientific name should be italicized (line 51).You didn't indicate figure 1 in the manuscript text; it should display in the correct position.