Case Report: The first reported case of pulmonary alveolar proteinosis with myasthenia gravis in a 27-year-old patient

Background: Pulmonary alveolar proteinosis is a very rare diffuse lung disease characterized by the accumulation of amorphous and periodic acid Schiff-positive lipoproteinaceous material in the alveolar spaces due to impaired surfactant clearance by alveolar macrophages. Three main types were identified: Autoimmune, secondary and congenital. Pulmonary alveolar proteinosis has been previously reported to be associated with several systemic auto-immune diseases. Accordingly, we present the first case report of pulmonary alveolar proteinosis associated with myasthenia gravis. Case: A 27-year-old female patient, ex-smoker, developed a dyspnea on exertion in 2020. The chest X-ray detected diffuse symmetric alveolar opacities. Pulmonary infection was ruled out, particularly COVID-19 infection. The chest scan revealed the “crazy paving” pattern. The bronchoalveolar lavage showed a rosy liquid with granular acellular eosinophilic material Periodic acid-Schiff positive. According to the lung biopsy results, she was diagnosed with pulmonary alveolar proteinosis. The granulocyte macrophage colony-stimulating factor autoantibodies were negative. Nine months later, she was diagnosed with bulbar seronegative myasthenia gravis, confirmed with the electroneuromyography with repetitive nerve stimulation showing significant amplitude decrement of the trapezius and spinal muscles. She was treated with pyridostigmine, oral corticosteroids and azathioprine. Given the worsening respiratory condition of the patient, a bilateral whole lung lavage was performed with a partial resolution of symptoms. Thus, this previously unreported association was treated successfully with rituximab, including improvement of dyspnea, diplopia and muscle fatigability at six months of follow-up. Conclusions: This case emphasizes on the possible association of auto-immune disease to PAP, which could worsen the disease course, as the specific treatment does not exist yet. Hence, further studies are needed to establish clear-cut guidelines for PAP management, particularly when associated to auto-immune diseases.


Introduction
Pulmonary alveolar proteinosis (PAP), also known as pulmonary alveolar phospholipoproteinosis, is a very rare chronic diffuse lung disease. 1 It is characterized by the accumulation of amorphous and Periodic acid-Schiff (PAS)positive lipoproteinaceous material in the alveolar spaces due to impaired surfactant clearance by alveolar macrophages. 2owever, the underlying lung structure is preserved. 2The lipoproteinaceous material is principally composed of abnormal surfactant phospholipids and apoproteins. 1The accumulated substances filling the alveoli results in damaging gas exchange. 1The classic symptoms are dyspnea and hypoxemia, ultimately leading to respiratory failure and death. 1 There are three main types of PAP: Autoimmune (previously named primary or idiopathic), secondary and congenital. 1 PAP has been previously reported to be associated with several systemic auto-immune diseases. 2Accordingly, we present the first case report of PAP associated with myasthenia gravis (MG).

Case report
This case reported was presented according to the CARE guideline. 327-year-old, North African female patient, had a medical history of allergic rhinosinusitis and asthma.She is working as a web editor, mainly as a work from home employee.Her tobacco consumption amounted to one pack per day over two years.She had no particular exposure to toxics.On July 2020, she had an acute onset of respiratory symptoms, which consisted of productive cough, dyspnea on exertion and fever.She was, empirically, treated as a respiratory infection with antibiotics.One month later, she remained with a dyspnea occurring with effort.The physical examination and the laboratory findings were unremarkable.On laboratory investigations, complete blood count, c-reactive protein, erythrocyte sedimentation rate, creatinine level and lactate dehydrogenase level were all normal.The chest X-ray detected diffuse symmetric alveolar opacities.Pulmonary infection was ruled out, particularly Coronavirus disease 2019.
The chest scan revealed bilateral ground-glass peri-broncho-vascular opacities with interlobular and intralobular septal thickening, defining the "crazy paving" pattern (Figure 1).The bronchoalveolar lavage showed a rosy liquid with granular acellular eosinophilic material PAS positive.The lung biopsy confirmed the diagnosis of PAP showing alveoli filled with eosinophilic, acellular, granular and PAS-positive material containing foamy macrophages and some cholesterol crystals (Figure 2).At the time of diagnosis, the granulocyte macrophage colony-stimulating factor (GM-Figure 1. Bilateral diffuse ground-glass opacities with interlobular and intralobular septal thickening, forming the "crazy paving" pattern.

REVISED Amendments from Version 1
We paid a great attention to our reviewer report and recommendations.Accordingly, we made some minor changes to our case report, and we submitted a new version.
Mainly, we added a data availability statement reporting the CARE guidelines 2013 supporting this case report, and we added the CARE guidelines reference inside the paper in the references list.We changed some keywords.Apart from few details, there were no major differences overall between this version and the previously published version.
CSF) autoantibodies were not available.Lung function tests were normal, apart from a declined diffusing capacity of lung for carbon monoxide (<68% of the predicted value).Laboratory results, including quantitative immunoglobulins, proteins electrophoresis and autoantibody screening (antinuclear antibodies, rheumatoid factor, anti-cyclic citrulline peptides, anti-thyroglobulin antibodies, antithymocyte globulin, p-antineutrophil cytoplasmic antibodies, extractable nuclear antigen antibodies) were normal, apart from an elevated c-antineutrophil cytoplasmic antibodies (50 U/ml with a normal range below 10 U/ml) and anti-Mi-2 antibodies (310 U/L with a normal range of 32-294 U/L) without clinical signification.
Nine months after the onset, the patient presented with asthenia, muscle fatigability and right diplopia with worsening of symptoms later in the day.An electroneuromyography with repetitive nerve stimulation showed a significant amplitude decrement of the trapezius and spinal muscles.The patient, thus was diagnosed with bulbar MG.The acetylcholine receptor antibodies were negative.The patient was treated with pyridostigmine (60 mg pill, three times a day, for life), oral corticosteroids (prednisone 50 mg/day, once a day, for 5 weeks followed by progressive degression to 15 mg/day) and azathioprine (50 mg pill, twice a day, for 8 months).At that time, GM-CSF autoantibodies were negative.During the follow-up period, the patient's respiratory condition worsened.She presented with oxygen desaturation at the level of 70% after walking 100 meters during the six-min walk test.Lung function tests degraded further, with a severe alteration of alveolocapillary diffusion (diffusing capacity of lung for carbon monoxide at 33% of the predicted value).After excluding the main differential diagnosis, the patient underwent a whole lung lavage (WLL).The left lung was washed with 10 liters of saline (Figure 3).Six weeks later, the right lung was washed with 20 liters (Figure 4).There was a partial resolution of symptoms.Following a multidisciplinary discussion, the patient was treated with rituximab (two intravenous injections of 1,000 mg, 14 days apart), after 6 weeks of azathioprine washout.An improvement of dyspnea, diplopia and muscle fatigability was noted at six months of follow-up.

Discussion
This case report empathizes the association between PAP and MG, and the worsening of the disease course.PAP is an ultra-rare alveolar filling process with an estimated prevalence of 6.87 per million in the general population. 1 PAP was first described by Rosen in 1958 and back to 2009, only 500 cases were reported in the literature. 1,4PAP occurs mainly in men with a sex ratio of two and a typical age of 40 to 50 years old in adults, which is contrasting with our case. 2About 50 to 80% of patients with PAP have a smoking history, as it was reported in our patient. 2The symptoms are non-specific, subacute and mild, resulting often in delaying the diagnosis by months, even by years. 4The positive diagnosis of PAP is suggested by the chest scan findings with the classically known "crazy paving" pattern. 4Bronchoalveolar lavage and the transbronchial biopsy with the characteristic features, establish the diagnosis of PAP. 4 In the present case, PAP was confirmed by the pathological typical results of a lung surgical biopsy.Based on the pathogenic mechanism, PAP can be grouped into three types. 1Firstly, primary PAP as the most frequent form found in 95% of patients, is an autoimmune disease caused by elevated levels of the GM-CSF autoantibodies. 1 In our patient, the GM-CSF antibodies were negative.However, their assessment was conducted under corticosteroids and azathioprine.The autoimmune hypothesis, though, was not definitely ruled out, especially when an associated auto-immune disease appeared during the course of PAP.Secondly, secondary PAP, occurring in 5% of patients, results from alveolar macrophage dysfunction caused by immune dysregulation, hematopoietic disorders, environmental exposure and pharmaceutical agents. 1 In our case there was no other associated underlying illness or exposure, which eliminated a secondary PAP.Thirdly, congenital PAP occurs due to genetic variations, usually observed in children, which is not our case. 1 In the current case, the onset of symptoms began after a respiratory infection that was very likely the initial trigger causing probably abnormal response in surfactant uptake. 5In the literature, there have been several cases of PAP associated with systemic auto-immune diseases; such as hemolytic anemia, polymyalgia rheumatica, ulcerative colitis, granulomatous polyangiitis, systemic lupus erythematosus  and dermatomyositis. 5,6In this regard, our case is noteworthy as it demonstrates the first case in the literature of PAP and MG association.
Our patient had a confirmed seronegative MG and was undergoing treatment with corticosteroids and an immunosuppressant.Whether the MG or its treatment has any bearing on the progression of PAP is questionable.Indeed, the worsening condition of the patient after MG treatment, suggested that the immunosuppressant therapy could be the exacerbating factor of PAP.This hypothesis is plausible, as it was reported in the literature.In fact, three cases of patients with collagen disease developing autoimmune PAP during the immunosuppressant therapy were reported. 6Nagasawa et al., outlined the development of autoimmune PAP, in a patient previously diagnosed with systemic lupus erythematosus, under glucocorticoid therapy and its worsening under immunosuppressive therapy. 7As for the treatment of PAP, WLL is the standard of care. 1 However no randomized controlled trials have been reported on WLL due to the extreme rarity of PAP. 1 New therapeutic strategies for PAP have emerged, including GM-CSF, rituximab and plasmapheresis. 1ituximab is an anti-CD20, already used in several autoimmune diseases with a proven efficiency. 8A clinical trial with rituximab, conducted by Kavuru et al., included 10 patients with PAP, had shown promise in seven out of nine patients. 8n our case, a bilateral WLL was performed with a partial resolution of symptoms.Considering the exercise intolerance persistence after WLL and taking into account the negative level of GM-CSF antibodies; our patient was treated with rituximab, which resulted in promising outcomes.This result supports the auto-immune PAP hypothesis.Yet, for a follow-up period as short as six months, there was not enough hindsight to assess the long-term effectiveness of rituximab.
In conclusion, this case emphasizes the possible association between auto-immune diseases and PAP, which could worsen the disease course, as the specific treatment does not exist yet.Hence, further observational studies and randomized controlled trials are needed to establish clear-cut guidelines for PAP management, particularly when associated with auto-immune diseases.

Consent
Written informed consent for publication of her clinical details and clinical images was obtained from the patient.aPAP is the most common presentation of PAP, and in 90% of cases it presents with GM-CSF neutralizing antibodies that produce dysfunction of the alveolar macrophage, limiting the clearance of pulmonary surfactant, causing deficiencies in gas exchange 1 .In this case, the anti GM-CSF antibodies are negative, so the diagnosis of aPAP is made by ruling out, as it cannot be related to secondary or tertiary PAP (according to the authors).Sometimes, an infectious disease can trigger a subsequent autoimmune disease.
The authors present the case following the international "CARE" consensus at the request of reviewer 1.However, these guidelines recommend a detailed and objective description of the patient's situation before and after the therapeutic intervention.The authors talk about improvement or worsening of clinical parameters without showing the objective value (for example, they talk about "improvement in dyspnea" but not about how many points they improve on the mRCO dyspnea scale).
The authors should construct a table showing the clinical and/or functional evolution parameters of the disease from the diagnosis of aPAP, worsening after diagnosis of MG and performing whole lung lavage (WLL), after WLL (treatment of choice), new worsening and after of the administration of rituxumab (although these are early results, they are useful for the scientific community given the status of experimental therapy) Examples of these parameters are respiratory function tests (FVC, FFEV1, RV, DLCO, KCO), sixminute walk test (theoretical distance, HR minute 6, O2 saturation minute 6), gas exchange tests (alveolar-arterial oxygen gradient, blood gas analysis ), modification in dyspnea measured on the mMRC scale, the need for complementary oxygen therapy, etc… Radiological evolution (chest xray, HRCT) can also be useful (not essential, if not available comment as limitation).An example of a similar case where these concepts are addressed, which enrich the clinical case for the expert reader, has been recently presented by my research team (in case it serves as a guide) 1 .
It is also important to indicate whether adverse effects have appeared associated with any of the treatments.
The CARE guidelines also consider the patient's perspective.Is it possible to ask the patient how she feels?In the case discussed by my research team 1 , after adequate treatment with inhaled GM-CSF, a patient with aPAP told us "I take a deep breath and I notice that my chest fills with air to the bottom, not like before."Without further ado, I congratulate you for the great work and for sharing it with the scientific community.

Is the case presented with sufficient detail to be useful for other practitioners? Yes
Competing Interests: No competing interests were disclosed.

Valentin Coirier
Rennes University Hospital, Rennes, France I read with interest the case-report "the first reported case of pulmonary alveolar proteinosis with myasthenia gravis in a 27-year-old patient.
The text is well written, with a clear description of the case.We can find typical characteristics of the disease, without any doubt about the diagnosis.We can ask why the biopsy has been performed because chest scan was typical, as was liquid from BAL.But images are nice, and interesting to describe the case.
Authors have taken account of the comments from the first reviewing.

Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes
Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?Yes

Is the case presented with sufficient detail to be useful for other practitioners? Yes
Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 22 August 2023 https://doi.org/10.5256/f1000research.154600.r198480© 2023 Ben Saad H.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Helmi Ben Saad
Faculty of Medicine of Sousse, Laboratory of Physiology, University of Sousse, Sousse, Tunisia The authors have addressed all my remarks.Good job.

Helmi Ben Saad
Faculty of Medicine of Sousse, Laboratory of Physiology, University of Sousse, Sousse, Tunisia I read with a great interest the case report entitled "Case Report: The first reported case of pulmonary alveolar proteinosis with myasthenia gravis in a 27-year-old patient".
The case report is very interesting.However, some minor changes should be applied before the acceptance of the paper: POINT 1.I strongly recommend applying the CARE guideline 1

POINT 3. Key words
Please avoid citing as keywords some terms previously used in the title or the abstract.Please opt for MeSH terms and classify the keywords in alphabetical order.
POINT 4. Case report section Line 17 page 3: In the sentence "The physical examination and the laboratory findings were unremarkable": please name the laboratory tests that were performed.Line 27 page 3: can you add the numerical values (and normal range) of c-antineutrophil cytoplasmic antibodies and anti-Mi-2 antibodies?Last line of page 3: change the sentence "of 70% after a 6-min walk of 100 meters" by "of 70% after walking 100 meters during the 6-min walk test".

POINT 5. Discussion
The first sentence should be a reminder of the main result (to take home message from this case report) Page 5 line 7: why 2009 why not 2023?In other terms, how many cases were published in PubMed until 2023?Add some limitations of this case-report: for example "assessment under corticosteroids and azathioprine", other limitations.

POINT 6. Consent (page 6)
Change "Written informed consent for publication of their clinical details and clinical images was obtained from the patient" By "Written informed consent for publication of her clinical details and clinical images was obtained from the patient."

POINT 1:
We applied carefully CARE guideline: We re-consulted the CARE guidelines of 2013.

○
We submitted the CARE checklist (as an appendix).

○
We Wrote (in the beginning of the section Case report, page 3/7): "This case reported was presented according to the CARE guideline".We added the reference inside the paper in the references list.

○ POINT 2:
We deeply regret our inattention concerning abbreviations misuse.
Example 1: We defined our abbreviation PAP referring to Pulmonary alveolar proteinosis in the abstract.Example 2: We defined "COVID-19" by changing the term to "Coronavirus disease 2019" (without use the abbreviations).

○
We classified the keywords in alphabetical order.

POINT 4:
Line 17 page 3: In the sentence "The physical examination and the laboratory findings were unremarkable": We named the laboratory tests that were performed.Line 27 page 3: We added the numerical value (and normal range) of c-antineutrophil cytoplasmic antibodies and anti-Mi-2 antibodies.Last line of page 3: We changed the sentence "of 70% after a 6-min walk of 100 meters" by "of 70% after walking 100 meters during the 6-min walk test".

POINT 5:
We added a first sentence in the discussion part as a reminder of the main result (to take home message from this case report).
Page 5 line 7: In PubMed, there was no systematic review of pulmonary alveolar proteinosis until 2023.There was no article or review specifying the number of cases published in PubMed until 2023.
We added another limitation to our case report: The follow-up period was short (only 6 months).There was not enough hindsight to assess the long-term effectiveness of rituximab.

POINT 6:
We changed "Written informed consent for publication of their clinical details and clinical images was obtained from the patient" by "Written informed consent for publication of her clinical details and clinical images was obtained from the patient."

Figure 4 .
Figure 4. Alveolar liquid derived from whole lung lavage of the right lung (20 L) gradually turning clearer.

Figure 3 .
Figure 3. Alveolar liquid derived from whole lung lavage of the left lung (10 L) gradually turning clearer.

References 1 .
Oterino-Moreira I, Linares-Asensio MJ, Sanz-Márquez S, Pérez-Encinas M: Response to inhaled granulocyte-macrophage colony-stimulating factor in patient with mild-to-moderate autoimmune pulmonary alveolar proteinosis-24 months of follow-up.Clin Respir J. 2023; 17 (10): 1077-1081 PubMed Abstract | Publisher Full Text scientific standard, however I have significant reservations, as outlined above.Reviewer Report 13 May 2024 https://doi.org/10.5256/f1000research.154600.r199233© 2024 Coirier V.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1 Reviewer
Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?Yes Is the case presented with sufficient detail to be useful for other practitioners?Yes Competing Interests: No competing interests were disclosed.Reviewer Expertise: Physiology and pulmonary function I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Version Report 05 January 2023 https://doi.org/10.5256/f1000research.139794.r158819© 2023 Ben Saad H.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abbreviations misuse: all abbreviations should be explained the first time they are usedunless it is a standard unit of measurement -and thereafter the use of abbreviations should be consistent throughout the paper.
○ POINT 2. Example 1: Abstract Please abbreviate Pulmonary alveolar proteinosis as PAP at first use (1 st line of the abstract) and therefore use always PAP. Example 2: Abstract and text Define COVID-19 (and do not use the abbreviations COVID).