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. 2023 Jun 5;17(6):e0011383. doi: 10.1371/journal.pntd.0011383

Evidencing leprosy neuronal inflammation by 18-Fluoro-deoxy-glucose

Patricia Sola Penna 1,*, Sergio Augusto Lopes De Souza 2, Paulo Gustavo Limeira Nobre De Lacerda 2, Izabela Jardim Rodrigues Pitta 3, Clarissa Neves Spitz 1,3,4, Anna Maria Sales 3, Flavio Alves Lara 5, Ana Caroline Siquara De Souza 1,3, Euzenir Nunes Sarno 3, Roberta Olmo Pinheiro 3,6,7, Marcia Rodrigues Jardim 1,3,4,6,7
Editor: Linda B Adams8
PMCID: PMC10270593  PMID: 37276237

Abstract

Background

Leprosy is caused by multiple interactions between Mycobacterium leprae (M. leprae) and the host’s peripheral nerve cells. M. leprae primarily invades Schwann cells, causing nerve damage and consequent development of disabilities. Despite its long history, the pathophysiological mechanisms of nerve damage in the lepromatous pole of leprosy remain poorly understood. This study used the findings of 18F-FDG PET/CT on the peripheral nerves of eight lepromatous patients to evaluate the degree of glucose uptake by peripheral nerves and compared them with clinical, electrophysiological, and histopathological evaluations.

Methods

Eight patients with lepromatous leprosy were included in this study. Six patients were evaluated up to three months after leprosy diagnosis using neurological examination, nerve conduction study, 18F-FDG PET/CT, and nerve biopsy. Two others were evaluated during an episode of acute neuritis, with clinical, neurophysiological, and PET-CT examinations to compare the images with the first six.

Results

Initially, six patients already had signs of peripheral nerve injury, regardless of symptoms; however, they did not present with signs of neuritis, and there was little or no uptake of 18F-FDG in the clinically and electrophysiologically affected nerves. Two patients with signs of acute neuritis had 18F-FDG uptake in the affected nerves.

Conclusions

18F-FDG uptake correlates with clinical neuritis in lepromatous leprosy patients but not in silent neuritis patients. 18F-FDG PET-CT could be a useful tool to confirm neuritis, especially in cases that are difficult to diagnose, such as for the differential diagnosis between a new episode of neuritis and chronic neuropathy.

Author summary

The lepromatous pole (LL) is the prototype of leprosy patients, contributing to its stigma. Nerve function impairment in LL patients may be clinically asymptomatic for an extended period, and despite its long history, lepromatous leprosy nerve damage pathophysiological mechanisms remain poorly understood. Tissues involved in infectious and inflammatory diseases are hypermetabolic and have increased glucose uptake by 18F-FDG. Our question was about how the uptake of this radiopharmaceutical would be in LL patients’ silent neuritis.

Our data demonstrated, for the first time, systemic peripheral nerve 18F-FDG incorporation using PET/CT whole-body images of a group of lepromatous patients with silent neuritis and acute neuritis. 18F-FDG PET-CT could be a useful tool to confirm neuritis, especially in cases that are difficult to diagnose, such as for the differential diagnosis between a new episode of neuritis and chronic neuropathy.

Introduction

Lepromatous leprosy (LL) is characterized by scattered skin lesions, which are more frequent in the cooler areas of the body with high bacterial load due to the host’s modest or absent cell-mediated immunity against the infectious agent Mycobacterium leprae (M. leprae) [1]. Peripheral neuropathy in patients with LL is characterized by the uninhibited multiplication of bacteria, predominantly in Schwann cells (SC), due to a series of immune and metabolic subversions of host cells imposed by the pathogen [2,3], which will, in the last instance, cause leprosy neuropathy. Nerve function impairment in patients with LL may be clinically asymptomatic for an extended period, even if microscopically involved, and may present clinical and electrophysiological evidence of extensive peripheral nerve damage from the onset of infection [4].

This condition of asymptomatic or oligosymptomatic sensory or sensory-motor nerve dysfunction that evolves indolently and is common in patients with LL is called silent neuritis [5,6]. Some authors reported that, in terms of nerve conduction studies, LL patients’ overall condition worsened, and abnormalities persisted despite improvements in skin lesions following multidrug therapy (MDT), even in patients without evident neuritis [4,7,8] or in those receiving corticosteroids [8]. Experimental in vivo and in vitro studies have shown that M. leprae invades SC early and can reprogram host gene expression [9].

Medeiros et al. (2016) evaluated in vitro and in vivo models of Schwann cells infected with M. leprae. They concluded that the bacillus alters the metabolism of Schwann cells, reducing mitochondrial activity and increasing glucose uptake by up to 50% [3]. Recently, this anaerobic shift of infected Schwann cells was found to be related to lipid accumulation, M. leprae’s evasion of the innate immune response, and axonal loss of function [10].

18-Fluoro-deoxy-glucose (18F-FDG) is a well-known radiopharmaceutical used in Positron Emission Tomography (PET/CT) that mimics glucose in the cell’s entry mechanisms, using glucose transposing proteins (Glut), just like non-radioactive glucose. Tissues involved in infectious and inflammatory diseases, with high expression of neutrophils and activated macrophages, demonstrate increased glucose uptake and 18F-FDG. Roy et al. (2018) and Shao et al. (2020) described case reports of acute neuritis in leprosy patients detected using 18F-FDG PET/CT [11,12].

Herein, we describe systemic peripheral nerve 18F-FDG incorporation using PET/CT whole-body images of eight lepromatous patients, correlating the degree of peripheral nerve glucose uptake with clinical, electrophysiological, and histopathological data.

This research study aimed to evaluate the extent of infection/inflammation and infer possible injury mechanisms in the peripheral nerves of LL patients with silent neuritis using an imaging method that mimics the entry of glucose into inflamed tissues.

Methods

Ethics statements

The research was conducted in compliance with the international compilation of human research standards, which was previously approved by the Ethics Committee of the Oswaldo Cruz Foundation (Approval number:3.152.162). All the patients provided written informed consent.

Patient selection

This is a case series of eight patients evaluated by the neurology team from the Souza Araújo Outpatient Clinic of the Leprosy Laboratory at the Oswaldo Cruz Foundation (Fiocruz) in Rio de Janeiro, Brazil.

From February 2019 to May 2022, 42 patients with the LL leprosy were admitted to the Souza Araújo outpatient clinic. Among these, 34 were excluded from the study: nine due to reactions related to leprosy; 10 had comorbidities associated with peripheral neuropathy, such as diabetes mellitus (eight patients), chronic alcoholism (one patient), and hypothyroidism (one patient); 12 patients did not accept to participate in the study; one was less than 18 years of age; one was pregnant; one was a case of recurrent leprosy.

Eight patients participated in the study; six were evaluated at the beginning of treatment and up to three months after diagnosis. The remaining two were assessed during clinical follow-up for episodes of acute neuritis.

Clinical and electrophysiological evaluation

All patients were diagnosed with the LL form of leprosy according to the Ridley and Jopling Classification based on the results of their slit-skin smears and skin histopathology. Patients underwent clinical examinations for leprosy diagnosis according to the protocol of the Leprosy Outpatient Unit of the Oswaldo Cruz Institute [13].

Six patients (cases 1–6) were in the initial phase of treatment. Patient 7 had already completed the MDT; he was submitted to neurological and nerve conduction evaluation at diagnosis but had been presenting Erythema Nodosum Leprosum (ENL) reactions since the beginning of the treatment, so it was initially excluded; furthermore, he developed acute neuritis two years after admission to the Souza Araújo Outpatient Clinic and was included for 18F-FDG PET/CT comparison. Patient 8 underwent treatment for six months and underwent a neurological examination one month following diagnosis; however, he did not accept participating in the study then. Six months later, when the patient experienced an acute neuritis episode, he was included in the 18F-FDG PET/CT evaluation.

Neurological examinations and nerve conduction studies were performed according to the protocol of the Leprosy Outpatient Unit of the Oswaldo Cruz Institute, published elsewhere [14]. The disability grade was recorded using the standard WHO grading criteria [15]. Nerve function impairment was defined as clinically detectable impairment of motor, sensory, and autonomic functions [16].

To evaluate the extent of nerve involvement, neuropathy was classified according to the number of impaired nerves and distribution of impairment in nerve conduction. Polyneuropathy was defined as the presence of diffused symmetrical peripheral nerve lesions. In addition, patients were diagnosed with mononeuropathy when a single nerve was affected and with multiple mononeuropathies when there was focal involvement of two or more nerves [17].

Based on the compound muscle and sensory nerve action potentials, the nerve segment lesion pathophysiology categories were defined by combining nerve conduction study parameters. In short, an axonal lesion was defined as either an isolated reduction in amplitude ≥ 30% of the reference values or a combined change in amplitude reduction of less than 30% associated with a reduction in conduction velocity of up to 60–75% of the reference values. Demyelination was verified as a ≥ 20% increase in latency, a > 35% reduction in conduction velocity, or a combined decrease in amplitude of up to 20% together with a 15–20% increased latency. Demyelinating lesions with axonal degeneration were determined by axonal and demyelinating lesions within the same nerve. A lesion was considered “no conduction” when action potentials could not be recorded [4].

PET/CT imaging evaluation

PET/CT was performed at the Nuclear Medicine Service of Clementino Fraga Filho University Hospital of the Federal University of Rio de Janeiro. All patients underwent examination following a 24 h low carb diet. After 60 min of intravenous administration of 0.14 mCi/kg of 18 F-fluorodeoxyglucose, the whole-body PET/CT examination was performed, and sequential images of computed tomography (16-channel CT) without contrast and PET were obtained, with the area of interest being a strip that extended from the cranial cap to the root of the thighs. In addition, the second group of images was acquired from the lower limbs. All images were reviewed in the transaxial, coronal, and sagittal planes and semi-quantitative analysis was performed using the target maximum standardized capture value (t-SUV max), considering the patient’s body mass as an index, and compared with the background SUVmax (bg-SUVmax).

Histopathological evaluation

The biopsied nerve samples were analyzed at the Leprosy Laboratory of Oswaldo Cruz Institute in Rio de Janeiro, Brazil. The sensory nerve was biopsied according to clinical and electrophysiological findings in six patients evaluated at the beginning of treatment (cases 1–6), not necessarily the same ones that showed 18F-FDG uptake. Furthermore, the following nerves were biopsied: dorsal cutaneous ulnar nerve on the dorsum of the hand (n = 4) and sural nerve at the ankle level (n = 2). Nerve samples were analyzed according to standard methods [18].

Results

Table 1 summarizes the demographic and clinical characteristics of the eight patients. Seven patients were male, and one was female; the ages ranged from 21 to 70 years (mean age, 42.6 years). The bacilloscopic index (BI) ranged from 3.5–5.75 (mean 5.08).

Table 1. Clinical characteristics of recruited patients.

Patient BI Symp on (y) Pain Par Thick Tact Ther Painf Motor Neuritis
1 5 No No No No No No No No No
2 5.5 2 Yes Yes Yes Yes Yes Yes Yes No
3 5.25 No No No Yes No Yes Yes No No
4 5.75 3 No Yes No Yes Yes Yes No No
5 3.5 2 Yes Yes No Yes Yes Yes Yes No
6 5.5 No info Yes Yes Yes Yes Yes Yes Yes No
7 5.5 No info Yes Yes Yes Yes Yes Yes Yes Yes
8 5 0 Yes No Yes Yes Yes Yes No Yes
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Abbreviations: BI, baciloscopic Index; Symp on (y), referred neural symptoms in years; No info, not informed; Par, Paresthesia; Thick—Thickening Tact, Tactile impairment; Ther, Thermal impairment; Painf, painful impairment; Motor, Motor impairment.

On neurological examination, six out of eight (75%) patients had symptoms of peripheral nerve damage (pain and paresthesia), two (25%) reported the onset of nerve symptoms before the dermatological injury (cases 4 and 7), two (25%) reported nerve symptoms and dermatological injury at the same time (cases 5 and 6), and two (25%) reported the onset of neurological symptoms after the dermatological lesions (cases 2 and 8). Patients 1 and 3 were asymptomatic at the time of examination.

Six patients evaluated at the beginning of MDT presented signs of peripheral nerve damage, regardless of symptoms

Of the six patients evaluated at the beginning of treatment (cases 1–6), four (66.6%) had symptoms related to peripheral nerve damage; paresthesia was the most common (66.6%). Neurological examination showed signs of peripheral nerve damage in five patients (83.3%). Thickening was observed in three of six patients (50%), sensory impairment was observed in five patients (83.3%), and motor impairment was observed in three patients (50%) (Table 1).

Table 2 summarizes the correlation between the eight patients’ clinical, electrophysiological, and PET/CT uptake findings.

Table 2. Correlation between clinical findings, NCS findings and PET uptake.

Clinical NCS t-SUV max/bg-SUV max Clinical NCS t-SUV max/bg-SUV max Clinical NCS t-SUV max/bg-SUV max
Patient 1 2 3
Nerve
R Median normal axonal no activ abnormal 0 no activ normal axonal no activ
R Radial normal normal no activ abnormal 0 no activ normal axonal no activ
R Ulnar normal axonal no activ abnormal demyel no activ abnormal axonal no activ
R Sural normal 0 no activ abnormal 0 no activ normal 0 no activ
R Peroneal normal axonal no activ abnormal 0 no activ normal normal no activ
L Median normal axonal no activ abnormal demyel no activ normal axonal no activ
L Radial normal normal no activ abnormal 0 no activ normal axonal no activ
L Ulnar normal axonal no activ abnormal demyel no activ normal axonal no activ
L Sural normal axonal no activ abnormal # no activ normal normal no activ
L Peroneal normal axonal no activ abnormal # 3.6/1.0 normal normal no activ
Patient 4 5 6
Nerve
R Median abnormal 0 no activ abnormal 0 no activ abnormal axonal no activ
R Radial abnormal 0 no activ abnormal 0 no activ abnormal 0 no activ
R Ulnar abnormal dem/ax.deg no activ abnormal dem/ax.deg no activ abnormal axonal 2.9/1.1
R Sural abnormal 0 no activ # # no activ abnormal 0 no activ
R Peroneal abnormal 0 no activ # # no activ abnormal demyel no activ
L Median abnormal 0 no activ abnormal 0 no activ abnormal demyel no activ
L Radial abnormal 0 3.6/0.9 abnormal 0 no activ abnormal 0 no activ
L Ulnar abnormal demyel 3.0/0.78 abnormal axonal no activ abnormal dem/ax.deg 2.8/1.2
L Sural abnormal 0 no activ # # no activ abnormal 0 no activ
L Peroneal abnormal 0 no activ # dem/ax.deg no activ abnormal 0 no activ
Patient 7 8
Nerve
R Median abnormal 0 no activ normal Normal no activ
R Radial abnormal 0 no activ normal Axonal no activ
R Ulnar abnormal dem/ax.deg no activ normal demyel 2.7/0.9
R Sural abnormal 0 no activ normal 0 2.4/1.4
R Peroneal abnormal 0 5.8/1.6 normal 0 2.4/0.8
L Median abnormal 0 no activ normal Normal no activ
L Radial abnormal 0 no activ normal Normal no activ
L Ulnar abnormal demyel no activ abnormal dem/ax.deg 3.3/0.9
L Sural abnormal 0 2.7/1.3 normal 0 2.4/1.4
L Peroneal abnormal 0 no activ normal 0 1.7/0.8
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Abbreviations: #, not realized; no activ, no activity; demyel, demyelinating; dem/ax. deg., demyelinating with axonal degeneration; t-SUV max/bg-SUV max—target maximum standardized capture value/ background.

Patients 1, 3, and 5 showed no uptake of 18F-FDG in any peripheral nerve but had diffuse electrophysiological alterations in a pattern of multiple mononeuropathies. The neurological examination was normal in patient 1; however, there were signs of peripheral nerve involvement in patients 3 and 5 (Fig 1).

Fig 1. Coronal and axial slices of 18F-FDG PET/CT show no uptake at the peripheral nerves (patients 1 and 3), such as the sural nerve (blue asterisk).

Fig 1

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Patients 2, 4, and 6 had 18F-FDG uptake in one to two peripheral nerves each, while nerve conduction studies and neurological examination findings demonstrated a more diffuse pattern of alteration, such as polyneuropathy (Fig 2).

Fig 2. Coronal and axial slices of 18F-FDG PET/CT show different levels of uptake at the peripheral nerves (blue asterisks), such as the sural and tibial nerve (patients 2 and 4), and ulnar and cutaneous nerve (patient 6), as seen at the color scale as orange to white.

Fig 2

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In patients 7 and 8, PET/CT was performed during an episode of acute neuritis. 18F-FDG uptake has been observed in the same symptomatic nerves and other clinically asymptomatic nerves, with nerve damage evidence in nerve conduction studies.

One year after MDT, Patient 7 developed worsening pain, paresthesia, and decreased loss of muscle strength in the right foot. All evaluated sensory and motor nerves were unexcitable to stimuli in nerve conduction studies. 18F-FDG PET/CT revealed hypermetabolism of radiolabeled glucose in the topography of the left sural and right deep peroneal nerves.

Patient 8 developed severe pain in the left ulnar path six months after the leprosy diagnosis. We observed bilateral lesions in the sensory and motor ulnar nerves clinically and through nerve conduction studies. In addition, the sensory nerves of the lower limbs were not excitable by nerve conduction studies. 18F-FDG PET/CT showed hypermetabolism of radiolabeled glucose in the bilateral ulnar, sural, and superficial peroneal nerves (Fig 3).

Fig 3. Maximum intensity projection (MIP).

Fig 3

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18F-FDG PET/CT shows high uptake (patient 8) in bilateral ulnar (blue arrows), sural, and superficial peroneal nerves.

The first six patients underwent nerve biopsy, which revealed chronic inflammatory neuropathy of leprosy etiology and positive acid-fast bacillus (AFB). Table 3 shows the relationship between 18F-FDG PET/CT uptake and the percentage of inflammatory infiltrates in the biopsied nerves.

Table 3. Correlation between biopsy findings, NCS and PET uptake.

Patient Nerve biopsied PET uptake NCS %infl.inf. fibrosis
1 Sural D No Inexcitable 8 Yes
2 Ulnar E No Inexcitable 60 Yes
3 Sural D No Inexcitable 30 Yes
4 Ulnar D No Inexcitable 40 Yes
5 Ulnar D No Inexcitable 100 Yes
6 Ulnar D Yes Inexcitable 100 Yes
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Abbreviations: NCS, Nerve conduction studies; %infl.inf., percentage of inflammatory infiltrate

Discussion

Rambukkana et al. (2002) demonstrated that nerve demyelination by M. leprae could occur even without the inflammatory process when the bacillus promotes Schwann cell reprogramming and alters nerve function through mechanisms that need to be clarified [19]. Masaki et al. (2013) suggested that leprosy neuropathy could occur due to Schwann cell dedifferentiation after infection, caused by loss of the SOX-10 transcription factor, for example, long before leukocyte infiltration and nerve fibrosis [9].

Medeiros et al. (2016) have demonstrated increased glucose uptake in infected Schwann cells in vitro and in vivo [3]. In this research project, we chose 18F-FDG PET/CT as an imaging resource to demonstrate glucose uptake in damaged nerve tissue of LL patients with silent neuritis and acute neuritis, since in vivo and in vitro previous studies have demonstrated this metabolic alteration.

We identified that LL patients with clinically and electrophysiologically diagnosed silent neuritis had an absence or a few 18F-FDG uptakes in their nerves. Apparently, 18F-FDG PET/CT was not sensitive enough to detect metabolic changes in infected Schwann cells in cases 1, 3 and 5, nor was it sensitive enough to demonstrate the extent of nerve damage, as demonstrated in neurophysiological examinations in patients 2, 4 and 6.

Roy et al. (2018) and Shao et al. [12] reported cases in which acute leprosy neuritis was detected using 18F-FDG PET/CT [10,11]. Our findings were similar to those of patients diagnosed with acute neuritis.

It is known that, once inside the cell, the 18F-FDG molecule is phosphorylated by hexokinase, transforming it into 18F-FDG-6-Phosphate. Thus, it cannot follow the glycolytic pathway or leave the cell, which causes its uptake. Inflammatory/infectious processes with high expression of neutrophils and activated macrophages also show increased 18F-FDG uptake. Therefore, we believe that further studies are necessary regarding silent nerve injuries, where the cellular inflammatory process seems different from that of acute neuritis.

These clarifications regarding the pathophysiology of silent neuritis are essential to developing new drugs to prevent permanent disabilities in these patients.

In conclusion, 18F-FDG uptake is related to acute neuritis in LL patients, and there is no 18F-FDG uptake among LL patients who present with silent neuritis. Furthermore, 18F-FDG PET-CT could be a valuable tool to confirm neuritis. In addition, in cases of already diagnosed patients who develop recurrent neuropathy, there is a possible tool to differential diagnosis between a new episode of neuritis and chronic neuropathy.

Acknowledgments

We thank the people who agreed to be study subjects, the team from the Leprosy Laboratory and outpatient clinic of the Instituto Oswaldo Cruz da Fiocruz, and the team from the nuclear medicine sector of Clementino Fraga Filho University Hospital. We would especially like to thank Cristiane Domingues, who was always attentive to the admitted LL patients.

Data Availability

Patient data are available in the Oswaldo Cruz Foundation Leprosy Laboratory database. Adress: Avenida Brasil, 4025-4011, Manguinhos, Rio de Janeiro - RJ Brazil. phone +55 21 25621588. Website: https://www.ioc.fiocruz.br/en/referencia/hanseniase. E-mail: asa.lahan@ioc.fiocruz.br PET/CT images are available in the database of the Nuclear Medicine Lab of the Federal University of Rio de Janeiro. at Hospital Universitário Clementino Fraga Filho Phone +55 21 39382362 Website: https://www.hucff.ufrj.br/.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011383.r001

Decision Letter 0

Mathieu Picardeau, Linda B Adams

24 Jan 2023

Dear Dr Penna,

Thank you very much for submitting your manuscript "Evidencing Leprosy Neuronal Inflammation by 18-Fluoro-deoxy-glucose" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Your manuscript has been reviewed by two experts, here and in the attachment. While they have found the manuscript interesting, they have raised some issues with the use of this technique. Please address each of their comments, both in an itemized rebuttal and in the manuscript itself. Special consideration should be given to the justification for the use of this technique in leprosy and its clinical applications.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Linda B Adams

Academic Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Section Editor

PLOS Neglected Tropical Diseases

***********************

Your manuscript has been reviewed by two experts, here and in the attachment. While they have found the manuscript interesting, they have raised some issues with the use of this technique. Please address each of their comments, both in an itemized rebuttal and in the manuscript itself. Special consideration should be given to the justification for the use of this technique in leprosy and its clinical applications.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Reject

Reviewer #2: The methods are generally satisfactory for a preliminary report. Some details need clarification:

The authors have made a reasonable attempt to assess histopathological findings in the nerves and to correlate these with the PET scans, but they did not (and probably could not) biopsy the same nerve sites that had been assessed by the scans. This is an inherent limitation in studying human peripheral nerves. Since leprosy neuritis and neuropathy may occur intermittently along a nerve, rather than diffusely, it is important that they acknowledge the fact that the sites are not identical and therefore that any correlations must be interpreted very cautiously.

“In short, an axonal lesion was defined as either an isolated reduction in amplitude ≥ 30% of the reference values”. Don’t you mean an isolated reduction in amplitude of < 30% . . .? or a 30% reduction . . .?

Histopathological examination -- “The sensory nerve was biopsied according to clinical or electrophysiological findings” Which sensory nerve?

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: Most of the results are clear and appropriate. some concerns:

Describing the time of onset of nerve vs dermatological symptoms, only 6 patients are accounted for:

2 (#4 & 7) with nerve onset first

2 (5&6) with nerve and skin onset at the same time

2 (2&8) with nerve onset after skin lesions

What about cases #1 and 3?

In Table 1, ‘Symp on” refers to duration of neural symptoms. What does ‘No” mean ?

In the legend for Table 2, please also include explanations for the terms used in the headings, e.g., “t-SUV, max/bg-SUV, max”

“In patients 7 and 8, PET/CT was performed during a neuritis episode, and 18F-FDG uptake was observed in the peripheral nerves in which the patients had symptoms and signs of neuritis, as well as in other nerves that showed no signs of neuritis on neurological examination, but with neurophysiological changes.” Does the final expression, “ with neurophysiological changes”, refer to the 18F-FDg uptake? If so, it will be less confusing to say so.

Also, if I understand this correctly, this means that some of the nerves with ‘no signs of neuritis’ had increased uptake. Plese comment on this.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The authors appear to have overstated the significance of some of their findings. Specifically, assertions regarding the assessment of Schwann cell activity/ uptake of FDG are overstated, since the methods do not appear to have sufficient sensitivity or resolution to distinguish increased uptake in Schwann cells vs inflammatory infiltrates, i.e., macrophages and lymphocytes.

Discussion

The first paragraph is somewhat confusing and should be edited to more concisely describe the overall findings.

e.g., “ . . . patients with LL present with sensory and motor nerve impairment despite the absence of 18F-FDG uptake in their nerves” – does this mean that they have previously had nerve injury, so they have impairment, but do not have active injury occurring at the time they present to the clinic?

“According to the present and previous studies, neuropathy in LL patients can occur as a result of Schwann cell dedifferentiation . . .” Nothing in this paper presents any evidence regarding Schwann cell dedifferentiation, and the methods do not address such dedifferentiation in any way. Therefore, this statement and reference should be omitted.

“Despite this, the nerve biopsy revealed an inflammatory process, even without clinical neuritis.” There are several possible explanations, but the most obvious is that the biopsy sites do not correspond exactly to the sites imaged by 18F-FDG uptake (see general comments). Therefore, any attempts to correlate these much be stated very cautiously.

“Apparently, 18F-FDG PET/CT was not sensitive enough to detect metabolic changes in infected Schwann cells.“ I do not see that the authors have determined that any of the uptake seen in their own study is specifically due to Schwann cells. Is the imaging of sufficient resolution and specificity to distinguish uptake by Schwann cells vs nearby macrophages or lymphocytes?

“We believe that further studies are necessary regarding silent nerve injuries, where the cellular inflammatory process seems to be different from that of acute neuritis . . .” This assumes that an inflammatory process is present in silent nerve injury, but that has not been determined. Alternatively, it could be a degenerative process rather than an inflammatory one.

“In addition, in cases of already diagnosed patients who develop recurrent neuropathy, there is a differential diagnosis between a new episode of neuritis and chronic neuropathy” The meaning and point of this sentence is not clear.

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Editorial and Data Presentation Modifications?

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Reviewer #1: (No Response)

Reviewer #2: Author summary

Typos and incomplete sentences in the following: “ . . . our question was about how the uptake of this radiopharmaceutical would be in a common silent neuritis in LL patients.d the uptake . . .”

The phrase real extension seems odd and I don’t understand what this means here.: ´. . . Our data demonstrated, for the first time, the real extension of peripheral nerve . . .’ I think you mean something else -- Perhaps you mean illustration of . . . or example of

Introduction

“Medeiros et.al. (2016) evaluated in vitro and in vivo models of Schwann cells . . .” This should be indicated as reference #3 instead of the year.

The expression “real extension” also appears in the last paragraph of the introduction.

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Summary and General Comments

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Reviewer #1: (No Response)

Reviewer #2: This is an interesting report regarding the use of 18FDG-PET as a means to assess physiological activity, in a small series of patients. The addition of this method to other tools for neurological assessment enables measurement of physiological activity in real time, a valuable new approach.

The peripheral nerve status of the patients has been carefully assessed and well documented using a variety of clinical assessments in addition to the PET scans. The finding of increased uptake during active neuritis is not surprising, but the capability to assess physiological activity in vivo during acute neuritis offers new possibilities in understanding this process. The finding of no uptake in patients with neuropathy but not acute neuritis also adds to our knowledge of neuropathy in leprosy.

The findings in acute neuritis do increase the evidence base regarding the inflammation in nerves in leprosy. It may be valuable to consider this in the broader context of the findings of FDG uptake in other inflammatory conditions such as arthritis, e.g., Graham RN, Panagiotidis E. [18F]FDG PET/CT in rheumatoid arthritis. Q J Nucl Med Mol Imaging. 2022 Sep;66(3):234-244.

It would be interesting to know if the authors believe that 18FDG-PET could be a useful tool to measure changes in inflammation during treatment of acute neuritis in leprosy, i.e., scanning before and during prednisone treatment.

--------------------

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Reviewer #2: Yes: David Scollard, M.D., Ph.D.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011383.r003

Decision Letter 1

Mathieu Picardeau, Linda B Adams

20 Apr 2023

Dear Dr Penna,

Thank you very much for submitting your manuscript "Evidencing Leprosy Neuronal Inflammation by 18-Fluoro-deoxy-glucose" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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This manuscript was sent to two highly qualified and knowledgeable reviewers. However, some of their comments were not addressed at all in the rebuttal; it is imperative to provide a response to every comment. In addition, each of their concerns must be addressed in the manuscript itself. In the rebuttal, please include the line numbers of the manuscript where each of the specific concerns were edited and clarified. Modifications to address their comments will improve the manuscript and reduce misunderstandings by other readers.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011383.r005

Decision Letter 2

Mathieu Picardeau, Linda B Adams

16 May 2023

Dear Dr Penna,

We are pleased to inform you that your manuscript 'Evidencing Leprosy Neuronal Inflammation by 18-Fluoro-deoxy-glucose' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Linda B Adams

Academic Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011383.r006

Acceptance letter

Mathieu Picardeau, Linda B Adams

1 Jun 2023

Dear Dr Penna,

We are delighted to inform you that your manuscript, "Evidencing Leprosy Neuronal Inflammation by 18-Fluoro-deoxy-glucose," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

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Paul Brindley

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PLOS Neglected Tropical Diseases

Associated Data

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    Data Availability Statement

    Patient data are available in the Oswaldo Cruz Foundation Leprosy Laboratory database. Adress: Avenida Brasil, 4025-4011, Manguinhos, Rio de Janeiro - RJ Brazil. phone +55 21 25621588. Website: https://www.ioc.fiocruz.br/en/referencia/hanseniase. E-mail: asa.lahan@ioc.fiocruz.br PET/CT images are available in the database of the Nuclear Medicine Lab of the Federal University of Rio de Janeiro. at Hospital Universitário Clementino Fraga Filho Phone +55 21 39382362 Website: https://www.hucff.ufrj.br/.

    Articles from PLOS Neglected Tropical Diseases are provided here courtesy of PLOS

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