Cytomorphological Patterns of Nerve Aspirates in Pure Neuritic Leprosy-A Single Centre Cross-Sectional Observational Study.

BACKGROUND: Pure neuritic leprosy (PNL) poses a diagnostic challenge because of absence of skin patches, inconclusive skin biopsies and nerve conduction studies. Nerve biopsy though the diagnostic gold standard, is invasive, requires expertise, and may not be feasible in all cases. Fine needle aspiration cytology (FNAC) of accessible thickened nerves can be utilized as a minimally invasive diagnostic modality in PNL. This study was carried out to describe cytomorphological patterns of nerve aspirates in patients of PNL for diagnosis and classification of leprosy and study its advantage, if any, over skin biopsy.
METHODS: Twenty-seven treatment naive clinically diagnosed patients of PNL were included in this cross-sectional study carried out from January 2017 to December 2018 at a tertiary care centre in Western India. FNAC was done from a clinically involved nerve and aspirates were evaluated for cytomorphological characteristics and the presence of Acid-Fast Lepra bacilli.
RESULTS: Nerve aspirates were diagnostic in 10 (37%) patients while 17 (63%) aspirates showed non-specific or no inflammation. Of the diagnostic aspirates, six (22.2%) were classified as tuberculoid leprosy, three (11.1%) as lepromatous and one (3.7%) as borderline leprosy. Mycobacterium leprae were demonstrated among three (11.1%) of these aspirates. In comparison, only three (11.1%) skin biopsies were diagnostic of leprosy with features of indeterminate spectrum. Remaining 24 skin biopsies showed normal histology in 20 (74.1%) cases to perivascular lymphocytic infiltrate in four (14.8%) cases.
CONCLUSION: Our study demonstrates that FNAC of clinically thickened nerves has a better diagnostic yield than skin biopsy in PNL and shows all spectrums of leprosy. It also offers the advantage of sampling major nerve trunks without the fear of residual neurological deficit. However, most of the smears were paucicellular and a negative aspirate does not rule out leprosy. Copyright:

INTRODUCTION

Pure neuritic leprosy (PNL) constitutes about 4–18% of leprosy patients in India with a higher prevalence in South India.[123] It is characterized by an area of sensory loss along the distribution of an involved nerve trunk with or without motor deficit, in the absence of any skin patch.[4]

Diagnosing and differentiating PNL from other causes of peripheral neuropathy is a challenge. Skin biopsy from area of sensory loss is usually inconclusive. Nerve involvement in leprosy is predominantly superficial and these thickened nerves are easily accessible for the sampling. Nerve biopsy in leprosy is not done routinely owing to its practical and ethical limitations, sampling errors, low sensitivity, and risk of permanent nerve damage.[567] In comparison, fine needle aspiration of a thickened nerve is a safer and lesser invasive diagnostic technique.[589] Only a handful of studies have evaluated the role of fine needle aspiration cytology (FNAC) of nerve in diagnosis of PNL.[910111213] Our study describes the cytomorphological patterns in the aspirates of FNAC of nerve in PNL and its reliability in diagnosis and classification of the disease.

MATERIALS AND METHODS

This prospective cross-sectional observational study was carried out over a period of two years from January 2017 to December 2018 in a tertiary care hospital in western India after approval from institutional ethics committee. All treatment naive clinically diagnosed cases of PNL above 15 years of age were included in the study after taking an informed written consent (in case of minor's, the consent was taken from the parent/guardian). During the study period, a total of 34 clinically diagnosed PNL patients reported to our centre. Of these, 27 fulfilled the inclusion criteria and were enrolled in the study. A detailed history and a clinical examination was done for all patients and data regarding age, sex, duration of disease, and presentation was recorded. Slit skin smear (SSS) was taken from four sites for calculation of Bacteriological index (BI). Skin biopsy was done from area with sensory involvement or area supplied by affected nerve.

FNAC of a superficial thickened nerve preferably a cutaneous sensory nerve was carried out. The most prominent part of nerve and most suitable site for needling was identified preferably over a firm bony base. Local infiltration of the area around the nerve with 0.5–1.0 ml of lignocaine improved patient compliance during the procedure and resulted in better yield of the smears. The nerve was fixed between the index finger and thumb of the left hand and a 22G needle fitted in 10 mL disposable plastic syringe was inserted along and parallel to the length of the nerve to cause minimum damage to it. Suction was applied and aspiration performed using a single-puncture, multidirectional technique. Suction was released before removing the needle from the tissue and the needle was then removed from the syringe, air aspirated and the needle refitted and forcefully expressed over the clean slides. A minimum of three smears were made for each aspiration. The first smear was fixed in a mixture of equal parts of 95% ethanol and ether for 30 min for Papanicolaou (PAP) stain. The second smear was air dried and stained with May Grünwald Giemsa (MGG) stain. The third slide was also air dried and stained with modified Ziehl Neelsen (ZN) stain using 5% sulphuric acid to demonstrate Acid fast bacilli leprae [AFB (L)]. All these smears were then studied for cytological details. The patient was assessed for any loss of motor function after the procedure. Both PAP and MGG stained smears were examined for presence of nerve fibres, Schwann cells, cellularity and infiltration by inflammatory cells, lymphocytes, epithelioid cells, giant cells, macrophages, granulomas and any caseous necrosis. The cellularity was further quantified into paucicellular (+), moderately cellular (++), and heavy cellular (+++). Nerve fragments, Schwann cells, and inflammatory cells were quantified as per their number into present (+), moderate in number (++), and numerous (+++). Smears stained by modified ZN stain were examined for the presence or absence of AFB (L) and BI noted. Cytological aspirates were classified into various spectrums as proposed by Vijaykumar et al.[8] [Table 1] Patients were classified as per Ridley Jopling criteria into tuberculoid (TT), borderline tuberculoid (BT), mid-borderline (BB), borderline lepromatous (BL), and lepromatous (LL) types.

Table 1

Cytomorphological findings of nerve aspirate and classification of Hansen’s disease as per Vijaikumar et al.[8]

Class	Cellular Aspirate	Cytomorphology of aspirate	BI
TT	Good	Cohesive epithelioid cell granuloma or lymphocytic cell collection· Predominantly epithelioid cells with predominant to moderate number of lymphocytes. Occasional giant cells and neutrophils	0-1+
BT	Same as TT	Same as TT	Same as TT
BB	Fair	Mixed cellularity of predominantly non foamy macrophages, moderate number of epithelioid cells and lymphocytes. Macrophage granuloma	2-3+
BL	Fair	Predominantly lymphocytes and moderate number of foamy macrophages	4-5+
LL	Fair to poor	Predominantly foamy macrophages and few lymphocytes	6+

TT: Tuberculoid leprosy, BT: Borderline tuberculoid leprosy, BB: Borderline leprosy, BL: Borderline lepromatous leprosy, LL: Lepromatous leprosy

RESULTS

Of the total 206 newly detected leprosy cases over two years in our centre, the proportion of clinically diagnosed PNL cases was 16.5% (34 in number). Of these 34 cases, 07 were excluded out (Unwilling for FNAC: 03, <15 years of age: 01, Treatment taken for leprosy: 03) from the study. Of the 27 patients in the study group, 24 (88.9%) were males and three (11.1%) were females. The mean age (±SD) of patients in our study was 34.7 ± 11.9 years and the duration of the symptoms from onset till first presentation varied between 01 month and 31 months (mean duration of symptoms - 8.6 months). Polyneuropathy was seen in 22 (81.5%) cases while five (18.5%) had mononeuropathy. The most common nerve involved in our study was left ulnar nerve in 16 (59.3%) cases followed by left radial cutaneous nerve in 12 (44.4%) cases.

SSS smear was negative in all 27 patients. Ulnar aspect of hand was the commonest site for skin biopsy in 16 (59.2%) patients. Twenty (74.1%) of the skin biopsies revealed no histopathological abnormality and four (14.8%) had non-specific perivascular lymphocytic infiltrate. Only three (11.1%) biopsies had histopathological findings suggestive of Indeterminate leprosy of which two (7.4%) had infiltrates around neurovascular bundle and one (3.7%) showed peri-neurovascular and peri-appendageal infiltrates along with AFB (L) in the nerve [Table 2].

Table 2

Histological findings in skin biopsies of study group (n = 27)

Histopathological findings	AFB (L) Seen	No. of cases	% of cases	Final diagnosis
Normal histology/ No abnormality	Not seen	20	74.1	No abnormality
Perivascular lymphocytic infiltrate	Not seen	4	14.8	Non specific finding
Peri neurovascular bundle infiltrate	Not seen	2	7.4	Indeterminate leprosy
Peri neurovascular bundle and peri appendageal infiltrate	1+	1	3.7	Indeterminate leprosy
Total		27	100	Indeterminate leprosy: 03

The common nerves chosen for FNAC in our study were the left radial cutaneous nerve and ulnar nerve in seven (25.9%) cases each. In order to ascertain if the aspirate was from a nerve, Schwann cells were looked for, present singly or in fascicles along with inflammatory infiltrate. Desired nerve tissue was aspirated in all the 27 cases.

Diagnostic cytomorphological patterns were observed in 10 (37%) nerve aspirates, four (14.8%) aspirates showed nonspecific inflammation and the remaining 13 (63%) showed a normal aspirate. [Table 3] Of the 10 diagnostic aspirates, six showed epithelioid cell granuloma, of which two aspirates had additional necrosis. Macrophage infiltrate was seen in three aspirates and two of these were positive for AFB (L) [Figures 1 and 2]. One aspirate showed a mixed infiltrate of epithelioid cell granuloma, few macrophages, necrosis, and AFB (L) [Figure 3]. Based on the cytomorphological findings, six (22.2%) FNAC aspirates were classified as tuberculoid leprosy, three (11.1%) as lepromatous leprosy (LL), and one (3.7%) as borderline leprosy. [Table 4]

Table 3

Cytomorphological findings of nerve aspirates and comparison with skin biospy (n = 27)

Case no	Age In years	Sex	Duration before diagnosis (months)	Cytomorphological findings#							Cytomorphological pattern on FNAC	Skin biopsy finding
				Cellularity	L	MP	EG	GC	Necr-osis	AFB (L) /BI
1.	35	M	05	++	+	++	-	-	-	++/ 5+	LL	Normal
2.	33	M	04	+++	++	-	++	+	-	-	Tuberculoid leprosy	Normal
3.	45	M	05	-	-	-	-	-	-	-	Normal	Normal
4.	33	F	24	++	+	-	+	-	-	-	Tuberculoid leprosy	Indeterminate
5.	21	M	01	+	+	+++	-	-	-	-	LL	Normal
6.	37	M	18	+	-	-	+	-	+	-	Tuberculoid leprosy	Indeterminate
7.	56	M	07	+	+	-	-	-	-	-	Non specific	Normal
8.	19	M	12	-	-	-	-	-	-	-	Normal	Normal
9.	52	M	04	-	-	-	-	-	-	-	Normal	Normal
10.	34	M	07	-	-	-	-	-	-	-	Normal	Normal
11.	30	M	05	++	+	-	-	-	-	-	Non specific	Normal
12.	27	M	12	+++	+	-	++	-	-	-	Tuberculoid leprosy	Normal
13.	18	M	12	-	-	-	-	-	-	-	Normal	Normal
14.	22	M	01	++	+	-	-	-	-	-	Non specific	Normal
15.	36	M	03	-	-	-	-	-	-	-	Normal	Normal
16.	32	F	12	++	+	-	++	-	-	-	Tuberculoid leprosy	Normal
17.	60	M	03	+	+	-	-	-	-	-	Non specific	Normal
18.	22	M	12	-	-	-	-	-	-	-	Normal	Normal
19.	33	M	31	-	-	-	-	-	-	-	Normal	Indeterminate
20.	28	M	07	-	-	-	-	-	-	-	Normal	Normal
21.	28	M	04	+	-	-	+	-	+	-	Tuberculoid leprosy	Normal
22.	25	M	03	+	-	-	-	-	-	-	Normal	Normal
23.	48	M	04	-	-	-	-	-	-	-	Normal	Normal
24.	49	M	07	-	-	-	-	-	-	-	Normal	Normal
25.	54	F	14	-	-	-	-	-	-	-	Normal	Normal
26.	25	M	07	++	++	++	++	+	+	++/2+	Borderline leprosy	Normal
27.	36	M	09	++	+	++	-	-	-	++/ 5+	LL	Normal

# Cellularity; +: Paucicellular, ++: Moderately cellular, +++: Heavy cellularity L/ MP/ EG/ GC; +: Present, ++: Moderate in number, +++: Numerous [M: Male, F: Female; L: Lymphocytes, M: Macrophage, EC: Epithelioid cell granuloma, GC: Giant cell, AFB (L): Acid-fast bacilli lepra, LL: Lepromatous leprosy]

Figure 1

FNAC of nerve with features of tuberculoid leprosy. (a) Case 2; FNAC of left ulnar nerve showing cohesive fragments of wavy neural cells with epithelioid cells and few lymphocytes (40 x; PAP smear). (b) Case 2; Aspirate showing giant cell (red arrow) (40 x; PAP smear). (c) Case 2; Well defined epithelioid cell granuloma (20 x; PAP smear). (d) Case 6; FNAC of right saphaneous nerve revealing “spread cheese” like necrosis of nerve without any inflammation (10 x; MGG)

Figure 2

FNAC of nerve with features of lepromatous leprosy. (a) Case 27; Highly cellular smear with wavy necrotic nerve tissue (red arrow) with polymorphs, lymphocytes, histiocytes, scattered foamy macrophages with granular dirty proteinaceous background (20 x; MGG). (b) Case 27; FNA of right greater auricular nerve showing necrotic aspirate with multiple non foamy histiocytes (red arrow), foamy cells (black arrow) and polymorphs (40 x; MGG). (c) Case 5; Multiple foamy histiocytes (20 x; MGG). (d) Case 1; FNA from left radial cutaneous nerve showing multiple AFB (L) [Red rectangle] (100 x; Modified ZN stain)

Figure 3

FNAC with features of borderline leprosy (Case 26). (a) FNAC of thickened right greater auricular nerve showing spindle elements of nerve with multiple well-defined epithelioid cell granuloma surrounded by lymphocytes with giant cell (yellow arrow) (10 x; MGG). (b) Wavy nerve with well-defined epithelioid cell granuloma surrounded by lymphocytes with giant cell (20 x; MGG). (c) Scattered histiocytes (yellow arrow) with several fragmented AFB (L) (red arrow) (100 x; Modified ZN stain). (d) Nerve fragments with necrosis and inflammatory infiltrate (red arrow) (10x; PAP smear)

Table 4

The cytomorphological patterns observed in FNAC of nerves (n = 27)

Cytomorphological pattern	n = 27	% of cases	FNAC diagnosis
Epithelioid cell granuloma only	4	14.8	Tuberculoid leprosy
Epithelioid cell granuloma + Necrosis	2	7.4	Tuberculoid leprosy
Macrophage granuloma only	1	3.7	Lepromatous leprosy
Macrophage granuloma + AFB (L)	2	7.4	Lepromatous leprosy
Epithelioid cell granuloma + Macrophages + Necrosis + AFB (L)	1	3.7	Borderline leprosy
Normal aspirate including nonspecific inflammatory infiltrate	17	63	Normal
Total	27	100.0	Tuberculoid leprosy: 06Lepromatous leprosy: 03Borderline leprosy: 01

Aspiration of the thickened nerves was well tolerated by the patients, barring some discomfort and tingling during the procedure. No iatrogenic loss of sensation or motor deficit was noticed in any patient post procedure.

DISCUSSION

World Health Organization's (WHO) classification of 1988 does not mention “neuritic leprosy” specifically. However, the recent revised WHO “Monitoring and Evaluation Guide to the Global Strategy” for Leprosy has now included “Pure Neuritis” in the case definition of MB leprosy specifically.[14] This indicates a global acceptance of PNL as a distinct type of leprosy.

The proportion of PNL in our study was 16.5% which was much higher than earlier reported proportion of 4.6% in this region.[1] It constitutes a significant proportion of all leprosy cases in other parts of India varying from 4.2% in Northern India to 17.7% in South India to 5.5% in western region.[2315] A recent 10 year retrospective study from Kerala has reported 8.5% cases of PNL among 879 leprosy patients.[16] Approximate 21% of all leprosy cases in Brazil are in the pure neuritic spectrum.[17]

As compared to other spectrum of Hansen's disease where both clinical signs and symptoms are recognizable earlier and histopathological diagnosis in form of skin biopsy is relatively easily accessible, the absence of skin lesions in PNL makes clinical diagnosis and histological confirmation a challenge leading to considerable delay in diagnosis.[1118]

The skin biopsy was diagnostic in only about a third of the cases of PNL.[19] In our study, the skin biopsy was diagnostic in three (11.1%) patients and suggestive of indeterminate leprosy. All these three patients had symptoms of PNL for more than 18 months before presentation. This reinforces the concept that PNL may be an early stage in the pathogenesis of the disease before the involvement of overlying skin. We could not find any data in literature comparing disease duration with skin biopsy findings in PNL.

Nerve biopsy is the gold standard for diagnosis of PNL, however complications and permanent nerve deficit may occur if done from a large functioning nerve.[9] Its use is further limited by sampling errors and low sensitivity.[18] FNAC of thickened nerve is a lesser invasive and relatively safer diagnostic modality than nerve biopsy in cases of PNL. In most of the previous studies, a sensory cutaneous nerve had been preferred for FNAC owing to its safety.[82021] In this study, we carried out FNAC of mixed/motor nerves in 14 (51.9%) patients without any adverse effects.

FNAC of the sensory cutaneous nerve has been studied in categorization of leprosy along the Ridley-Jopling (RJ) scale.[82021] RJ classification helps in understanding the immunology of the leprosy patient, thereby assessing in prognosis and possible complications of the disease. Though Vijaykumar et al. reported a 100% diagnostic yield from FNAC nerve in PNL cases, the reported yield by various authors ranges from 38.7% to 66.6%.[89111213] We found FNAC of nerve in PNL to be diagnostic in 10 (37%) cases out of 27 cases. In remaining 17 (63%) cases, the aspirates were paucicellular, and no conclusive diagnosis on cytology was possible.

In tuberculoid spectrum, there is type 1 cytokine response leading to a strong T cell and macrophage activation resulting in a localized infection. Perineurium is breached by T cells which destroy Schwann cells and axons. This results in fibrosis of the epineurium, replacement of the endoneurium with epithelioid granulomas, and caseous necrosis occasionally.[418] Such involvement of nerve and presence of epithelioid granuloma within the nerve is pathognomonic of tuberculoid leprosy. Bacilli are either absent or few in number.[8] In LL spectrum, nerve enlargement and damage occurs from neural invasion by lepra bacilli. Bacilli invade Schwann cells, leading to foamy degenerative myelination and axonal degeneration.[4] Nerve aspirate is characterized by numerous lepra bacilli and foamy macrophages that infiltrate the nerve.[8] In BL leprosy, findings vary with the amount of T cell reaction against M. leprae. Well-formed epithelioid granuloma surrounded by lymphocytes is seen with a good T cell response. Dermal nerve destruction is less severe than in tuberculoid leprosy. However, in case of a weaker immune response, granuloma formation and lymphocytic exudate is less well marked. Dermal nerves are largely spared with presence of proliferation of their Schwann cells. If the immune response is much weaker, cytomorphological findings approach toward lepromatous spectrum.[820]

Table 5 highlights cytomorphological patterns obtained in previous studies in comparison to this study. In our study, cohesive infiltrates of epithelioid cells and lymphocytes were seen in the tuberculoid spectrum, whereas toward the lepromatous pole, macrophages were predominant with lack of cohesion of cells. As proposed by Jardim, the presence of a non-specific inflammatory infiltrate on nerve aspirates is not diagnostic of leprosy. However, it does not rule out leprosy.[22] Polymerase chain reaction (PCR) when combined with nerve aspirates in PNL can increase the diagnostic utility.[1213]

Table 5

Comparison of FNAC findings with previous studies

Cytomorphological findings	This study (n = 27)	Naik et al.[21] (n = 25)	Prasoon et al.[20] (n = 23)
ECG	4 (14.8%)	6 (24%)	6 (26.1%)
ECG + Necrosis	2 (7.4%)	3 (12%)	1 (4.3%)
Necrosis+ lepra bacilli	-	4 (16%)	3 (13.10%)
Macrophage granuloma only	1 (3.7%)	-	-
Macrophage granuloma + AFB (L)	2 (7.4%)	-	-
ECG + Macrophages + Necrosis + AFB (L)	1 (3.7%)	-	-
Lepra bacilli only	-	2 (8%)	12 (52.2%)
Necrosis + lepra bacilli	-	-	1 (4.3%)
Lymphocyte, macrophage infiltration	-	4 (16%)	-
FNAC positivity/ Number of cases	10/27; all PNL cases	19/25; 08 PNL cases	23/23; 08 PNL cases

ECG: Epithelioid cell granuloma; AFB (L): Acid fast bacilli (Lepra); PNL: Pure neuritic leprosy

Necrosis was seen in three (11.1%) aspirates in our study and has been reported by other workers.[2223] Caseation necrosis because of a heightened delayed hypersensitivity response is well known in nerve abscesses in tuberculoid spectrum.[24] Necrosis seen in one patient of Borderline leprosy in our study could be because of ischemic necrosis resulting from intraneural compression of vasculature by an expansile granuloma.

One of the nerve aspirates revealed concomitant presence of epithelioid cells, macrophages, AFB (L) and necrosis which was classified as borderline leprosy. The cytomorphological pattern was suggestive of a downgrading reaction from borderline tuberculoid (BT) to mid-borderline (BB)/Borderline Lepromatous.[8] Nerve involvement is common in lepra reaction. Type 1 reaction may either be a reversal reaction or a downgrading reaction, as seen in borderline cases. Reversal reaction is characterized by edema, reduced number of bacilli, and increase in number of epithelioid cells, giant cells, and lymphocytes. While in downgrading reaction, an increase in number of bacilli and replacement of defensive cells by macrophages is seen. Type 2 lepra reaction is seen in LL and occasionally in BL leprosy. It may be associated with neuritis. There is edema, infiltration by neutrophils, occasionally vasculitis with fragmented and granular lepra bacilli.[20]

In our study, the complete spectrum of leprosy from tuberculoid to lepromatous leprosy was seen and we could classify the cases as Paucibacillary (PB) and Multibacillary (MB) on FNAC. We could not differentiate between Tuberculoid (TT) and BT leprosy on cytomorphological patterns as FNAC classified these PNL cases as PB, as in earlier studies.[8202325] However, a negative aspirate does not entirely rule out leprosy.[8] We did not find any correlation between number of thickened nerves and spectrum of leprosy as opposed to studies which found AFB (L) positivity more in polyneuropathy cases.[1213]

FNAC proved to be a better modality in both diagnosing as well as classifying PNL cases when compared to skin biopsy. FNAC was diagnostic in 10 (37%) cases, whereas skin biopsy could identify three (11.1%) cases only. Moreover, two out of these three cases were classified as tuberculoid leprosy on FNAC, whereas skin biopsy was suggestive of indeterminate leprosy. The nerves being an immune privileged site, M. leprae in nerves may not be detected immunologically leading to a higher bacillary density inside nerve lesions when compared to skin histology.[26] This emphasizes the importance of FNAC of nerve in accurate classification of the cases as higher spectrum was seen in nerve FNAC than the skin biopsy. However, it requires expertise to get good aspirates from the nerves. Here, in 63% cases, the aspirates were paucicellular, and no conclusive diagnosis on cytology was possible.

Though, almost all patients had discomfort and tingling sensation while FNAC of nerve was being done, no iatrogenic loss of sensation or motor deficit was noticed in any patient who underwent nerve aspiration.

PNL constitutes a significant proportion of all leprosy cases. Skin biopsy in this spectrum is usually inconclusive. The only conclusive evidence to diagnose PNL is the nerve biopsy of the involved nerve but this modality is associated with sampling errors and low sensitivity and may result in permanent nerve deficit especially if done from a motor nerve. Cytomorphological examination of aspirates from a thickened nerve is a minimally invasive diagnostic technique with easy learning curve. It is more reliable than skin biopsy for diagnosis and accurate classification of PNL and can reduce the requirement of nerve biopsy. Addition of PCR to the cytomorphological aspirates would increase the diagnostic yield. The limitations of our study were lack of comparison of FNAC of nerve with nerve biopsy. Resource constraints at our centre limited the use of PCR of the nerve aspirates.

Ethics committee/Institutional review board's permission

We also declare that the study was assessed and approved by the institutional ethics committee/institutional review board and that the letter of approval is available with us for examination.

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Nil.

Conflicts of interest

There are no conflicts of interest.