Received: 14 April 2023 Revised: 8 June 2023 Accepted: 16 June 2023

DOI: 10.1111/iwj.14296

ORIGINAL ARTICLE

Risk factor analysis for diabetic foot ulcer-related

amputation including Controlling Nutritional Status score
and neutrophil-to-lymphocyte ratio

Yandan Zhu 1 | Hongtao Xu 2 | Yuzhen Wang 2 | Xia Feng 1 |

Xinyu Liang 1 | Liying Xu 2 | Zhiqiang Liang 2 | Zhongjia Xu 2 | Yawen Li 2 |
Yi Le 2 | Manchen Zhao 2 | Jianfei Yang 2 | Ji Li 2 | Yemin Cao 2

1
Shanghai University of Traditional
Chinese Medicine, Shanghai, China
2
Shanghai Traditional Chinese Medicine
Integrated Hospital, Shanghai University
of Traditional Chinese Medicine,
Shanghai, China
Correspondence
Jianfei Yang, Shanghai Traditional
Chinese Medicine Integrated Hospital,
Shanghai University of Traditional
Chinese Medicine, Shanghai, 200082,
China.
Email: yangjianfeizy@163.com
Ji Li, Shanghai Traditional Chinese
Medicine Integrated Hospital, Shanghai
University of Traditional Chinese
Medicine, Shanghai, 200082, China.
Email: 13917975752@163.com
Yemin Cao, Shanghai Traditional Chinese
Medicine Integrated Hospital, Shanghai
University of Traditional Chinese
Medicine, Shanghai, 200082, China.
Email: dr-cao@163.com
Abstract
Diabetic foot ulcer often leads to amputation, and both nutritional status and
immune function have been associated with this process. We aimed to investi-
gate the risk factors of diabetic ulcer-related amputation including the Control-
ling Nutritional Status score and neutrophil-to-lymphocyte ratio biomarker.
We evaluated data from hospital in patients with diabetic foot ulcer, perform-
ing univariate and multivariate analyses to screen for high-risk factors and
Kaplan–Meier analysis to correlate high-risk factors with amputation-free sur-
vival. Overall, 389 patients underwent 247 amputations over the follow-up
period. After correction to relevant variables, we identified five independent
risk factors for diabetic ulcer-related amputation: ulcer severity, ulcer site,
peripheral arterial disease, neutrophil-to-lymphocyte ratio and nutritional sta-
tus. Amputation-free survival was lower for the moderate-to-severe versus mild
cases, for the plantar forefoot versus hindfoot location, for the concomitant
peripheral artery disease versus without and in the high versus low
neutrophil-to-lymphocyte ratio (all p < 0.01). The results showed that ulcer
severity (p < 0.01), ulcer site (p < 0.01), peripheral artery disease (p < 0.01),
neutrophil-to-lymphocyte ratio (p < 0.01) and Controlling Nutritional Status
score (p < 0.05) were independent risk factors for amputation in diabetic foot
ulcer patients and have predictive values for diabetic foot ulcer progression to
amputation.

KEYWORDS
Controlling Nutritional Status score, diabetic foot ulcer, lower extremity amputation,
neutrophil-to-lymphocyte ratio

Yandan Zhu and Hongtao Xu contributed equally to this study, as first authors.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2023 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.

4050 wileyonlinelibrary.com/journal/iwj Int Wound J. 2023;20:4050–4060.

ZHU ET AL. 4051

Key Messages
• diabetic foot ulcer is the leading cause of non-traumatic lower extremity
amputation, and ulcer development and complications have been correlated
with both nutritional status and neutrophil-to-lymphocyte ratio
• this case–control study aimed to evaluate the correlations of these two fac-
tors with amputation risk while performing an overall risk factor analysis
• we found five independent risk factors for diabetic foot ulcer-related ampu-
tations: ulcer severity, ulcer location, concomitant peripheral artery disease,
neutrophil-to-lymphocyte ratio and nutritional status (evaluated using the
Controlling Nutritional Status score)

1 | INTRODUCTION for DFU-related amputation, including NLR and

Diabetic foot ulcer (DFU) is among the most serious com-
plications of diabetes. Ulcerations of the distal lower
extremity, along with deep tissue destruction and necro-
sis, are the manifestations of damage induced by a combi-
nation of neurological, peripheral vascular and
biomechanical abnormalities in patients with diabetes.1
The global prevalence of DFU is 6.3%, with a wide varia-
tion between countries and regions, and ranges from
1.5% to 16.6%.2 DFU is associated with amputation. The
Chinese guidelines for the prevention and treatment of
the diabetic foot (2019 edition)3 reported the following
amputation rates for Chinese patients with DFU: 19.03%
for total, 2.14% for major and 16.88% for minor amputa-
tions. Diabetic ulcer-related amputation seriously affects
patients' quality of life and adds heavy burdens to both
individuals and society. Prediction and management of
the risks for amputation are, therefore, essential for
improving the prognosis of DFU. The development of
DFU is closely linked to nutritional status. According to
statistics, approximately 60% of affected patients suffer
from malnutrition.4 Malnutrition is influenced by many
factors. The nutrition of patients with DFU is worsened
by individual complications and comorbidities.5 Malnu-
trition affects immune function and increases the inci-
dence of infection6 and risk of amputation. The
neutrophil-to-lymphocyte ratio (NLR) is a novel marker
of immune–inflammatory responses. Recent studies have
shown that the NLR is predictive of DFU complications
and amputations7 and it could be a potential new thera-
peutic target for DFU.8
Several recent studies have investigated the risk fac-
tors for DFU-related amputation. However, a few
reports are available regarding the relationships among
nutritional status, NLR and amputation risk. The
Controlling Nutritional Status (CONUT) score is an
objective nutritional assessment that is simple, easy to
administer and suitable for all populations.9 This study
is a case–control trial designed to explore the risk factors
CONUT scores. The intent is to provide a reference for
physicians to screen high-risk patients and to individu-
alise treatment.
2 | MATERIALS AND METHODS
2.1 | Study participants
We selected patients with a primary diagnosis of DFU
admitted to our hospital between 1 January and
31 December 2021. The inclusion criteria were the fol-
lowing: (a) fulfilment of the diagnostic criteria for DFU
established by the International Working Group on the
Diabetic Foot (IWGDF), Wagner grades 1–5, with unilat-
eral onset; (b) age 30–90 years; (c) complete and reliable
clinical history; and (d) cooperation regarding scheduled
follow-up visits. The exclusion criteria were the follow-
ing: (a) diagnosis of type I diabetes or a specific type of
DFU; (b) patients with severe immunodeficiency;
(c) concomitant malignancy or other chronic wasting dis-
eases such as hepatitis or tuberculosis; (d) patients who
were pregnant or breastfeeding; (e) mental or intellectual
impediments to participation; (f) mortality during hospi-
talisation or over follow-up.
2.2 | Related definitions
The diagnostic criteria for DFU refer to the relevant reg-
ulations in the 2019 IWGDF guidelines. The diagnostic
criteria are as follows: current or prior diagnosis of dia-
betic foot infection, ulceration or tissue destruction, usu-
ally accompanied by lower limb neuropathy and/or
peripheral arterial disease (PAD) in the lower extrem-
ity.1 The foot lesions were graded according to the Wag-
ner Assessment System (grades 0–5).10 We divided the
cohort according to the Wagner classification as mild
(grades 1–2) and moderate-to-severe (grades 3–5) groups.
4052
The ulcer site was assessed as plantar forefoot or hind-
foot according to the position relative to the metatarsals.
Comorbidity was considered present for patients taking
relevant therapeutic drugs or for a history of relevant
conditions. Relevant conditions included hypertension:
taking antihypertensive medication, history of hyperten-
sion or systolic/diastolic blood pressure ≥140/90 mmHg
(1 mmHg = 0.133 kPa) on admission; cerebrovascular
accident (CVA): history of any events causing neurologi-
cal deficit, whether persistent or resolved; coronary
heart disease (CHD): history of angina pectoris or myo-
cardial infarction, any positive cardiac stress test results
or pathological signs on coronary angiography; PAD:
ABI <0.9, or moderate-to-severe stenosis or occlusion of
blood vessels in the lower limbs confirmed by lower limb
computed tomography angiography or colour Doppler
ultrasonography11; chronic kidney disease (CKD): glo-
merular filtration rate (eGFR) <60 mL/min/1.73 m2 as
defined by the National Kidney Disease Outcomes Qual-
ity Initiative (NKDOQI) guidelines12; current alcohol or
tobacco use: less than 1 month of abstinence at admis-
sion; previous alcohol or tobacco use: former user and
currently abstinent for at least 1 month; non-traumatic
amputation: previous amputation owing to DFU or
chronic lower limb ischaemia and amputation: the
removal of any part of the lower limb through the joint
or bone, with amputations below the ankle (including
toes and metatarsals) considered minor and above the
ankle major.13 For patients with multiple admissions for
DFU, only data from the first admission were included.
Follow-up continued until 30 June 2022.
2.3 | Sample size estimation
The general principle of sample size estimation for
observational studies is that the sample size for positive
outcome events should be at least 5–10 times the num-
ber of study variables.14 The number of amputations in
this study was 247, and the number of variables ranged
from 5 to 19, which largely met the statistical
requirements.
2.4 | Data collection and processing
General demographic information included the patients'
age, sex, height, weight, body mass index (BMI), and
alcohol and smoking history. Other information collected
included disease status, including duration of diabetes,
comorbidity, DFU duration, ulcer site and Wagner grad-
ing. DFU comorbidities included hypertension, CHD,
CVA, CKD and PAD. Laboratory indicators included
ZHU ET AL.
white blood cells, neutrophils, lymphocytes, haemoglobin
(HB), C-reactive protein (CRP), platelets, fibrinogen,
D-dimer, total protein (TP), albumin, prealbumin (PA),
triglycerides (TG), total cholesterol, high-density lipopro-
tein (HDL), low-density lipoprotein (LDL), fasting
plasma glucose (FPG), haemoglobin A1c (HbA1c), esti-
mated glomerular filtration rate (eGFR), uric acid, pro-
teinuria, erythrocyte sedimentation rate (ESR) and brain
natriuretic peptide (BNP). The receiver operating charac-
teristic (ROC) curve was used to determine the optimal
threshold for NLR, and patients were put into high- and
low-NLR groups. Using the CONUT score,9 albumin
scored 0 for the range 35–45 g/L, 2 for 30–34.9 g/L, 4 for
25–29 g/L and 6 for <25 g/L. Lymphocytes scored
0 for ≥1.6  109 L 1, 1 for 1.2  109–1.599  109 L 1, 2
for 0.8  109–1.199  109 L 1 and 3 for <0.8  109 L 1.
Total cholesterol scored 0 for >180 mg/dL, 1 for
140–180 mg/dL, 2 for 100–139 mg/dL and 3 for
<100 mg/dL. The scores of these three indicators were
added together to give a total CONUT score, where 0–1 is
normal, 2–4 is mild malnutrition, 5–8 is moderate malnu-
trition and 9–12 is severe malnutrition. Patients with
DFU were put into three groups according to the CONUT
score: normal nutrition (scores 0–1, C0), mild malnutri-
tion (scores 2–4, C1) or moderate-to-severe malnutrition
(scores 5–12, C2) groups.
2.5 | Statistical analyses
The Epidata 3.1 software was used to create a database of
clinical information on DFU patients. Continuous vari-
ables were expressed as mean ± standard deviation
(SD) for normally distributed variables, and the t-test was
used for comparison between the two (amputated
vs. non-amputated) groups. Variables with a non-normal
distribution were expressed as the median (lower quar-
tile, upper quartile), and the Mann–Whitney U test was
used for comparison between the groups. Categorical var-
iables were expressed as cases and percentages and com-
pared using the chi-square test or Fisher's exact
probability method. The variables were initially screened
by one-way analysis of variance ( p < 0.05); then, a step-
wise regression method was used to screen potential
influencing factors, and other variables were introduced
to correct for the influencing factors before conducting a
multivariate logistic regression analysis. Survival analysis
was performed using the Kaplan–Meier method, and dif-
ferences between the groups were compared using the
log-rank test. For those with missing data values, multi-
ple interpolations were used to fill in the data, depending
on the value type. Statistical significance was assessed
with p < 0.05.
ZHU ET AL.
3 | R E SUL T S
3.1 | Patient selection
Overall, 600 patients were admitted to our hospital from
1 January 2021 to 30 December 2021 with a primary diag-
nosis of DFU. After excluding four patients who died dur-
ing hospitalisation, the data of 596 patients with DFU were
analysed. One hundred and seventy-eight patients were
excluded after further screening according to inclusion cri-
teria, exclusion criteria and incomplete clinical informa-
tion. Of these, 27 patients were lost to follow-up, and two
patients died unexpectedly over follow-up. Ultimately,
389 patients with DFU were included in our analysis.
3.2 | General information of patients
with DFU
Of the 389 patients with DFU, 292 (75.1%) were male and
97 (24.9%) were female, with a male-to-female ratio of
3.01:1. The BMI is 23.44 (21.48, 24.62) kg/m2. Patient age
ranged from 32 to 90 years, and the median age was
68 (60, 74) years. Current and previous smokers accounted
for 139 (35.7%) and never-smokers for 250 (64.3%).
Current and previous drinkers were 61 (15.6%) and never-
drinkers were 328 (84.4%) patients. Amputations were
necessary in 247 and not required in 142 patients, yielding
an amputation rate of 64.5%. This included 195 minor
(50.1%) and 52 major (13.4%) amputations. There was
combined hypertension in 221 (56.8%), CVA in 54 (13.8%),
CHD in 98 (25.2%), PAD in 154 (39.6%) and CKD in
61 (15.7%) patients. The mean follow-up period for
389 patients was 5 months (range, 2–12 months).
3.3 | General and clinical characteristics
of patients with DFU
The proportion of patients with PAD was higher in the
amputation group than in the non-amputation group
(p < 0.01), and the differences between the groups were
not statistically significant for any other indicators
(p > 0.05) (Table 1).
3.4 | Conditions of foot ulcer in patients
with DFU
Of the 389 patients with DFU, 81 (20.8%) were in the
mild group and 308 (79.2%) in the moderate-to-severe
group. Combined plantar forefoot ulcers were present in
287 (73.8%) and combined hindfoot ulcers in 102 (26.2%).
4053
The proportion of patients with moderate-to-severe, plan-
tar forefoot ulcers was higher in the amputation group
than in the non-amputation group ( p < 0.01) (Table 2).
3.5 | Laboratory indicators of patients
with DFU
Patients in the amputation group had higher white blood
cells and platelets than those in the non-amputation
group (p < 0.01); those in the amputation group also had
higher levels of CRP, fibrinogen, D-dimer, HbA1c, BNP
and ESR than those in the non-amputation group
(p < 0.01). Levels of PA, HDL, TG and uric acid were
lower in the amputation group than in the non-
amputation group ( p < 0.01), whereas the proportion of
patients with high NLR, proteinuria, low HB and high
CONUT score was higher in the amputation than in the
non-amputation group (p < 0.01). Differences in
the remaining indicators were not statistically significant
between the two groups (p > 0.05) (Table 3).
3.6 | Multivariable dichotomous logistic
regression analysis to predict risk factor for
amputation
The variables described in Sections 3.3, 3.4 and 3.5 with
p < 0.05 were further screened for relevance using step-
wise regression by including them in a multivariate
dichotomous logistic regression analysis, after correcting
for the effects of age, BMI and duration of diabetes and
DFU. The results showed that ulcer severity (p < 0.01),
ulcer site ( p < 0.01), PAD ( p < 0.01), NLR ( p < 0.01) and
CONUT score ( p < 0.05) were independent risk factors
for amputation (Table 4).
3.7 | NLR ROC curve
The 389 patients with DFU were divided into a high NLR
group (NLR ≥3.594, 188 patients) and a low NLR group
(NLR <3.594, 201 patients). The cutoff was based on the
optimal threshold of the ROC curve for NLR (area under
the curve AUC value = 0.7239; sensitivity = 0.7465;
specificity = 0.6154; 95% CI = 0.6721–0.7757) (Figure 1).
3.8 | Kaplan–Meier curve of amputation-
free survival
Kaplan–Meier survival analysis was performed to investi-
gate the impact of risk factors on amputation.
4054 ZHU ET AL.

TABLE 1 General and clinical characteristics of patients with DFU in amputation and non-amputation groups.

Amputation Non-amputation
group group
Characteristics N = 247 N = 142 p-value
Age, year 67.3 ± 0.7 66.5 ± 1.0 0.504
Male, n (%) 189 (76.5) 103 (72.5) 0.382
2
BMI, kg/m 23.39 (21.26, 24.49) 23.59 (21.69, 25.13) 0.073
Smoking, n (%) 0.089
Current and previous 96 (38.9) 43 (30.3)
Never 151 (61.1) 99 (69.7)
Drinking, n (%) 0.938
Current and previous 39 (15.8) 22 (15.5)
Never 208 (84.2) 120 (84.5)
Duration of diabetes, years 17 (10, 20) 18 (10,20) 0.693
Duration of diabetic foot ulcer, n (%) 0.288
<1 months 81 (32.8) 56 (39.4)
1–3 months 128 (51.8) 62 (43.7)
>3 months 38 (15.4) 24 (16.9)
History of lower extremity amputation, n (%) 16 (6.5) 12 (8.5) 0.469
Comorbidity, n (%)
Hypertension 143 (57.9) 78 (54.9) 0.570
CVA 36 (14.6) 18 (12.7) 0.602
CHD 66 (26.7) 32 (22.5) 0.360
PAD 120 (48.6) 34 (24.0) <0.01
CKD 34 (13.7) 27 (19.0) 0.170

Note: Values are presented as mean ± SD, median (lower quartile, upper quartile) or number (%).
Abbreviations: BMI, body mass index; CHD, coronary heart disease; CKD, chronic kidney disease; CVA, cerebrovascular accident; DFU, diabetic foot ulcer;
PAD, Peripheral arterial disease.

T A B L E 2 Conditions of foot ulcer in patients with DFU in was 95.1% in the mild group, and the median
amputation and non-amputation groups. amputation-free survival time was 3 ± 0.486 (2.408,
3.952) months in the moderate-to-severe group.
Amputation
group Non-amputation
Amputation-free survival was lower in the plantar fore-
Conditions N = 247 group N = 142 p value foot group than in the hindfoot group (p < 0.01)
(Figure 2B). Median amputation-free survival time was
Ulcer severity, <0.01
n (%)
4 ± 0.648 (95% CI = 2.730–5.270) and 20 ± 5.796 (95%
CI = 8.639–31.361) months in the plantar forefoot and
Mild (1, 2) 5 (2.0) 76 (53.5)
hindfoot groups, respectively. Amputation-free survival
Moderate-to- 242 (98.0) 66 (46.5)
was lower in the PAD group than in the non-PAD group
severe (3–5)
(p < 0.01) (Figure 2C). The median amputation-free sur-
Ulcer site, n (%) <0.01 vival times were 4 ± 0.688 (2.652, 5.348) and 10 ± 2.884
Plantar forefoot 200 (81.0) 87 (61.3) (4.348, 15.652) months in the PAD and non-PAD groups,
Hindfoot 47 (19.0) 55 (38.7) respectively. Amputation-free survival was lower in the
Abbreviation: DFU, diabetic foot ulcer.
high NLR group than in the low NLR group (p < 0.01)
(Figure 2D). The 6-month amputation-free survival
rate in the low NLR group was 62.1%, and the
Amputation-free survival was lower in the moderate- median amputation-free survival time in the high NLR
to-severe group than in the mild group ( p < 0.01) group was 3 ± 0.302 (95% CI = 2.408–3.592) months.
(Figure 2A). The 6-month amputation-free survival rate Amputation-free survival was higher in the group with
ZHU ET AL. 4055

TABLE 3 Laboratory indicators of patients with DFU in amputation and non-amputation groups.

Amputation Non-amputation
Indicators group, N = 247 group, N = 142 p value
9
White blood cells, 10 /L 8.6 (6.5, 12.4) 6.9 (5.5, 9.3) <0.01
Lymphocyte, 109/L 1.45 (1.10,18.81) 1.63 (1.27,2.11) <0.01
High NLR, n (%) 152 (61.5) 36 (25.4) <0.01
HB<120 g/L, n (%) 176 (71.3) 71 (50.0) <0.01
9
Platelets, 10 /L 305.1 ± 7.0 272.5 ± 9.0 <0.01
CRP, mg/L 43 (5, 106) 2 (1, 20) <0.01
Fibrinogen, g/L 4.37 (3.69, 4.95) 3.51 (3.09, 4.37) <0.01
D-dimer, ng/mL 271.0 (166.0, 466.0) 207.0 (111.3, 334.0) <0.01
TP, g/L 63 (58, 68) 65 (61, 68) 0.133
PA, g/L 0.14 (0.07, 0.21) 0.22 (0.15, 0.27) <0.01
HDL, mmol/L 0.83 (0.70, 1.04) 1.01 (0.83, 1.16) <0.01
LDL, mmol/L 2.36 (1.69, 2.94) 2.38 (1.78, 3.14) 0.264
TG, mmol/L 1.13 (0.90, 1.44) 1.26 (1.00, 1.78) <0.01
FPG, mmol/L 8.0 (5.5, 11.0) 7.7 (5.2, 10.3) 0.314
Uric acid, μmol/L 260 (209, 353) 303.5 (234.5, 382.75) <0.01
Proteinuria (%) 119(48.2) 36(25.4) <0.01
HbA1c (%) 9.50 (7.80, 11.10) 8.75 (7.20, 10.30) <0.05
BNP, pg/mL 107 (47, 232) 57.5 (29.75, 110.75) <0.01
ESR, mm/H 79 (48, 96) 46.5 (17, 81) <0.01
CONUT, n (%) <0.01
0–1 (CO) 29 (11.7) 38 (26.8)
2–4 (C1) 81 (32.8) 72 (50.7)
5–12 (C2) 137 (55.5) 32 (22.5)

Note: Values are presented as mean ± SD, median (lower quartile, upper quartile) or number (%).
Abbreviations: BNP, brain natriuretic peptide; CONUT, Controlling Nutritional Status; CRP, C-reactive protein; DFU, diabetic foot ulcer; ESR, erythrocyte
sedimentation rate; FPG, fasting plasma glucose; HbA1c, haemoglobin A1c; HB, haemoglobin; HDL, high-density lipoprotein; LDL, low- density lipoprotein;
NLR, neutrophil to lymphocyte ratio; PA, prealbumin; TG, triglyceride; TP, total protein.

T A B L E 4 Multivariable dichotomous logistic regression 1.0

analysis of DFU amputation.

Variables OR 95% CI p value 0.8

Ulcer severity 59.804 20.299–176.190 <0.01

Sensitivity

0.6
Ulcer site 0.130 0.061–0.277 <0.01
PAD 4.443 2.142–9.216 <0.01 0.4
NLR 3.811 1.885–7.707 <0.01 AUC : 0.7239
95% CI : 0.6721–0.7757
CONUT score 2.063 1.317–3.230 <0.05 0.2 P<0.01

Abbreviations: CONUT, Controlling Nutritional Status; DFU, diabetic foot

ulcer; NLR, neutrophil to lymphocyte ratio; PAD, peripheral arterial disease. 0.0
0.0 0.2 0.4 0.6 0.8 1.0
1 - Specificity
normal nutritional status (C0) than in the groups with
mild malnutrition (C1) and moderate-to-severe malnutri- F I G U R E 1 NLR ROC curve of DFU patients. DFU, diabetic
tion (C2), and the difference in survival curves between foot ulcer; NLR, neutrophil to lymphocyte ratio; ROC, receiver
the three groups was statistically significant (C0 vs. C1: operating characteristic curve.
4056 ZHU ET AL.

(A) (B)
100 100
Plantar forefoot

Probability of Survival
Probability of Survival 80 80
Hind plantar

P<0.01
Mild DFU
60 60
Moderate-to-severe DFU
P<0.01
40 40

20 20

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time(mouths) Time(mouths)

(C) (D) 100

100
non-PAD low NLR
high NLR

Probability of Survival
Probability of Survival

PAD
80 80 P<0.01
P<0.01

60 60

40 40

20 20

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time(mouths) Time(mouths)

(E) 100 C0

C1
Probability of Survival

80 C2

P<0.05
60

40

20

0
0 5 10 15 20 25 30
Time(mouths)

F I G U R E 2 Amputation-free survival for the amputation cohort. (A) The KM survival curves indicate survival for the amputation cohort
with mild DFU and moderate-to-severe DFU. (B) The KM survival curves indicate survival for the amputation cohort with plantar forefoot
and hindfoot. (C) The KM survival curves indicate survival for the amputation cohort with non-PAD and PAD. (D) The KM survival curves
indicate survival for the amputation cohort with low NLR and high NLR. (E) The KM survival curves show major amputation-free survival
for the amputation cohort with C0, C1 and C2. C0, normal nutritional status group; C1, mild malnutrition group; C2, moderate-to-severe
malnutrition group; DFU, diabetic foot ulcer; NLR, neutrophil to lymphocyte ratio.

p < 0.05; CO vs. C2: p < 0.01; C1 vs. C2: p < 0.01) for C1 and C2 were 9 ± 2.935 (95% CI = 3.248–14.752)
(Figure 2E). The 6-month amputation-free survival rate months and 3 ± 0.323 (95% CI = 2.367–3.633) months,
for CO was 70.4%. Median amputation-free survival times respectively.
ZHU ET AL.
4 | DISCUSSION
DFU is a global healthcare problem, with a 5-year sur-
vival rate of 44% after related amputation.15 To date,
numerous studies have reported risk factors for amputa-
tion in DFU. However, the results of each predictor have
not been consistent across studies, and this variability
may be due to the different study designs and popula-
tions. Our study further explored the clinical and bio-
chemical factors associated with the risk of DFU-related
amputation by including CONUT score and NLR. After
screening potential high-risk factors for amputation using
univariate and multivariate analyses, we further per-
formed multivariate logistic regression analysis using
stepwise regression and after correction to the effects of
other factors. We identified five independent risk factors
for amputation, that is, ulcer severity, ulcer site, PAD,
NLR and CONUT score.
Wagner grading was used to assess ulcer depth, osteo-
myelitis and necrosis/gangrene on a scale ranging from
0 to 5. Almost all DFU-related amputation risk studies
have highlighted the reliability of the Wagner classifica-
tion in predicting amputation.16,17 Because of the ulcer
severity that increases with the Wagner classification
grade, more extensive wounds often require amputation.
In our study, the risk of amputation was significantly
increased in moderate-to-severe cases (Wagner grade
3–5) (OR = 59.804, 95% CI = 20.299–176.190), and the
median amputation-free survival time was 3 months. Pic-
well et al.18 found that the most common ulcer site of
DFU was the forefoot (56%) through analysis of 1000
patients in the Eurodiale study. Lee et al.19 found that
forefoot DFU is a strong risk factor for amputation. The
blood supply to the forefoot region, with its distal loca-
tion, is often inadequate. Thus, forefoot DFU is often
treated with minor amputation, including partial or com-
plete toe amputation.20 Our results supported this find-
ing, with an amputation rate of 50.1% (n = 195) for
forefoot ulcer (OR = 0.130, 95% CI = 0.061–0.277) and a
median amputation-free survival time of 4 months. PAD,
a marker of atherosclerosis, has been widely discussed as
an independent risk factor for DFU-related amputation.17
One study of cardiovascular disease risk factors found
that the relative risk of cardiovascular death in patients
with PAD was 1.69.21 PAD can lead to intermittent clau-
dication, rest pain, refractory ulcer and gangrene.22 It can
also reduce wound antibiotic concentrations and increase
the proliferation of multi-drug-resistant bacteria, which,
in turn, increase the chances of amputation.23 In a study
by Ugwu et al.,24 the presence of PAD increased the rate
of DFU-related amputation by nearly threefold. In this
study, patients with concomitant PAD had a significantly
increased risk of amputation (OR = 4.443, 95%
4057
CI = 2.142–9.216) and a median amputation-free survival
time of 4 months.
Malnutrition is common in patients with DFU and
has a significant impact on prognosis. Hong et al.25
assessed all-cause mortality in 771 patients with type
2 diabetes and DFU using various nutritional indices and
found that all were valid predictors of all-cause mortality.
In Scotland, Chamberlain et al.26 found an association
between malnutrition and the risks of amputation and/or
all-cause mortality in patients with diabetes. There are
many potential contributing factors to the development
of malnutrition in patients with DFU. These patients
have reduced nutritional intake and absorption due to
poor appetite, poor dietary structure and the frequent
presence of multiple chronic comorbidities.27 Prolonged
non-healing of the wound, localised bleeding, oozing and
a persistent inflammatory state can lead to hypoprotei-
naemia by increasing capillary permeability and promot-
ing protein degradation.28 In addition, reduced mobility
in patients with DFU may lead to disuse muscular atro-
phy, which would decrease the net muscle protein con-
tent.29 Therefore, patients with DFU tend to have varying
degrees of malnutrition. Compared with patients with
mild DFU (Wagner grades 1–2), patients with moderate-
to-severe DFU (Wagner grades 3–5) have deeper ulcer,
require more nutrients such as protein from the body for
wound repair and have a more pronounced poor nutri-
tional status.25 Therefore, patients with DFU require
prompt nutritional assessment. Owing to the lack of a
consensus definition for malnutrition, there are various
methods for assessing it, such as BMI, serum albumin
and nutritional assessment tools. BMI or albumin levels
alone are no longer considered reliable indicators of
nutritional status.30,31 A variety of clinical nutrition
assessment tools, such as the mini nutrition Assessment
(MNA), subjective global assessment (SGA) and nutri-
tional risk screening 2002 (NRS 2002), are not always
easy to implement in clinical practice. The CONUT score
is an objective nutritional assessment that uses three
objective biomarkers, total lymphocyte count, total cho-
lesterol level and serum albumin level, to assess the
nutritional status in terms of protein metabolism,
immune function and lipid metabolism.9 The CONUT
score is a valid indicator of mortality32 and foot ulcer
healing.33 In this study, patients were divided into three
groups according to the CONUT score: normal nutri-
tional status (C0), mild malnutrition (C1) and moderate-
to-severe malnutrition (C2). The relationship between
CONUT scores and amputation was evaluated, and the
CONUT score was an independent factor affecting ampu-
tation (OR = 2.063, 95% CI = 1.317–3.230). In addition,
the 6-month amputation-free survival rate was 70.4% in
the normal nutritional status group, and the median
4058
amputation-free survival time was longer in the mild ver-
sus the moderate-to-severe malnutrition group (9 vs.
3 months). Therefore, the CONUT score may be a poten-
tial biomarker for predicting DFU-related amputation.
As nutritional status is closely related to the immune
system function, patients with poor nutritional status are
more susceptible to infection,34 which may help explain
the high level of infection-related complications and mor-
talities. An NLR is a novel immune-inflammatory
response marker that can be used to assess the suppres-
sive and excitatory activities of the immune system.35
Activated neutrophils infiltrate the vessel walls and
release a variety of protein hydrolases, including elastase,
which mediates both basement membrane degradation
and vascular endothelial damage.36 Lymphocytes act in
the opposite way and are able to play anti-inflammatory
and protective roles for the endothelium; however, their
numbers are reduced by an inflammatory and high-
glucose environment.37 Chen et al.38 showed that higher
NLR may be a reliable predictive biomarker of mortality
in patients with DFU-related amputations. Demirdal
et al.7 found that the NLR was higher in amputated ver-
sus non-amputated patients and that the NLR could be
used to predict the DFU-related amputation risk. Our
study also confirmed this result. This was accomplished
by dividing the enrolled cases into high- and low-NLR
groups based on the optimal NLR threshold from the
ROC curve. The results suggest high NLR was an inde-
pendent factor affecting amputation (OR = 3.811, 95%
CI = 1.885–7.707), and amputation-free survival was
lower in the high than in the low NLR group (p < 0.01).
Poor blood glucose control is considered a risk factor
for amputation in patients with DFU. However, results
reported in relevant studies are inconsistent. Shatnawi
et al.39 found that high HbA1c levels were an important
risk factor and a significant predictor of DFU-related
amputation. In contrast, Moon et al.19 reported that low
HbA1c levels are associated with major amputations in
patients with DFU. Chronic hyperglycaemia disrupts
wound healing in DFU, but hypoglycaemia may also
cause a stress response and induce immune disorders
that slow wound healing.40 In this study, univariate anal-
ysis showed higher HbA1c levels in the amputation ver-
sus the non-amputation group (p < 0.05), but the final
logistic regression analysis did not show a significant
difference.
5 | C ON C L U S I ON
The results showed that ulcer severity ( p < 0.01), ulcer
site (p < 0.01), PAD ( p < 0.01), NLR (p < 0.01) and
CONUT score (p < 0.05) were independent risk factors
ZHU ET AL.
for amputation in DFU patients and have predictive
values for DFU progression to amputation.
6 | LIMITATION
First, because of the limitations of the inclusion and
exclusion criteria for this study, ultimately, 211 of
600 patients were eliminated from the study. Undeniably,
this has some limitations. Second, this study was a retro-
spective trial, and because of limited data availability,
many potential risk factors were not analysed. It was also
difficult to determine causal relationships between the
risk factors and the amputation variables. Third, this
study lacked information on subjective nutritional indica-
tors and reduction in skeletal muscle mass. Finally, selec-
tion bias could not be ruled out. Our hospital is a tertiary
referral centre, and patients who visit or are referred to
our hospital may already have deteriorating DFU. There-
fore, the results of this study may not apply to the general
population. Finally, this study calculated survival rates
over a relatively short period. More research is needed in
future.
A C KN O WL ED G EME N T S
We are very grateful for the support of the doctors at the
Department of Vascular Disease at the Shanghai Tradi-
tional Chinese Medicine Integrated Hospital, Shanghai
University of Traditional Chinese Medicine, to complete
this study.
F U N D I N G IN F O R M A T I O N
This study was partially supported by the 13th Five-Year
Clinical Key Specialties (Traditional Chinese Medicine
Surgery) of Shanghai Municipal Health Commission,
National Natural Science Foundation of China (Grant
No. 82174382), Shanghai Health Commission Clinical
Research Special (Grant No. 20224Y0387), Shanghai
Hongkou District Health Commission Traditional
Chinese Medicine Research Project Grant (Grant No.
HKQ-ZYY-2021-10).
C O N F L I C T O F I N T E R E S T S T A TE M E N T
All authors declare that they have no financial or other
conflicts of interest in relation to this research and its
publication.
DA TA AVAI LA BI LI TY S T ATE ME NT
The data that support the findings of this study are avail-
able from the corresponding author on reasonable request.
ORCID
Yandan Zhu https://orcid.org/0000-0001-8673-1186
ZHU ET AL.
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How to cite this article: Zhu Y, Xu H, Wang Y,
et al. Risk factor analysis for diabetic foot ulcer-
related amputation including Controlling
Nutritional Status score and neutrophil-to-
lymphocyte ratio. Int Wound J. 2023;20(10):
4050‐4060. doi:10.1111/iwj.14296