SMJ

E-ISSN 2228-8082

Volume 74, Number 12, December 2022

Siriraj Medical Journal

The world-leading biomedical science of Thailand

By Kantnatt Charatcharoenwitthaya, et al.

ORIGINAL ARTICLE REVIEW ARTICLE

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THAILAND SECTION 1954

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E-mail: sijournal92@gmail.com

Siriraj Medical Journal SMJ‌

Volume 74, Number 12, December 2022

ORIGINAL ARTICLE

828 Proinflammatory Cytokines Associated with InvA and PagC Genes of Salmonella typhi

Isolated from Patients Undergoing Cholecystectomy

Ali J. Eidan

836 Impact of the COVID-19 Pandemic on Tertiary-care University Dermatology Outpatient Clinic and Dermatology Procedures

Kingkaew Wamaphutta, et al.

844 Are Technical Skills Assessed Using Medical Knowledge Associated with Non-technical Skill Knowledge in Anaesthesia Resident Training?

Maliwan Oofuvong, et al.

857 Anxiety, Depression and Cognitive Emotion Regulation Strategies in Psychiatric Patients during the COVID-19 Pandemic

Woraphat Ratta-apha, et al.

865 Clinical Characteristics and Treatment Outcomes of Patients with Primary Ocular Adnexal Lyphoma at Siriraj Hospital

Sumalee Vangveeravong, et al

874 Peripheral Atherosclerotic Profile in Type 1 Diabetic Patients: Lipid Ratios as a Predictive Marker of Asymptomatic Patients

Walid Hassene HAMRI, et al.

883 Effects of Different Durations of 9-Square Dance Exercise Versus Treadmill Exercise on the Physical Fitness and Quality of Life of Healthy Volunteers: A Pilot Randomized Controlled Trial

Sarunthorn Puttipaibool, et al.

REVIEW ARTICLE

891 Radiologist’s Role in Artificial Intelligence Era

Sornsupha Limchareon, et al.

895 The Impact of Lockdown during COVID-19 Pandemic on Physical and Mental Health of Adolescents

Kantnatt Charatcharoenwitthaya, et al.

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Ichizo Nishino (National Institute of Neuroscience NCNP, Japan)

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Sara Schwanke Khilji (Oregon Health & Science University, USA) Matthew S. Dunne (Institute of Food, Nutrition, and Health, Switzerland) Marianne Hokland (University of Aarhus, Denmark)

Marcela Hermoso Ramello (University of Chile, Chile) Ciro Isidoro (University of Novara, Italy)

Moses Rodriguez (Mayo Clinic, USA)

Robert W. Mann (University of Hawaii, USA)

Wikrom Karnsakul (Johns Hopkins Children’s Center, USA)

Frans Laurens Moll (University Medical Center Ultrecht, Netherlands) James P. Dolan (Oregon Health & Science University, USA)

John Hunter (Oregon Health & Science University, USA) Nima Rezaei (Tehran University of Medical Sciences, Iran) Dennis J. Janisse (Subsidiary of DJO Global, USA)

Folker Meyer (Argonne National Laboratory, USA)

David Wayne Ussery (University of Arkansas for Medical Sciences, USA) Intawat Nookaew (University of Arkansas for Medical Sciences, USA)

Victor Manuel Charoenrook de la Fuente (Centro de Oftalmologia Barraquer, Spain)

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(Swedish University of Agricultural Sciences, Sweden)

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Anuwat Pongkunakorn (Lampang Hospital, Thailand) Nopporn Sittisombut (Chiang Mai University, Thailand)

Vasant Sumethkul (Ramathibodi Hospital, Mahidol University, Thailand) Yuen Tanniradorm (Chulalongkorn University, Thailand)

Saranatra Waikakul (Siriraj Hospital, Mahidol University, Thailand) Pa-thai Yenchitsomanus (Siriraj Hospital, Mahidol University, Thailand) Surapol Issaragrisil (Siriraj Hospital, Mahidol University,Thailand) Jaturat Kanpittaya (Khon Kaen University, Thailand)

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Statistician: Saowalak Hunnangkul (Mahidol University, Thailand)

Nopphol Pausawasdi (Siriraj Hospital, Mahidol University, Thailand) Supakorn Rojananin (Siriraj Hospital, Mahidol University, Thailand) Jarupim Soongswang (Siriraj Hospital, Mahidol University, Thailand) Suttipong Wacharasindhu (Chulalongkorn University, Thailand) Prapon Wilairat (Mahidol University, Thailand)

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Proinflammatory Cytokines Associated with InvA and PagC Genes of Salmonella typhi Isolated from Patients Undergoing Cholecystectomy

Ali J. Eidan, Ph.D.

Department of Basic Science, Faculty of Nursing, University of Kufa, Al-Najaf, Iraq.

ABSTRACT

Objective: Cholecystectomy is the surgical removal of the gallbladder. The gallbladder is often colonized by Salmonella during typhoid fever. The association between cytokine and virulence genes of Salmonella is not well known. The current study aims to identify the association between proinflammatory cytokines, TNF-α and IFN-γ, and two virulence genes, InvA and PagC, extracted from S. typhi isolates obtained from patients undergoing cholecystectomy. Materials and Methods: One hundred and fifty clinical specimens, including gallbladder tissues and blood, were collected from patients undergoing cholecystectomy at AL-Sadder Medical City and AL-Furat General Hospital/ Iraq from December 2019 to September 2020. A Monoplex PCR technique was used to detect InvA and PagC, and an enzyme-linked immunosorbent assay was used to measure serum levels of TNF-α and IFNγ.

Results: From a total of 66 S. typhi isolates, the prevalence of InvA and PagC was (84.8% and 69.6%) respectively. Even after adjusting for potential confounding factors, serum levels of TNF-α and INF-γ were statistically significantly higher in PagC detected in S. typhi isolates compared to PagC not detected (p-value<0.001), but there was no significant difference between InvA detected in S. typhi isolates and InvA not detected (p-value > 0.05).

Conclusion: The virulence genes of S. typhi, especially PagC, may be considered a potent inducer of proinflammatory cytokine secretion. Further studies are needed to present the patient‘s additional clinical course according to cytokine level.

Keywords: Typhoid; cytokine; TNF-α; IFN-γ; InvA; PagC (Siriraj Med J 2022; 74: 828-835)

INTRODUCTION

Cholecystectomy operations have been developed from open to laparoscopic surgery, which helps in reducing postoperative pain, returning faster to daily activities, and shortening the recovery period.1,2 It remains the most effective treatment option for Salmonella enterica serovar typhi (S. typhi) carriers with gallstones.3 S. typhi is a human-restricted pathogen that causes typhoid fever, a disease transmitted primarily through the fecal-oral route. Typhoid fever is considered to be one of the most important and under-reported diseases in the developing

world. Based on the most recent global estimates, 9.9 and 24.2 million incident cases and 75,000–208,000 typhoid-related deaths occur annually.4 In Asia, the incidence was more than 100 cases per 105 people per year, mostly among travelers; large outbreaks of

S. typhi that are multidrug resistant have been observed in both urban and rural areas where access to sanitary facilities, food, and water are limited.5 In Iraq, Mousa et al. estimated the incidence of typhoid fever to be 34.2 cases/105 individuals in 2012.6

Corresponding author: Ali J. Eidan

E-mail: alij.abosaibee@uokufa.edu.iq

Received 29 August 2022 Revised 16 October 2022 Accepted 19 October 2022 ORCID ID:http://orcid.org/0000-0001-5545-7380 http://dx.doi.org/10.33192/Smj.2022.97

All material is licensed under terms of the Creative Commons Attribution 4.0 International (CC-BY-NC-ND 4.0) license unless otherwise stated.

Typhoid fever is associated with a wide range of clinical symptoms from asymptomatic to severe, including fever, headache, malaise, and perforation of the ileum as complications of ileal ulceration. When some people are exposed to S. typhi, they become carriers, as they keep bacteria in the gallbladder that are responsible for storing and excreting bile from the liver to the small intestine.7 Abdominal ultrasound screening showed an ulcerative inflammatory reaction with fluid accumulation and an obvious thickening of the gallbladder in more than 59% of patients with typhoid fever, which may be these features in response to a local infection.8

With the advent of molecular biology, including DNA fingerprinting and plasmid profiling, many vivo studies investigate gene variations of S. typhi that help bacterial organisms to adhere and survive in different environmental niches, as strains that lack the invasion machinery have drastically reduced capacity to colonize, spread9 and/or drive inflammation10 in mouse models. To our knowledge, live attenuated S. typhi strains delivered orally to humans or mice infected with S. typhimurium, which causes an illness similar to typhoid, have both been extensively used to study immune responses. These investigations showed a paradoxical phenomenon whereby the excessive synthesis of tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) by T cells reduces the protective immune response to S. typhi antigens.11 The current study aims to identify the association between proinflammatory cytokines, TNF-α and IFN-γ, and two virulence genes, InvA and PagC, extracted from

S. typhi isolates obtained from patients undergoing cholecystectomy.

MATERIALS AND METHODS

Following the acceptance of the Nursing Faculty‘s Ethical Review Board at the University of Kufa in Iraq (N: 10/05 on 02/September/2019) for our proposal, a case-control study was conducted in AL-Sadder Medical City and AL-Furat General Hospital/Iraq, from December 2019 to September 2020.

Subjects

One hundred and fifty patients undergoing either laparoscopic or open cholecystectomy were included as the study group. The patients were admitted to the hospital before 24 hours of surgery for preoperative investigations and assessment. Before surgery, no antimicrobial substances were prescribed while 3rd generation cephalosporins were given intra-operatively. Thirty individuals were included as the control group without suffering from acute fever or calcular cholecystitis. All subjects gave their written informed consent before enrollment.

Sample collection

A five mL blood sample was obtained from each participants by venous puncture and placed in a plain tube. After a blood clot, centrifugation at (3 x 103 rpm) for 20 minutes was used to separate the serum. All sera were stored at -20°C and had not been thawed before being analyzed. Gallbladder tissues taken from each patient after surgery were used for culture and immediately frozen at -20°C for DNA extraction.

Isolation and Identification of S. typhi

Sections of the gallbladder‘s tissue were injected into a Stewart transport medium (Hampshire, UK). In a sterile mortar, (1g) of gallbladder tissue was crushed and pestled in (1ml) saline, then it was cultured on selenite cystine medium (Hampshire, UK) and incubated for 24 hrs. at 37°C. For identification of individual colonies, they were subcultured on MacConkey agar, Salmonella Shigella agar, and Xylose Lysine Deoxycholate agar, as well as gram staining, IMViC test, triple sugar iron test, motility, lysine, and urease decarboxylase activities, were performed. The final identification was detected using an automated VITEK-2 compact system (BioMérieux –France). After these identification tests, we obtained 66 isolates of S. typhi out of 150 collected clinical specimens, and the remaining samples (84) were excluded from the current study.

By using the slide agglutination method, Salmonella antisera were performed for serotype and to determine corresponding antigens of S. typhi (somatic, flagellar, and virulence (Vi)) in the healthy control group according to the product‘s instructions.

Molecular Study

Genomic deoxyribonucleic acid (DNA) was extracted by using a DNA extraction kit (promega, USA), and a Monoplex PCR technique was performed to determine InvA and PagC. The forward and reverse primers used in this study for the InvA were 5'-ACA GTG CTC GTT TAC GAC CTG AAT-3' and 5'-AGA CGA CTG GTA

CTG ATC GAT AAT-3', resulting in a 260-bp product.12 PagC forward and reverse primers were 5'-CGC CTT TTC CGT GGG GTA TGC-3' and 5'-GAA GCC GTT

TAT TTT TGT AGA GGA GAT GTT-3', respectively, yielding a 454-bp product.13

For PCR reaction, a 20µL tube was used, which contained 5 μl of master mix (Taq DNA polymerase, Tris-HCL pH9, dNTPs, 30 mM KCL,1.5 mM MgCl2,

and Track dye from Bioneer company-Korea), 5μl DNA template, 2.5μl form each forward and reverse primer, and 5μl deionized water. PCR programming conditions for InvA were 35 cycles of initial denaturation at 94°C

for 2 min, denaturation at 95°C for 1 min, annealing at 58°C for 1 min, elongation at 75°C for 1 min, and final elongation at 72°C for 10 min. PCR programming conditions for PagC were 25 cycles of initial denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 66.5°C for 30 s, elongation at 72°C for 2 min, and final elongation at 72°C for 10 min.

Proinflammatory cytokines

TNF-α was measured in serum samples using an enzyme-linked immunosorbent assay Kit (ab46087, UAS). IFNγ was measured in serum samples using an enzyme-linked immunosorbent assay Kit (ab174443, UAS). According to each standard curve and the dilution factor, the TNF-α and IFNγ concentrations in each plate were calculated.

Statistical analyses

A descriptive analysis of the qualitative variables was carried out, presenting the frequencies, median and interquartile range. A statistically significant difference was assessed by Chi- square test (X2), student‘s t test, and Kruskal-Wallis test. For post hoc comparisons, the Dunnett C technique was applied. A general linear model analysis of covariance was conducted for adjusting for potential confounding factors. The statistical tests were executed using the GraphPad Prism version 8 software. Statistical significance was determined at a p-value < 0.05.

RESULTS

Demographic data of S. typhi- infected patients:

Demographic and clincal data of S. typhi-infected patients are summarized in Table 1.

The S. typhi-infected patients included 41 females and 25 males, with a mean age of 49.7±10.3 years (range, 32-65 years). The healthy individuals included 17 females and 13 males with a mean age of 50.6±8.7 years (range, 36-63 years). The clinical features of S. typhi-infected patients were 59 cases of pain, 51 cases of anorexia, 43 cases of nausea, 44 cases of vomiting, and 57 cases of fever, while the study did not find any of these features in healthy individuals.

For smoking and drinking alcohol, we found 27 cases of smoking and only two cases of drinking alcohol in patients, while there were six smokers and no drinkers in the control group. For other diseases, there were 15 patients who had other diseases and were taking different drugs. Furthermore, total white blood cells (WBCs) count were higher in patients than in the control group. As shown in Table 1, with the exception of age, gender, and drinking alcohol, the S. typhi-infected patients and

control group significantly differed in demographic and clinical features (p-value <0.05).

Monoplex PCR Detection of Virulence Genes of

S. typhi Isolates:

From a total of 66 S. typhi isolates, the prevalence of virulence genes was 56 (84.8%) for InvA and 46 (69.6%) for PagC (Fig 1).

Fig 1. Prevalence of InvA and PagC, in S. typhi isolates which were collected from patients undergoing cholecystectomy by monoplex PCR assay.

By agarose gel electrophoresis, InvA and PagC were demonstrated in Fig 2A & 2B, respectively.

Fig 2. Ethidium bromide-stained agarose gel showing monoplex PCR for detection of the (A) InvA and (B) PagC of S. typhi. For both genes, lane 1: molecular weight marker and lanes 1 to 8: amplification products.

TABLE 1. Demographic and clinical data of participants (S. typhi-infected patients and healthy control).

0.612

<0.001**

Nausea

Yes 43 65.15% - - X2 = 35.40

Vomiting Yes

No

44

22

66.67%

33.33%

-

30

-

100%

X2 = 36.92

df = 1

<0.001**

No 23 34.85% 30 100% df = 1

<0.001**

Fever

< 38 9 13.64% 30 100% X2 = 4.514

Smoking Yes

No

27

39

40.91%

59.09%

6

24

20%

80%

X2 = 3.997

df = 1

0.046*

>= 38 57 86.36% - - df = 1

0.034*

Alcohol drink

0.566

0.004**

0.004**

Abbreviations: RUQ= Right upper quadrant; WBCs=White blood cells

*= significant; **= high significant

Proinflammatory cytokines findings

A general linear model analysis was conducted with age and total WBCs count as continuous predictors; gender, abdominal pain, anorexia, nausea, vomiting, fever, smoking, drinking alcohol, drugs, and other diseases as categorical predictors; and pro-inflammatory cytokines as dependent variables. Following their adjustment as covariates, the serum levels of TNF-α was statistically significantly different in S. typhi-infected patients and healthy control groups (X2=50.46, df=2, p-value <0.001) (Fig 3).

Moreover, Fig 4 shows that, like TNF-α, the serum level of INF-γ was statistically significantly different in

200

175

150

125

100

75

50

TNF-α (Pg\ml)

✱ ✱ ✱ ✱

NS ✱ ✱ ✱ ✱ NS

S. typhi-infected patients and healthy control group (X2=46.99, df=2, p-value <0.001).

According to InvA gene detection, the post hoc comparsion revealed that the serum level of TNF-α was statistically significantly higher in InvA detected in

S. typhi isolates (143.1, (97.5-149.8)) compared to healthy control (85,1 (79.9-91.4)) (X2=36.14, df=2, p-value <0.001),

and it was statistically significantly higher in InvA not detected (143.4, (136.4-148.1)) compared to healthy control (X2=42.0, df=2, p-value <0.001), but there was no significant difference between InvA detected and InvA not detected (X2=-5.88, df=2, p-value >0.05) (Fig 3). Like TNF-α, the post hoc comparsion of InvA revealed that the serum level of INF-γ were statistically significantly higher in InvA detected in S. typhi isolates (21.2, (13.7-22.5)) compared to healthy control (11.6 (9.9-14.6)) (X2=27.9, df=2, p-value <0.001), and it was

35

30

25

20

15

10

5

IFN-γ (Pg\ml)

✱ ✱ ✱ ✱

NS ✱ ✱ ✱ ✱ NS

Fig 3. Boxplot of serum TNF-α levels in S. typhi-infected patients based on detection of virulence genes, InvA and PagC, and healthy control,**p-value < 0.001.

InvA detected

InvA not detected

Healthy Control

PagC detected

PagC not detected

Healthy Control

Fig 4. Boxplot of serum INF-γ levels in S. typhi-infected patients based on detection of virulence genes, InvA and PagC, and healthy control,

**p-value < 0.001.

statistically significantly higher in InvA not detected (21.9, (21.6-22.1)) compared to healthy control (11.6 (9.9-14.6)) (X2=41.2, df=2, p-value <0.001), but there was no significant difference between InvA detected and InvA not detected (X2=-13.24, df=2, p-value >0.05) (Fig 4).

According to PagC gene detection, the post hoc comparsion revealed that the serum level of TNF-α was statistically significantly higher in PagC detected in

S. typhi isolates (148.1, (142.1-151.2)) compared to PagC not detected (93.4, (80.4-144.2)) (X2=31.75, df=2, p-value

<0.001), and it was statistically significantly higher in PagC detected compared to healthy control (85,1 (79.9- 91.4)) (X2=48.78, df=2, p-value <0.001), but there was no significant difference between PagC not detected and healthy control with (X2=17.03, df=2, p-value >0.05) (Fig 3). Like TNF-α, the post hoc comparsion of PagC revealed that the serum level of INF-γ was statistically significantly higher in PagC detected in S. typhi isolates (21.8, (21.5-23.2)) compared to PagC not detected (12.6, (9.9-21.5)) (X2=31.78, df=2, p-value <0.001), and it was statistically significantly higher in PagC detected compared to healthy control (11.6 (9.9-14.6)) (X2=46.3, df=2, p-value

<0.001), but there was no significant difference between PagC not detected and healthy control (X2=14.5, df=2, p-value >0.05) (Fig 4).

DISCUSSION

This study detect two virulence genes, InvA and PagC, in S. typhi isolates which were collected from patients undergoing cholecystectomy. Out of 66 S. typhi isolates, the findings of monoplex PCR of studied virulence genes were 56 (84.8%) InvA detected, and 46 (69.6%) PagC detected. A previous study showed that S. typhi isolates possess several genes for pathogenicity, such as InvA, PagC, SpaN, OrgA, PrgH, InvJ, which give specific virulence traits and may deviate from the typical pattern of S. typhi, because they may be gained by horizontal transfer from another organism.14

In most strains of S. typhi isolated from humans and animals, the InvA has been reported to be very important for pathogenicity as this gene encodes a protein responsible for adhesion and invasion of S. typhi into epithelial cells of the infected host.15 In addition to the above virulence gene, the PagC is also very important for bacterial adhesion, toxin transfer, and surviving with infected host cells through stimulating bacteria to form a vesicle called outer membrane vesicles (OMVs), spherical membranous compounds that are secreted from the surfaces of virtually all gram-negative microorganisms. High expression of PagC accelerates S. typhi-OMVs

formation.16 In consistency with the rseults of our study, previous studies have observed a high prevalence of InvA and PagC in S. typhi.17,18 On the basis of the results of this study, these two virulence genes, InvA and PagC, could be very useful genes for the fast determination of Salmonella pathogen.

Furthermore, despite S. typhi infection, our findings show that TNF-α release was significantly higher in PagC detected in S. typhi isolates than in PagC not detected, and INF-γ release was also significantly higher, but there was no significant difference between InvA detected in

S. typhi isolates and InvA not detected, indicating that this phenomenon extends to other cytokine molecules. These findings could be attributed to strong defense responses, such as immune cell proliferation and excretion of T helper 1 (Th1) cytokines after expression of PagC. This supports the findings of two studies from Iraq and Indonesia.19,20 Following in vitro incubation of the peripheral blood mononuclear cells (PBMCs) with isolated flagella from

S. typhi, Wyant et al.21 have demonstrated a decrease in the number of cells expressing the lipopolysaccharide (LPS) receptor CD14. According to the same research group,22 PBMCs that have been coincubated with flagella of S. typhi have a diminished response to mitogens and antigens. Patients with typhoid fever and cholecystitis show alterations in the total number of circulating white blood cells (WBCs).23,24 However, the majority of participants in the study described here had total WBCs that were within the normal range at the time of enrollment. It remains to be proven whether this process manifests in vivo during typhoid fever and is the cause of the reported reduction in proinflammatory cytokine release. The serum of typhoid fever patients may, however, include soluble molecules that prevent or decrease PagC-induced cytokine release, as has been observed for LPS-induced TNF-α release in mycobacterial infections.25 LPS-induced IL-1beta and TNF-α production were inhibited by IL-6 and IL-1 receptor antagonist in vitro, and these two cytokines can both be increased in the blood of individuals with typhoid fever.26

TNF-α is a proinflammatory cytokine that is released when bacterial engulfment by macrophages occurs. Infected macrophages become S. typhi antigen presenters to Th1, which secretes IFN- γ and interleukin-12. IFN- γ stimulates many effector activities of mononuclear phagocytes, such as monocytes and macrophages, in order to kill intracellular pathogens.27 A study discovered that incubating peripheral blood mononuclear cells for four days with S. typhi flagella raises TNF-α to 2000 pg/ml.28 Depending on Salmonella flagellum expression by fliC gene, the ability of salmonella to enhance production of

TNF-α and IFN-γ by human monocytes was shown.29 So, the LPS is not the only component of gram-negative microorganisms that enhances cytokine production, but also on the bacteria‘s genes. According to the results of this study, infection by PagC -involved S. typhi is more harmful than infection by PagC -not involved S. typhi, because excessive production of inflammatory cytokines, such as TNF-α, and the subsequent release additional inflammatory mediators might lead to decreased blood pressure, shock, and even death.30 The present findings have limiting factors; the sample size was small to draw any firm conclusions on the relationship between cytokines and virulence genes, and due to a deficiency of laboratory supplies, we were unable to complete all analyses with other virulence genes.

CONCLUSION

The virulence genes of S. typhi, especially PagC, may be considered a potent inducer of systemic inflammatory cytokine secretion. Further studies are needed to present the patient‘s additional clinical course according to cytokine level.

ACKNOWLEDGMENTS

The author would like to thank all the staff of AL- Sadder Medical City and AL-Furat General Hospital in Al-Najaf province for their help on the current manuscript.

Conflict of interest: none

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9. Bourgeois JS, Wang L, Rabino AF, Everitt J, Alvarez MI, Awadia S, et al. ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice. PLoS Pathog. 2021;17(7):e1009713.

10. Akiyama T, Khan AA, Cheng CM, Stefanova R. Molecular characterization of Salmonella enterica serovar Saintpaul isolated from imported seafood, pepper, environmental and clinical samples. Food Microbiol. 2011;28(6):1124-8.

11. Bhuiyan S, Sayeed A, Khanam F, Leung DT, Rahman Bhuiyan T, Sheikh A, et al. Cellular and cytokine responses to Salmonella enterica serotype Typhi proteins in patients with typhoid fever in Bangladesh. Am J Trop Med Hyg. 2014;90(6):1024-30.

12. Ajayi A, Smith S, Ibidunni BS, Coulibaly KJ, Funbi JT, Adeleye AI. Serotype Distribution and Virulence Profile of Salmonella enterica Serovars Isolated from Food Animals and Humans in Lagos Nigeria. Microbiology and Biotechnology Letters. 2019; 47(2):310-6.

13. Skyberg JA, Logue CM, Nolan LK. Virulence genotyping of Salmonella spp. with multiplex PCR. Avian Dis. 2006;50(1):77- 81.

14. Marcus SL, Brumell JH, Pfeifer CG, Finlay BB. Salmonella pathogenicity islands: big virulence in small packages. Microbes Infect. 2000;2(2):145-56.

15. Kumar A, Balachandran Y, Gupta S, Khare S, Suman. Quick PCR based diagnosis of typhoid using specific genetic markers. Biotechnol Lett. 2010;32(5):707-12.

16. Lu J, Li L, Pan F, Zuo G, Yu D, Liu R, et al. PagC is involved in salmonella pullorum OMVs production and affects biofilm production. Vet Microbiol. 2020;247:108778.

17. Srisanga S, Angkititrakul S, Sringam P, Le Ho PT, Vo ATT, Chuanchuen R. Phenotypic and genotypic antimicrobial resistance and virulence genes of Salmonella enterica isolated from pet dogs and cats. J Vet Sci. 2017;18(3):273-81.

18. Torkan S, Bahadoranian M, Khamesipour F, Anyanwu M. Detection of virulence and antimicrobial resistance genes in Escherichia coli isolates from diarrhoiec dogs in Iran. Arch Med Vet. 2016;48:181-90.

19. Aburesha RA MM, Abd Al-Kareem K , Almayahi BA, Al-Jumaa ZM. Determination IL6, INF-γ Levels in Typhoid Fever Patients. Prensa Med Argent. 2020;106(3):1-6.

20. Febriza A, Natzir R, Hatta M, As’ad S, Budu, Kaelan C, et al. The Role of IL-6, TNF-a, and VDR in Inhibiting the Growth of Salmonella Typhi: in vivo Study. The Open Microbiology Journal. 2020;14:65-71.

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Impact of the COVID-19 Pandemic on Tertiary- care University Dermatology Outpatient Clinic and Dermatology Procedures

Kingkaew Wamaphutta, B.Ed., Chowalit Thasen, B.Sc., Chudapa Sereeaphinan, M.D., Pichanee Chaweekulrat, M.D., Waranya Boonchai, M.D.

Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

ABSTRACT

Objective: This study aimed to determine the changes in patient characteristics, disease prevalence, and dermatology procedures during the COVID-19 pandemic.

Materials and Methods: We retrospectively reviewed the demographic data, diagnoses, and dermatology procedures of the patients who visited an outpatient dermatology clinic between March 1, 2018, and December 31, 2021. Changes in the number of patients, the patterns of diagnosis, and the dermatology procedures were analyzed.

Results: The number of patients decreased at the start of each of the 4 COVID-19 waves that occurred during the study period. Older adults increased their dermatology visits during the pandemic. The skin disease with the highest proportional increase in the frequency of visits was cutaneous T-cell lymphoma. The proportion of noncosmetic procedures significantly increased, in contrast to a decrease in the proportion of cosmetic procedures.

Conclusion: The COVID-19 pandemic significantly affected outpatient dermatology by changing the setting and frequency of patient visits to the outpatient clinic, the pattern of the diseases, and the dermatology procedures.

Keywords: COVID-19; Dermatology procedure; Dermatology; Outpatient department (Siriraj Med J 2022; 74: 836-843)

INTRODUCTION

Coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by Severe Acute Respiratory Syndrome Coronavirus-2. First reported in Wuhan, China, in late 2019,1 it rapidly spread globally and was declared a pandemic by the World Health Organization in March 2020.2,3 The first patient in Thailand was reported on January 12, 2020. An outbreak occurred in March 2020 due to several clusters in Bangkok’s boxing venues and nightclubs, and the disease began spreading throughout Thailand.4,5 To control the infection, the Thai government implemented a public health emergency decree on March 26, 2020.5

The COVID-19 pandemic has dramatically affected personal lifestyles, governing and political systems, and national economies. It has also greatly impacted healthcare systems through the need to control the disease while allocating finite medical resources to COVID-19 and non-COVID-19-related illnesses.6-8 Access to medical services sometimes became limited, leading to appointment cancellations, and face-to-face visits have been adversely affected by social distancing and stay-at-home policies introduced by governments and businesses.9 The various COVID-19 restriction policies have also disrupted the provision of dermatology services.2,9 Skin diseases are usually nonfatal and involve few emergency conditions.

Corresponding author: Waranya Boonchai E-mail: waranya.boonchai@gmail.com

Received 7 October 2022 Revised 25 October 2022 Accepted 26 October 2022 ORCID ID:http://orcid.org/0000-0002-6673-6534 http://dx.doi.org/10.33192/Smj.2022.98

All material is licensed under terms of the Creative Commons Attribution 4.0 International (CC-BY-NC-ND 4.0) license unless otherwise stated.

Therefore, we anticipated that the COVID-19 pandemic would result in a decline in outpatient visits or a shift to teledermatology, especially with mild cases of skin disease. Previous European studies demonstrated a significant decline in patient visits to dermatology outpatient departments (OPD) and an increase in patients missing appointments during the pandemic.7,9,10 There were also effects on the prevalence of certain skin diseases11-13 and the performance of minor versus major dermatology procedures.14,15 We aimed to evaluate the changes in patient characteristics, disease prevalence, and dermatology procedures during the COVID-19 pandemic in Thailand, which has different medical settings and legislation from Europe.

MATERIALS AND METHODS

Study design and participants

This single-center study was conducted at the Department of Dermatology, Faculty of Medicine, Siriraj Hospital, Mahidol University. The hospital is the largest tertiary care referral hospital in Thailand. Before this research began, the Siriraj Institutional Review Board approved its protocol (Si 571/2021). We retrospectively reviewed the electronic registration database to identify patients visiting the dermatology OPD between March 1, 2018, and December 31, 2021. All consecutive patients in the study period were selected for the analysis. Details were retrieved of the number of patients, their demographic data (age and sex), skin diagnoses (per the International Classification of Diseases [ICD]-10), and dermatology procedures (per the ICD-9 system). The number of COVID-19 cases in Thailand was obtained from Thailand’s Ministry of Public Health website.16 The primary outcome of the study is the changes in the proportion of visit from common diseases in dermatology outpatient unit before and during COVID-19 era. The secondary outcome is the changes in patients’ demographic data, dermatological procedures before and during COVID-19 era.

Sample size consideration

The sample size was calculated with an equation for detecting the difference of two independent proportions. From the literature review, contact dermatitis is common diseases during COVID-19 pandemic; therefore, the number of visits for contact dermatitis was used for sample size calculation. Using reported frequency of contact dermatitis of 5.8% in 1,165 and 9.9% in 717 outpatient dermatology visits before and during COVID-19 pandemic, respectively11, alpha error of 5%, the desired power of 90% and a predetermined allocation ratio of 1:1, the estimated sample size is 1,215 before COVID-19 era and 751 visits during COVID-19 era.

Data collection

The proportions of patients, diagnoses, and procedures from the pandemic period (March 2020-December 2021) were compared with the corresponding values for the preceding (March 2018-February 2020). The changes in the monthly number of patients and procedures were evaluated, and collinearity with the number of new COVID-19 cases in Thailand was assessed.

Statistical analysis

Data were analyzed using SPSS version 18 (SPSS Inc. Released 2009. PASW Statistics for Windows. Version

18.0. Chicago: SPSS Inc.). The proportion of visit from diseases and procedures before and during the COVID-19 pandemic were compared using either the Chi-squared or Fisher’s exact test. A probability (P) value of less than

0.05 was considered statistically significant. Continuous data were reported using mean or median. Categorical data were reported using frequency and percentage.

RESULTS

Between March 2018 and December 2021, 200 368 patients visited the Siriraj dermatology OPD. The average number of patients during the pre-pandemic period-4925 patients/month-declined by approximately 30% to 3423 patients/month during the pandemic. Women patients predominated, with a female-to-male ratio of 2:1; however, the degree of female preponderance significantly decreased during the COVID-19 period. The most common patient age group in both the pre-pandemic and pandemic periods was 18- to 44-year-olds (40.7% and 40.1%, respectively). The proportion of older patients (> 65 years) significantly increased during the pandemic (Table 1).

The relationships between the number of new COVID-19 cases in Thailand and the number of patients and dermatology procedures conducted at the dermatology OPD are illustrated in the Figure. Although patient- case and dermatology-procedure numbers declined significantly in every wave of COVID-19, they returned to near normal approximately 1 month after each COVID-19 peak. Changes in the number of dermatology procedures performed during the pandemic corresponded with the variations in the number of OPD cases.

Table 2 classifies skin diseases according to ICD-10 and details percentage changes in the frequency of OPD visits for treatment before and during the pandemic. The diseases are presented in 3 groups: significantly increased visit frequency, significantly decreased visit frequency, and unchanged visit frequency. In terms of the degree of frequency, the top-3 skin diseases before and during the pandemic were identical: unspecified dermatitis,

TABLE 1. Demographic data of patients visiting Siriraj Hospital’s dermatology outpatient department

Abbreviation: COVID-19, coronavirus disease 2019

Number of visits

TABLE 2. Distribution of diagnosed skin diseases before and during COVID-19 pandemic

Number of visits

TABLE 2. Distribution of diagnosed skin diseases before and during COVID-19 pandemic (Continued)

* A P value < 0.05 indicates statistical significance (chi-squared test).

Abbreviations: BCC, basal cell carcinoma; COVID-19, coronavirus disease 2019; CTCL cutaneous T-cell lymphoma; ICD, International Classification of Diseases; SCC, squamous cell carcinoma; SJS, Stevens–Johnson syndrome; SPTCL, subcutaneous panniculitis-like T-cell lymphoma; TEN, toxic epidermal necrolysis

vitiligo, and psoriasis. The skin disease with the highest percentage increase in frequency was cutaneous T-cell lymphoma, followed by Kaposi sarcoma, subcutaneous panniculitis-like T-cell lymphoma, other bullous disorders, and mycosis fungoides. Conversely, the diseases with the highest reductions in visit frequency were mastocytosis, sweet syndrome, leprosy, hemangioma, and varicella.

Regarding dermatology procedures, there was a marked decrease in the number of procedures conducted at the OPD following the COVID-19 outbreak. Overall, there was a 37% decline, from a mean of 2211 to 1382 procedures/month (Table 3). However, the fall was not uniform across all procedures. The proportion of noncosmetic procedures significantly rose (85.27% to

86.67%), whereas the proportion of cosmetic procedures fell (14.73% to 13.33%). The noncosmetic procedure with the highest frequency increase was radical excision of skin lesions, while the melanocyte-keratinocyte transplantation procedure demonstrated the largest frequency decline. Regarding cosmetic procedures, platelet-rich plasma injections saw the greatest frequency increase, and the use of iontophoresis fell the most.

DISCUSSION

This study demonstrated the influences of the COVID-19 pandemic on the number of patient visits and procedures performed at a dermatology OPD. The total number of patients visiting the OPD decreased,

Number of procedures

Percentage

TABLE 3. Number of dermatology procedures before and during COVID-19 pandemic

*, Not statistically significant

Abbreviations: 5-FU, 5-fluorouracil; COVID-19, coronavirus disease 2019; ICD, International Classification of Diseases; MTX, methotrexate

consistent with reports from other countries.10,17 In addition, another study showed a more pronounced loss of patients from a hospital’s dermatology OPD than its other departments.18 The explanation could be that, given most dermatology conditions’ nonfatal and nonemergency nature, the risk of acquiring a COVID-19 infection outweighed the perceived necessity for an OPD visit.10,18 The Thai government’s COVID-19 prevention policies also included interregional travel restrictions. The policies impeded or prevented patients in rural areas from accessing face-to-face medical consultations at tertiary healthcare centers in other provinces. Moreover, international surveys revealed that over 50% of dermatologists had been reassigned from dermatology departments to fields more directly related to COVID-19 to mitigate staff shortages resulting from the pandemic. However, the relocations of dermatologists created staff shortfalls in dermatology OPDs.9,19 For a limited period, Thailand’s COVID-19 prevention policies also placed severe restrictions on the conduct of cosmetic procedures throughout many areas of Thailand, including Bangkok, where our hospital is situated.

The COVID-19 pandemic also influenced the age and sex distribution of patients visiting the dermatology OPD. The proportion of older adults became significantly higher during the pandemic, which accords with another study.5,12 However, the majority of patients at our dermatology clinic were teenagers and adults, who could be asymptomatic carriers of COVID-19. Therefore, to minimize the risk of COVID-19 transmission, nonemergency dermatology visits should be temporarily suspended or serviced through alternative methods, such as teledermatology.20,21 Moreover, the number of female patients significantly decreased during the pandemic. This dissimilarity might reflect different levels of awareness among men and women of the hazards of COVID-19.

Certain skin conditions saw an increase in the frequency of visits during the pandemic. This was particularly the case with diseases with high severity or urgent situations, such as cutaneous T-cell lymphoma (ICD-10; C84.8, C86.3, C84) and vesiculobullous diseases (ICD-10; L10-13). Among the most commonly diagnosed diseases, the frequency of dermatitis, psoriasis, and urticaria significantly increased during the pandemic. For urticaria and psoriasis, the increase in prevalence might have been related to COVID-19-associated stress and anxiety, which would have precipitated these diseases.12,22,23 Psychological stress affects or exacerbates many skin disorders, including psoriasis and urticaria but also atopic dermatitis, seborrheic dermatitis, vesiculobullous disorder (pemphigus, pemphigoid), and viral infections

(herpes zoster).24-26 Our results also revealed an increase in the frequency of these diseases after the pandemic, consistent with previous studies.5,13 Moreover, the increased frequency of urticaria and dermatitis might have been related to mass vaccination programs against COVID-19. Urticaria is the most common cutaneous adverse reaction (34.44%) associated with COVID-19 vaccine administration, followed by eczematous skin reaction (6.95%).27 Eczema levels might also rise due to the extended mask wearing and increased attention to hand hygiene occurring during the COVID-19 era.10-13 Reactivation of varicella-zoster virus has been reported in association with COVID-19 infections because of weakened immune systems and the administration of different COVID-19 vaccines.28,29 There are also many literature reports of acute telogen effluvium after recovery from COVID-19 infection.30 Our findings showed an increased telogen effluvium frequency, supporting previous observations.

Particular skin conditions showed significantly lower visit frequencies. The decreases were possibly due to the nature of the diseases (not emergent, gradual onset, or rare). Alternatively, the skin conditions might have mainly had cosmetic issues, so hospital visits could be suspended. Vitiligo and acne were among the most commonly diagnosed skin diseases that had significantly decreased visit frequencies during the pandemic. Moreover, patients might have felt more comfortable seeking treatment for these conditions at primary or secondary healthcare facilities than at a crowded tertiary healthcare center, where they would have a higher risk of contracting COVID-19. Teledermatology is another treatment channel that had a significant uptake during the pandemic. Commonly occurring ambulatory dermatoses like dermatitis and acne were reported to be more amenable to being assessed and managed via teledermatology.31 Furthermore, in Thailand, patients can easily buy medications for skin conditions over the counter without a prescription. Our study also found that the frequency of sexually transmitted diseases (STDs) such as syphilis and anogenital herpes infection significantly decreased during the pandemic. Previous studies in several countries reported lower incidences of STDs during the pandemic.32-34 Possible factors contributing to the apparent fall in STD-case numbers are the introduction of strict social distancing measures (which reduced sexual risk behaviors) and a decline in STD screening services and resources for controlling STDs.33,34

In our study, the number of overall dermatology procedures decreased, and this occurred for both noncosmetic and cosmetic procedures. Similarly, there was a reduction

Fig 1. Changes in the number of patients and dermatology procedures during the COVID-19 pandemic

Abbreviations: COVID-19, coronavirus disease 2019; OPD, outpatient department

in scheduled and performed procedures in Italy after its lockdown, resulting from appointment cancellations by patients or the development of COVID-19 infections.15 During the COVID-19 period, health authorities were obliged to prioritize medical activities and decrease nonurgent visits because healthcare systems had rapidly become overwhelmed. Most dermatology activities were suspended. However, life-saving or time-dependent dermatology procedures (such as radical excisions of skin lesions, biopsies, and resections of malignant lesions) continued to be performed at high levels. Conversely, the melanocyte-keratinocyte transplantation procedure for vitiligo was the least performed procedure, possibly because vitiligo is a prolonged condition. The present study found a proportional increase in noncosmetic procedures, with a corresponding fall in the number of cosmetic procedures. This change in proportions resulted from the hospital’s protocol of limiting nonurgent procedures at our center, such as laser and hair transplantation. Among cosmetic procedures, the number of platelet-rich plasma injections had the highest increase, given that they were a substitute for hair transplantation surgery. However, the number of botulinum toxin and filler procedures significantly rose during the pandemic. The increasing levels of virtual socializing might have led to dissatisfaction of individuals with their onscreen appearance, resulting in a heightened interest in some cosmetic procedures.35 A similar situation was observed with the demand for facial plastic surgery procedures.36

There are some limitations to the study. First, since the data was collected from a single tertiary center, it may lack generalizability on the national scale or in other settings such as primary or secondary care. Second, due to the study’s retrospective nature, some data relating to patients’ decisions to avoid OPD visits may not have been captured.

In conclusion, the COVID-19 pandemic affected outpatient dermatology by changing the setting and numbers of patients visiting the OPD, the pattern of the diseases, and the dermatology procedures. The study presents Thailand’s COVID-19 circumstances that affected our dermatology practices.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Miss Julaporn Pooliam of the Faculty of Medicine Siriraj Hospital, Mahidol University, for assistance with the statistical analyses. The authors are also indebted to Mr. David Park for the English-language editing of this paper.

Conflicts of interest: All authors have no conflicts of interest or financial support to declare.

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Are Technical Skills Assessed Using Medical Knowledge Associated with Non-technical Skill Knowledge in Anaesthesia Resident Training?

Maliwan Oofuvong, M.D., Ph.D.*, Ngamjit Pattaravit, M.D., Orarat Kanjanawanichkul, M.D., Sirikarn Siripruekpong, M.D., Kanjana Nuanjun, R.N., Boonthida Suwannarat, R.N.

Department of Anesthesiology, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand.

ABSTRACT

Objective: We examined the association between midterm examinations and anaesthetists’ non-technical skills (ANTS) knowledge using multiple choice questions (MCQs) for anaesthesia training.

Materials and Methods: A prospective cross-sectional study was implemented based on two cohort studies conducted in November 2017 and November 2019 at the university hospital in southern Thailand. Each cohort consisted of MCQs, short answer questions (SAQs), objective structured clinical examinations (OSCEs), and MCQs after ANTS simulation workshops during the midterm examinations. The main exposure variable was the midterm examination (MCQs/ SAQs/ OSCEs) whereas the ANTS MCQs were the outcome. The potential predictors were the residents’ role, sex, PGY (1-3), and competency. Associations between midterm examinations and ANTS MCQ scores were analysed using Pearson’s correlation coefficients (r) and multivariate linear regression analysis, and presented as beta coefficient (β) and 95% confidence limit (CL).

Results: Forty-eight anaesthesia residents were recruited for the study. After adjusting for PGY, knowledge score, and attitude evaluated by the staff, OSCE was found to be significantly associated with the pretest ANTS scores (β [95% CL]= 1.02 (0.06, 1.98)) and MCQ scores were significantly associated with the posttest ANTS scores (β [95% CL]= 0.14 (0.04, 0.24)). SAQ scores had negative associations with the pretest (β [95% CL]=-0.11 (-0.21, -0.01)) and posttest (β [95% CL]=-0.16 (-0.27, -0.05)) ANTS scores. It was found that there was a significant relationship between midterm examinations and posttest ANTS scores (r=0.52).

Conclusion: Technical skills using OSCE and midterm MCQ examination scores were associated with non-technical skill knowledge in anaesthesia training.

Keywords: Technical skill; anaesthesia’s non-technical skill knowledge; multiple choice questions; short answer questions; objective structured clinical examination (Siriraj Med J 2022; 74: 844-856)

INTRODUCTION

Anaesthetists’ non-technical skills (ANTS) are a crucial component of anaesthesia resident training because they emphasise communication skills and the ability to work in a multidisciplinary team during anaesthesia practise.1 It is also part of the anaesthesia training

requirement in conjunction with the technical skills required to answer multiple choice questions (MCQs), short answer questions (SAQs), objective structured clinical examinations (OSCEs), and oral examinations. Because it is a behavioural marker tool, at least two evaluators are required to assess residents’ behaviour during the

Corresponding author: Maliwan Oofuvong E-mail: oomaliwa@gmail.com

Received 23 July 2022 Revised 5 November 2022 Accepted 6 November 2022 ORCID ID:http://orcid.org/0000-0001-5840-5965 http://dx.doi.org/10.33192/Smj.2022.99

All material is licensed under terms of the Creative Commons Attribution 4.0 International (CC-BY-NC-ND 4.0) license unless otherwise stated.

simulation workshop to accurately evaluate performances related to 15 elements of ANTS.2 Some predictors – such as the number of evaluators, residents’ skill level range, and the observational process – can affect the reliability of the evaluators.3 Other predictors associated with higher non-technical skills were trainees’ experience, recent training in the life support protocols,4 and higher technical skills.5,6 Most studies have demonstrated the correlation between technical and non-technical skills in simulation workshops.5,6 To date, no studies have reported other evaluated tools besides the behaviour maker tool to examine the association between non-technical and technical skills in anaesthesia. Not all institutes like ours might be able to evaluate ANTS behaviour in all residents in one time period workshop; therefore, through midterm examinations, we applied the ANTS assessment by using the debrief session and evaluating ANTS knowledge to ensure ANTS comprehension and understanding. Moreover, we examined whether the technical skills assessed using medical knowledge during midterm examinations were associated with ANTS knowledge, based on the MCQ scores of residents who either participated in or observed the ANTS simulation workshop.

MATERIALS AND METHODS

This prospective observational study was conducted after receiving approval from the Ethics Committee, Faculty of Medicine, Prince of Songkla University on 1 October 2019 (REC 62-265-8-1). We recruited anaesthesia residents from different post-graduate years (PGY-1, PGY-2, and PGY-3) who attended a simulation-based ANTS training workshop at the simulation centre, Faculty of Medicine, Prince of Songkla University, in November 2017 and November 2019. Since the workshop was a part of the anaesthesia training programme, written informed consent from the participants was waived by the Ethics Committee.

Standard operating procedure

Midterm examinations held annually in the month of November and consist of MCQs, SAQs, and OSCEs (Fig 1). Residents’ performance in terms of knowledge, skill, and attitude are simultaneously evaluated by the anaesthesia staff. The simulation ANTS workshop has been held biennially since 2015. All residents were informed of the schedule of the workshop one month in advance. The three case scenarios were based on the dominant ANTS category themes. Meanwhile, situation awareness (SA), teamwork and communication (TW), task management (TM), and decision making (DM), were developed by

three different anaesthesia staff members for each theme. For 2019, the scenario ‘Missing tooth’ for the TW theme, the scenario ‘Anaesthesia emergency service’ for the TM theme, and the scenario ‘Decision making in on call resident’ for the DM theme, were created by SS, MO, and NP, respectively. All the residents (PGY-1, PGY-2, PGY-3) received the ANTS material one week before the workshop. The manikins and workstations were prepared and checked to ensure readiness one day before the workshop. On the day of the workshop, the residents received a ten-minute briefing on overall concepts of the ANTS by MO and completed a pretest consisting of 20 MCQs (Appendix 1). Each scenario consisted of a 30-minute simulation workshop and a 30-minute debriefing session. One or two residents from PGY-1, PGY-2, or PGY-3 were randomly selected to participate in the scenario theme (Fig 1), while the remaining residents observed the sessions through a monitor in the observer room. Each debriefing session consisted of de-rolling and applying the ANTS, and was facilitated by the anaesthesia staff who created the respective scenarios. After finishing the workshop, all residents completed the posttest (Appendix 1). An anaesthesia staff member (MO) discussed the MCQ answers with the residents after the posttest.

Operational definition

Scenario participant was defined as a first-, second- or third-year resident who participated as a team leader or team member in the scenario. Observer was defined as a first-, second- or third-year resident who observed the session from the observation room. Technical skills in anaesthesia training were represented by MCQ, SAQ, and OSCE scores during the same period of the simulation ANTS training (midterm examinations in November 2017 and November 2019).

Outcome of the study and outcome measurement

The primary outcome of the study was the posttest MCQ score after the ANTS workshop. The secondary outcome was the pretest MCQ score before the ANTS workshop. The pretest and posttest MCQs consisted of the same 20-item questions. The reliability of these pretest and posttest MCQ items were 0.95 and 0.98, respectively, as assessed by two anaesthesiologists (NP and SS). The MCQ consisted of 80% (16/20 items) application taxonomy-related questions and 20% (4/20) recalled taxonomy-related questions, with the ratio of ANTS content being 25% in SA (5/20 items), 25% in TM (5/20 items), 30% in TW (6/20 items), and 20% in DM (4/20 items).7

Fig 1. Midterm examinations of anaesthesia training.

Note: PGY: post-graduate year, ANTS: anaesthetists’ non-technical skills, MCQ: multiple-choice question, SAQ: short answer question, OSCE: objective structured clinical examination.

Predictors and potential confounders

The main exposure variable was the midterm examination, which comprised 180 MCQs, 12 SAQs, and 24 OSCEs (Appendix 2). The average score of the anaesthesia residents’ competency for each year in terms of knowledge, skills, and attitude was also evaluated by all anaesthesia staffs before the examination (Appendix 2). Potential predictors were the residents’ role in the scenario (participant/observer), sex, PGY (1-3), and percentage of self-study prior to attending the workshop.

The step of statistical analysis

First, the basic comparison test to compare the residents’ characteristics and the midterm examination scores as well as MCQ ANTS scores among PGY (1-3) were performed. Continuous variables were presented using the median and interquartile range for non-normally distributed data and using mean and standard deviation (SD) for normally distributed data. Categorical variables were presented using frequency and percentage, and were compared using Fisher’s exact test or Pearson’s

APPENDIX 1

MCQ ANTS pretest and posttest

1. You are an anesthesiologist on call and handed over a case of hypotension. The inotrope was given but the hypotension persisted. You did not notice that the line of inotrope was clamped. Which ANTS category is related to this event?

   1. Task management

   2. Teamwork & communication

   3. Situation awareness

   4. Decision-making

2. You are an anesthesiologist and a team leader of a fire situation in OR. You need to discuss with the surgeon, a chairman of the surgery department, to rapidly manage a patient. The surgeon has refused to abandon surgery until he has completed the operation. Which key element would you deal with in this situation?

   1. Providing & maintaining standards

   2. Co-ordinating activities with team

   3. Using authority & assertiveness

   4. Recognising & understanding

3. According to question 2, which ANTS category was referred to?

   1. Task management

   2. Teamwork & communication

   3. Situation awareness

   4. Decision-making

4. You are a third-year resident working in the emergency room. There is a pregnancy case with fetal distress; however, all 3 emergency rooms are occupied. Which ANTS category applies in this situation?

   1. Task management

   2. Teamwork & communication

   3. Situation awareness

   4. Decision making

5. From question 4, you managed the pregnancy case with fetal distress by taking the first available elective room. To which key element does this situation referred?

   1. Identifying & utilizing resources

   2. Co-ordinating activities with the team

   3. Reconising & understanding

   4. Balancing risks & selecting options

6. What is the key element of situation awareness?

   1. Providing & maintaining standards

   2. Assessing capabilities

   3. Recognising & understanding

   4. Identifying options

7. What is the key element of decision making?

   1. Planning & preparing

   2. Supporting others

   3. Anticipating

   4. Re-evaluating

      
      

      
      

      
      

      APPENDIX 1 (Continued)

      
      

8. Which is FALSE regarding the key element of teamwork & communication?

   1. Identifying & utilizing resources

   2. Co-ordinating activities with the team

   3. Exchanging information

   4. Using authority & assertiveness

9. Failure of which ANTS category could lead to communication failure between two colleagues?

   1. Task management

   2. Teamwork & communication

   3. Situation awareness

   4. Decision making

10. What tool is the most important for the simulation of Anaesthetists' Non-Technical skills (ANTS) training?

    1. Simulation center

    2. Standardized patient

    3. Real-life case scenario

    4. Feedback or debriefing

11. You handed over the details, except for the information of morphine allergy, of a case of total abdominal hysterectomy with bilateral salpingo-oophorectomy to your colleague who was on call. This mistake could refer to which ANTS category(ies)?

    1. Teamwork & communication

    2. Teamwork & communication and task management

    3. Teamwork & communication and situation awareness

    4. Teamwork & communication and decision making

12. After placing an epidural catheter for painless labor at the labor room, you noticed that there was no basic monitoring. You managed this by informing an anesthetist nurse to palpate a radial pulse and a nurse aid to urgently get a monitor from the OR. What ANTS category are you concerned with the most?

    1. Task management

    2. Teamwork & communication

    3. Situation awareness

    4. Decision making

13. The anesthesiologist in charge did not have any plan for postoperative pain control for patient receiving total abdominal hysterectomy with bilateral salpingo-oophorectomy. The patient developed severe pain at the PACU. This error did not refer to which ANTS category?

    1. Situation awareness

    2. Teamwork & communication

    3. Task management

    4. Decision making

14. A surgeon would like to perform surgery in a case of immediate post-cardiac arrest in the OR. As the anesthesiologist in charge, you managed to discuss with him regarding the risk of performing surgery. Which ANTS category best reflects your action?

    1. Situation awareness

    2. Teamwork & communication

    3. Task management

    4. Decision-making

       
       

       
       

       APPENDIX 1 (Continued)

       
       

15. You are an anesthesia resident who administered the wrong type of packed red cell to a patient with massive bleeding. Before you transfused the packed red cell, you checked only the patient's name without checking the hospital number nor cross-checking with an anesthetist nurse. This mistake refers to which ANTS category?

    1. Teamwork & communication

    2. Decision making

    3. Situation awareness

    4. Task Management

16. The mistake from question 15 was relevant to which key element of that particular ANTS category?

    1. Balancing risks & selecting options

    2. Providing & maintaining standards

    3. Co-ordinating activities with the team

    4. Anticipating

17. You were a team leader of advanced cardiac life support (ACLS) resuscitation. You noticed that your team member could not perform chest compression effectively, so you managed to exchange a chest compressor with a mask ventilation member. Your decision correlates with which key element of ANTS?

    1. Co-ordinating activities with the team

    2. Exchanging information

    3. Using authority & assertiveness

    4. Assessing capabilities

18. You are giving anesthesia to a patient with a history of difficult airway. After the patient loses consciousness, you cannot ventilate him, so you call a senior anesthesiologist staff to stand by in the room. Your action could refer to which ANTS category(ies)?

    1. Situation awareness

    2. Situation awareness and teamwork & communication

    3. Situation awareness and task management

    4. Situation awareness and decision making

19. An anesthesia resident who was on call was about to intubate a non-difficult airway patient when he overheard a CPR code. The resident arranged for an anesthetist nurse to do the intubation instead. The resident then informed the anesthesiologist staff by phone while he ran to the CPR scene. His action was relevant to which key element?

    1. Prioritising

    2. Recognising & understanding

    3. Co-ordinating activities with the team

    4. Balancing risks & selecting options

20. As an anesthesiologist staff, you informed an anesthetist nurse to administer intravenous succinylcholine to a child. You suspected the child might develop a laryngospasm since there was no reading of oxygen saturation on the monitor. After you checked the oxygen saturation probe, you found that it was not connected to the child’s finger. This error was relevant to which ANTS category?

    1. Task management

    2. Teamwork & communication

    3. Situation awareness

    4. Decision making

APPENDIX 2

Combination of midterm examination score of resident training and resident’s competency evaluated by staff in 2017 and 2019

Abbreviation: PGY: post-graduate year

chi-square test. Continuous variables were compared using Kruskal–Wallis tests and analysis of variance, as appropriate. Second, to determine the degree of correlation between midterm examinations and MCQ ANTS scores, Pearson’s correlation coefficients (r) were used. Third, the r used among the main exposures and outcomes in the second step was considered a univariate analysis, and therefore, a directed acyclic graph (DAG) was used to select the potential confounders that could affect the main exposures and the outcomes into the multivariate model. Then, the DAGitty software version 3.0. was applied. Potential confounding variables including resident competencies (knowledge, skills, and attitude) suggested by the DAG were then selected for a multivariate linear regression model and were retained irrespective of their statistical significance.8,9 The outcomes were tested to ensure that they fit the residual of linear distribution assumption. The association between the posttest/pretest MCQ scores and midterm examinations was presented as an adjusted beta coefficients with 95% confidence limits (CL) and considered significant if the F-test p values were <0.05. Finally, the effect modification between the potential predictors and midterm examinations on the outcomes were evaluated for each final model.

Sample size calculation

The sample size was calculated based on the correlation between midterm MCQ scores and posttest ANTS scores. A correlation coefficient (r) of 0.4, which represented a medium level of correlation, was used to calculate the required sample size of 47 patients under a significance

level of 0.05. Since a total pool of 24 residents were available from PGY-1 to PGY-3, we used data from two cohorts – collected in 2017 and in 2019 – to include in this study project.

RESULTS

Forty-eight anaesthesia residents were recruited from among the two cohorts of ANTS training in 2017 and 2019, including 16 PGY-1, 16 PGY-2, and 16 PGY- 3 residents. Table 1 compares the characteristics of the various three-year anaesthesia residents, which reveals no significant differences. Table 2 compares the ANTS pretest/posttest scores, midterm examination scores, and anaesthesia residents’ competency as evaluated by the staff. There were no significant differences between the ANTS pretest/posttest scores and midterm examination scores among groups; however, the skill competency increased by year of resident training (P<0.001).

Table 3 shows the correlation between midterm examination scores, residents’ competency as evaluated by staff, and MCQ ANTS scores. The correlation between the midterm examination scores and pretest/posttest ANTS scores was low (r<0.3). SAQ scores had a negative correlation with posttest ANTS scores but were not significantly different (r=-0.24, P=0.10).

Analysis of pretest MCQ score

Nine variables (sex, PGY, self-study, knowledge score, skill competency, attitude, MCQ score, SAQ score, and OSCE) as suggested by a previous literature review were related to ANTS scores.4,5,7 There was an effect

TABLE 1. Comparison of characteristics of anaesthesia residents.

*Numbers in the table represent frequency (%) unless stated otherwise.

TABLE 2. Comparison of ANTS pretest and posttest scores, midterm examination scores, and anaesthesia residents’ competency as evaluated by staff.

*Mean (standard deviation) and P value by analysis of variance. †Median (interquartile range) and P value by Kruskal–Wallis test. Abbreviations: ANTS: anaesthetists’ non-technical skills, MCQ: multiple choice question, SAQ: short answer question, OSCE: objective structured clinical examination

TABLE 3. Correlation between midterm examination scores of anaesthesia training, residents’ competency as evaluated by staff, and multiple choice question ANTS scores.

P value by Pearson's correlation coefficients (r), OSCE: Objective structured clinical examination.

modification between sex and midterm MCQ scores in the pretest MCQ scores (P<0.05). Three potential biasing variables (PGY, knowledge score, and attitude) of the total effect of midterm examinations indicated by the DAG (Fig 2A) were included as the minimally sufficient adjustment set with midterm examination variables (MCQ*sex/SAQ/OSCE) (Table 4). Among males, MCQ scores had a negative correlation with pretest ANTS scores (P=0.024). Regardless of sex, SAQ scores had a

negative correlation with pretest ANTS scores (P=0.033) whereas OSCE was positively correlated with pretest ANTS scores (P=0.038).

Analysis of posttest MCQ scores

Eleven variables (sex, PGY, self-study, resident roles, knowledge score, skill competency, attitude, pretest ANTS score, MCQ score, SAQ score, and OSCE) suggested by the previous literature review were related to ANTS scores.4,5,7

Fig 2. Hypothesised causal relationship between midterm examinations and pretest (A) and posttest (B) non-technical skill scores.

Note: PGY: post-graduate year, ANTS: anaesthetists’ non-technical skills, MCQ: multiple choice question, SAQ: short answer question, OSCE: objective structured clinical examination.

There was no evidence of effect modification between sex and any other variables in relation to the midterm examinations in the posttest MCQ model. Three potential biasing variables (PGY, knowledge score, and attitude) of the total effect of midterm examinations indicated by the DAG (Fig 2B) were included as the minimally sufficient

adjustment set with midterm examination variables (MCQ/SAQ/OSCE) (Table 4). The midterm MCQ scores showed a positive correlation with ANTS posttest scores (P=0.007) whereas the SAQ scores showed a negative correlation with ANTS posttest scores (P=0.004).

TABLE 4. Multiple linear regression by total effect model predicting relative probability of pretest and posttest ANTS scores based on midterm examinations.

Male (ref= female)

8.14 (-0.11, 16.4)

8.04 (-0.07, 16.15)

0.052

Main exposure variables

Pretest ANTS score† Crude β

(95% CL)

Adjusted β

(95% CL)

P value*

MCQ score 0.07 (0, 0.14) 0.14 (0.04, 0.24) 0.006

SAQ score

0 (-0.07, 0.07)

-0.11 (-0.21, -0.01)

0.033

MCQ score: male -0.12 (-0.23, 0) -0.13 (-0.24, -0.02) 0.024

r of final model

0.38

Main exposure variables

Posttest ANTS score‡ Crude β

(95% CL)

Adjusted β

(95% CL)

P value*

OSCE score 0.5 (-0.17, 1.18) 1.02 (0.06, 1.98) 0.038

SAQ score

-0.07 (-0.16, 0.01)

-0.16 (-0.27, -0.05)

0.004

MCQ score 0 (-0.07, 0.07) 0.14 (0.04, 0.24) 0.007

r of final model

0.52

OSCE score -0.51 (-1.37, 0.36) -0.25 (-1.29, 0.8) 0.634

*F-test. β: Beta coefficients, CL: confidence limits, MCQ: multiple choice question, SAQ: short answer question, OSCE: objective structured clinical examination, ref: reference.

†Minimally sufficient adjusted set suggested by a directed acyclic graph: post-graduate year of anaesthesia residents, knowledge score, attitude competency.

‡Minimally sufficient adjusted set suggested by a directed acyclic graph: post-graduate year of anaesthesia residents, knowledge score, attitude competency.

DISCUSSION

We examined whether technical skills in anaesthesia training using midterm MCQ/SAQ/OSCE assessments were associated with non-technical skill knowledge based on the pretest/posttest MCQ ANTS simulation workshop. According to the one-day workshop with three ANTS scenarios for each midterm examination, only one or two residents, and not all, could participate in each workshop. The rest were observed in the observer room. Instead of assessing their ANTS behavior one- by-one in the workshop, the debrief session, which was attended by all the residents, was performed to ensure a better and understanding of ANTS scenarios. Therefore, an assessment of ANTS knowledge using MCQs was feasible and helped the residents understand more about the ANTS elements incorporated in their

learning by performing them daily. Therefore, the residents gradually gained non-technical skills during the training. Since we used two prospective cohorts (2017 and 2019), eight residents who were PGY1 in 2017 become PGY3 in 2019. Approximately 17% (8/48) of the residents might have been familiar with pretest MCQ ANTS. To minimise this bias, PGY was one of the adjusted sets for confounders in the final multivariate model of pretest and posttest MCQ ANTS. Our findings from Table 3 reveal that the relationship between MCQ/SAQ/OSCE scores and pretest/posttest MCQ ANTS scores (univariate analysis) were low (r<0.3), but the final multivariate linear regression models revealed a significant relationship not only between midterm examinations and pretest ANTS scores (r=0.38) but also between midterm examinations and posttest ANTS scores (r=0.52).

This proves that the relationship between technical and non-technical skills in anaesthesia is not simply linear due to the environment and many other circumstances during training, such as sex, PGY, self-study, residents’ competencies, and residents’ roles during ANTS workshop. This non-linear network can help researchers interpret the complex relationships within it.10,11 Therefore, DAG was performed to construct the causal relationship between the two anaesthesia skills and to minimise confounding variables. We used midterm examinations (MCQ/SAQ/ OSCE) to represent technical skill in anaesthesia training, which was similar to what was done in Raksamani’s study.12 The difference was that the MCQs, which comprised all four ANTS categories (SA/TM/TW/DM), was implemented instead of assessing ANTS behaviours among residents to correspond with non-technical skills. Moreover, to minimise the measurement bias, technical skills, as assessed by midterm examinations and the ANTS workshop, were performed in the same evaluation period (Fig 1).

Before the ANTS workshop, we found that OSCE had a positive correlation with pretest MCQ ANTS scores. This may be attributable to the fact that OSCE demonstrates clinical skills/competency in a standardised medical scenario, which represents routine clinical practise.13 Thus, this skill may have been applied in the scenario in the pretest MCQ before the workshop. Furthermore, our results revealed that midterm MCQ scores had a positive correlation with pretest MCQ scores but, among males, it had negative correlation. Regardless of sex, the reason for a positive correlation between the midterm MCQ scores and pretest ANTS MCQ scores could be similar types of questions. However, compared to females, MCQ scores revealed a reverse association with pretest MCQ scores among males. A previous study revealed that males had higher pretest MCQ scores than females.7 Therefore, it was found that sex could somehow affect the correlation between midterm MCQ scores and pretest ANTS scores and was considered a common confounding variable in other models.14,15

After the ANTS workshop, there was no effect modification between the midterm examinations and posttest ANTS scores. We found midterm MCQ scores to have an obvious positive correlation with posttest ANTS scores (P<0.007). Perhaps, the ANTS lecture before the workshop promoted ANTS basic knowledge simultaneously, while enhancing ANTS skills through different case scenarios (SA/TW/TM/DM) during the workshop and debriefing sessions. This possibly led to a higher positive correlation between midterm MCQ scores and posttest ANTS score. A previous study revealed that participants or observers can gain similar knowledge of

ANTS based on the pretest/posttest MCQ scores.7

The reasons we implemented the 20-item MCQs to represent non-technical skills in anaesthesia training were as follows: first, multiple choice tests can be an effective and simple way to measure learning.16 Second, assessments can be completed quickly, providing students with prompt feedback. Third, MCQs can assess knowledge and cognition and cover large topics in the curriculum.17 Moreover, the application of MCQs can vary from simple recall to problem solving. Well-written MCQs can go beyond testing rote facts and might measure higher cognitive abilities.18 However, from our results (Table 2), it was found that the posttest MCQ ANTS scores among residents (14.3) improved compared to pretest scores (12.6) but not greatly. This might be because the nature of four-answer choices makes it difficult to decide the best answer among four ANTS categories from each ANTS situation, especially an ANTS training beginner. Therefore, we discussed the appropriate answer with the residents after the posttest to stimulate higher cognitive function during clinical practise.

Surprisingly, SAQ scores revealed a negative correlation with pretest MCQ ANTS scores, and this finding was confirmed by posttest MCQ ANTS scores when SAQ scores indicated a more significant negative correlation. This could be because SAQs need to be related to a common or specific topic depending on the objective of the curriculum subjects whereas the ANTS MCQ test is broader and non-specific to a particular component of ANTS. Moreover, SAQs are more similar to essay questions that tend to be more difficult than MCQs since there are more cues to stimulate memory and because recall rather than recognition is required.19 A good attitude, which was our most important confounder, is necessary in anaesthesia training and could be the key to becoming proficient in both technical and non-technical anaesthesia skills, especially the learners’ attitudes towards medical ethics education.20

Strengths and limitations of the study

We formed a multivariate analysis model using DAG to adjust indicated confounders in the final model in order to determine whether the midterm examinations were independent predictors of the outcomes. The limitations of the study are as follows: first, the assessment of ANTS knowledge was used instead of ANTS behavioural skills of each resident owing to the limitation of time for the midterm ANTS workshop. Second, because we used two cohorts from two different years, the periods of training might have affected the midterm examination scores because the training environment changes with time.

CONCLUSION

Our findings revealed that technical skills based on midterm MCQ and OSCE assessments were associated with non-technical skill knowledge in anaesthesia. An assessment of ANTS knowledge during the workshop can help residents comprehend detailed ANTS information and foresee adverse situations in advance. Residents can gain non-technical skills by performing routine anaesthesia practise by collaborating with other anaesthesia personnel (nurse anaesthetists/anaesthesia technicians/anaesthesia staffs/surgeons) daily. A good attitude, which was our most important confounder, is necessary in anaesthesia training and could be the key to becoming proficient in both technical and non-technical anaesthesia skills. A further research regarding non-technical skill knowledge and non-technical skill behaviour of all residents should be performed to improve our ANTS training workshop.

ACKNOWLEDGMENTS

We would like to thank Editage for assistance with editing the manuscript.

Funding: Faculty of Medicine, Prince of Songkla University, Hat Yai, Thailand.

Conflict of interest: The authors have no conflicts of interest.

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16. Farley JK. The multiple-choice test: writing the questions. Nurse Educ 1989;14:10-2, 39.

17. Salih KMA, Alshehri MAA, Elfaki OA. A comparison between students’ performance in multiple choice and modified essay questions in the MBBS pediatrics examination at the College of Medicine, King Khalid University, KSA. Journal of Education and Practice 2016;7:116-20.

18. Scully D. Constructing multiple-choice items to measure higher-order thinking practical assessment. Assessment, Research, and Evaluation [serial on the Internet]. 2017 May [cited 2022 Jul 5]; 22(4): [about 14 p.]. Available from:https://scholarworks. umass.edu/cgi/viewcontent.cgi?article=1355&context=pare

19. Farooqui F, Saeed N, Aaraj S, Sami MA, Amir M. A comparison between written assessment methods: multiple-choice and short answer questions in end-of-clerkship examinations for final year medical students. Cureus 2018;10:e3773. doi: 10.7759/cureus.3773.

20. Sathirareuangchai S. Iramaneerat C. Medical students’ and interns’ attitudes toward medical ethics education in a Thai medical school. Siriraj Med J 2016;68:97-103.

Anxiety, Depression and Cognitive Emotion Regulation Strategies in Psychiatric Patients during the COVID-19 Pandemic

Woraphat Ratta-apha, M.D., Ph.D.*, Nichkamol Kittipavara, M.D.*, Varaporn Sripirom, M.N.S.**, Chia-Chun Hung, M.D., Ph.D.***,****, Tony Szu-Hsien Lee, Ph.D. ***,****,*****, Pornjira Pariwatcharakul, M.D.*, Kamonporn Wannarit, M.D.*, Panate Pukrittayakamee, M.D.*, Netnapa Promsuwong, B.N.S.**, Jingswat Sirikunchoat, M.D.*, Sirirat Kooptiwoot, M.D.*

*Department of Psychiatry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, **Psychiatric Outpatient Department

Unit, Siriraj Hospital, Bangkok, Thailand, *** Center for Addiction Prevention and Policy Research, National Taiwan Normal University, Taipei, Taiwan, **** Continuing Education Master's Program of Addiction Prevention and Treatment, National Taiwan Normal University, Taipei, Taiwan,

*****Department of Health Promotion and Health Education, National Taiwan Normal University, Taipei, Taiwan.

ABSTRACT

Objective: The coronavirus disease 2019 (COVID-19) pandemic likely impacted emotional regulation and mood states. The present study aimed to investigate the perceived risk, stigma, and emotional regulation strategies of psychiatric patients, as well as the association between these characteristics, cognitive emotion regulation strategies (emotional suppression and cognitive reappraisal), and anxiety and depressive symptoms during the COVID-19 pandemic.

Materials and Methods: The present cross-sectional study of 282 patients with anxiety and mood disorders was conducted during the COVID-19 pandemic in 2020. Participants completed questionnaires that investigated anxiety (hospital anxiety and depression scale [HADS]), depression (patient health questionnaire [PHQ-9]), and cognitive emotion regulation strategies (emotion regulation questionnaire [ERQ]). Descriptive statistics were used to assess the data. The t-test, chi-square test, and Mann-Whitney U test were used to compare the differences between the two groups, with cut-off scores of 11 in the HADS and nine in the PHQ-9.

Results: Most participants were female (78.4%), and the median age was 31 years. A total of 23.8% of participants reported having anxiety symptoms, and 24.8% and 54.3% of participants reported having depression on the HADS-D and PHQ-9, respectively. HADS-A, HADS-D, and PHQ-9 were found to be significantly associated with emotional regulation style (P=0.002, P=0.005, P=0.006) but not with perceived risk and stigma. Patients with anxiety or depression tended to use expressive suppression more often than cognitive reappraisal.

Conclusion: This study found that mood states were associated with cognitive emotional regulation strategies during the COVID-19 pandemic. Patients with anxiety or depression tended to use expressive suppression more often than cognitive reappraisal.

Keywords: COVID-19; coronavirus; cognitive emotional regulation; anxiety; depression (Siriraj Med J 2022; 74: 857-864)

Corresponding author: Sirirat Kooptiwoot E-mail: skooptiwoot@gmail.com

Received 13 October 2022 Revised 7 November 2022 Accepted 9 November 2022 ORCID ID:http://orcid.org/0000-0001-6427-4738 http://dx.doi.org/10.33192/Smj.2022.100

All material is licensed under terms of the Creative Commons Attribution 4.0 International (CC-BY-NC-ND 4.0) license unless otherwise stated.

INTRODUCTION

Coronavirus disease 2019 (COVID-19) spread rapidly following its emergence in 2019 in Wuhan, Hubei, China.1 The World Health Organization (WHO) designated the coronavirus disease a public health emergency after only a few months, and the number of verified cases and associated fatalities continues to increase.2 The present pandemic presents a serious worldwide problem. Thailand was the first country outside of China to diagnose a case of COVID-19 on January 17, 2020.3

The current global crisis has affected healthcare systems, global economic outlooks, and the delivery of education, as well as people’s daily lives, habits, and physical and mental health, whereby psychological distress due to a fear of infection, depression, anxiety, and stigma have been reported.4,5 Anxiety and fear may have a negative impact on one’s mental health, particularly those with psychological vulnerability. Furthermore, other pre- existing social, cultural, and economic vulnerabilities can also play a part.6 When a stressful event occurs, certain groups, such as women, children and the elderly, are at greater risk of complications.7

Patients with psychiatric disorders are also vulnerable. When a large stressor or unexpected event occurs, psychiatric symptoms may be worsened or prolonged.8 Previous research in Thailand found that outpatients with a mental health condition had a strong understanding of COVID-19, good preventive practices, and a low level of anxiety about COVID-19.9

An individual’s emotional regulation, which can be defined as “the process that individuals influence which emotions they have, and how they experience and express these emotions”, appears to contribute to a stress reaction.10 The current study concentrated on the two most important emotion management strategies: cognitive reappraisal and emotional suppression. Cognitive reappraisal necessitates the adaptive application of cognitive effort to modify a situation’s evaluation, which alters the emotional response before it fully occurs. Conversely, expressive suppression is a response-focused technique that involves a behavioral attempt to inhibit the display of an already occurring emotional response.11,12 During the COVID-19 pandemic, previous studies revealed that emotional suppression was associated with poorer psychological well-being, such as increased depressive symptoms, psychological distress, and reduced emotional well-being.13-15

To the best of our knowledge, concerns regarding mood symptoms and emotional regulation in patients with psychiatric disorders during the pandemic have not been thoroughly studied in Thailand. Most research has

focused on the overall population or a specific community during the pandemic. The current study has two goals: 1) to investigate the psychological impact, perceived risk, stigma, and emotional regulation strategies of patients with psychiatric problems during the COVID-19 pandemic, and

2) to investigate the relationship between the characteristics, emotion regulation strategies, anxiety, and depressive symptoms. The authors expect that those psychiatric patients who employ emotion regulation techniques that favor emotional suppression over cognitive reappraisal will have higher PHQ-9 and HADS scores.

MATERIALS AND METHODS

Participants

The current study included psychiatric patients who visited the outpatient unit of the psychiatric clinic at Siriraj Hospital in April and May 2020. Participants had to be at least 18 years old, diagnosed with a mood or anxiety disorder, have a good comprehension of the Thai language, and be willing to participate in the study. Individuals with severe or emergency medical or psychiatric disorders, such as unstable vital signs, psychotic symptoms, or suicidal behavior, were excluded. The study was approved by the Siriraj Research Affairs and Siriraj Institutional Review Board (SIRB), Faculty of Medicine Siriraj Hospital, Mahidol University (Si 659/2020). The study was performed in accordance with the principles of the Declaration of Helsinki. Some parts of the questionnaire obtained for the present study were translated and conducted under the approval of the “Preventive measure and psychology stress on COVID-19” across Japan, Korea, Taiwan, and Thailand” (REC Number:

202003HS002).

Procedure and measurement

The study team recruited 282 patients with mental disorders according to the inclusion criteria listed above. The participants completed a questionnaire that assessed their demographic data and questionnaire sets. To comply with the COVID-19 pandemic policy, the authors offered a paper-based questionnaire with a set of envelopes to be returned by post and a paper with a quick response code for an online questionnaire. Participants were free to answer in any way that they saw fit. The questionnaires were completed anonymously.

Instruments

The questionnaire consisted of three sections. The first section collected demographic information about the respondents, including sex, age, occupational status, and educational status. The second section consisted

of questions about perceived risk and stigma that applied the questions from the questionnaire of the project “Preventive measure and psychology stress on COVID-19 by medical staff and students across Japan, Korea, Taiwan, and Thailand”. The third section included three structured scales: the Thai version of the patient health questionnaire (PHQ-9), the hospital anxiety and depression scale (HADS), and the emotion regulation questionnaire (ERQ).

Patient health questionnaire (PHQ-9)

The PHQ-9 is a self-report of depressive symptoms comprised of nine items based on the DSM-IV diagnostic criteria for major depressive episodes.16 Lotrakul et al. translated the original version to Thai and used it as a screening tool for depression. With a cut-off score of nine or higher, the Thai version of the PHQ-9 exhibited acceptable internal consistency (Cronbach’s alpha=0.79).17

Hospital anxiety and depression scale (HADS)

The HADS is a questionnaire used for screening anxiety and depressive disorders.18 The HADS consists of 14 items, including the HADS-Anxiety Subscale (HADS-A) and the HADS-Depressive Subscale (HADS-D). A score equal or greater than 11 indicated anxiety or depression. The Thai version of the HADS-A has a sensitivity and specificity of 100% and 86%, respectively, and the HADS-D has a sensitivity and specificity of 85.7% and 91.3%, respectively.19

Emotion regulation questionnaire (ERQ)

Gross and John developed the ERQ, a 10-item self- reported questionnaire intended to examine respondents’ inclination to manage their emotions in two styles: cognitive reappraisal (six items) and expressive suppression (four items).11 Higher subscale mean scores suggest greater use of a specific emotion control style. Khumrod and Soonthornchaiya translated and then evaluated the Thai version of the ERQ to assess its validity and reliability (2017). In the Thai version, the content validity index of the ERQ was 0.80, and its reliability was 0.82.20

Statistical analysis

The data were analyzed using SPSS (version 21.0; IBM SPSS, Armonk, NY, USA). Descriptive statistics were used to characterize the demographic data, agreement of perceived risk, and stigma among all participants. Descriptive analyses included the means, standard deviations, medians, and interquartile ranges (for non-normally distributed data). Using the HADS cut-off score of 11 and the PHQ-9 cut-off score of nine, the data were classified

into two groups: the participants who had anxiety or depression (HADS-A ≥ 11, HADS-D ≥ 11, or PHQ-9 ≥ 9) and those who did not. The t-test, chi-square test, and Mann-Whitney U test were conducted to compare the differences in characteristics, ERQ, perceived risk, and stigma between the two groups. Pearson’s correlation was used to investigate the correlation between HADS-A, HADS-D, and PHQ-9. A probability level of p < 0.05 was considered to be statistically significant.

RESULTS

Basic characteristics

This cross-sectional study included 282 participants. Of these, 221 (78.4%) were females, and 61 (21.6%) were males. The median age was 31 years (median [(Q1-Q3) = (25-41)]). the employment rate was 251 (89.0%), and the majority of participants (229; 81.2%) had graduated from college or university (Table 1). The primary diagnoses were depressive disorders, bipolar disorder, and anxiety disorder or insomnia disorder in 45.7%, 14.9%, and 39.4%, respectively. Overall, 23.8% of participants reported having anxiety symptoms (HADS-A positive; HADS-A ≥ 11), while 24.8% and 54.3% of participants reported having depression according to the HADS-D (HADS-D ≥ 11) and PHQ-9 (PHQ-9 ≥ 9), respectively. The median age of the participants with anxiety and depression was significantly different between the two groups (HADS-A, p<0.001; HADS-D, p=0.002; PHQ-9,

p<0.001). Participants who were HADS-A, HADS-D, or PHQ-9 positive were younger than those who were negative. No other differences were observed between the two groups (Table 1).

Emotional regulation style: Comparison between styles by HADS and PHQ-9

Participants preferred cognitive reappraisal over expressive suppression (72.3% vs 27.7%) (Table 2). When anxiety symptoms were used to compare emotional regulation styles, the HADS-A was found to be significantly associated with emotional regulation style (P=0.002; OR [95% CI] = 2.51 [1.39-4.52]). Participants who were anxious (HADS-A positive) preferred expressive suppression (42.4%), whereas those who were not anxious (HADS-A negative) preferred cognitive reappraisal (72.3%). HADS-D was significantly associated with emotional regulation style in participants with depression (P=0.005; OR [95% CI] = 2.29 [1.27-4.13]). Participants with depression (HADS-D positive) preferred expressive suppression (40.9%), whereas those without depression (HADS-D negative) preferred cognitive reappraisal (76.8%). The PHQ-9 was also significantly associated with emotional

Ratta-apha et al.

HADS-A

HADS-D

PHQ-9

TABLE 1. Characteristics of psychiatric patients grouped by the hospital anxiety and depression scale-anxiety subscale (HADS-A), depression subscale (HADS-D), and patient health questionnaire-9 (PHQ-9)

* Mann-Whitney U test

TABLE 2. Emotional regulation style in psychiatric patients grouped by the hospital anxiety and depression scale-anxiety subscale (HADS-A), depression subscale (HADS-D), and patient health questionnaire-9 (PHQ-9)

regulation style (P=0.006; OR [95% CI] = 2.20 [1.24- 3.88]). Participants with depression (PHQ-9 positive) used expressive suppression (34.5%), whereas participants without depression (PHQ-9 negative) used cognitive reappraisal (80.7%).

Perceived risk and stigma

Regarding perceived risk, 58.2% (n=163) participants agreed with the statement “It is possible that I might be infected by COVID-19,” 63.0% (n=177) agreed with the statement “It is possible that my neighbors, colleagues,

and friends might be infected by COVID-19,” and 87.5% (n=246) agreed with the statement “It is possible that others might be infected by COVID-19”.

Regarding stigma, 11.0% (n=31) of participants agreed that they felt fear/afraid, 25.3% (n=71) avoidance, 3.2% (n=9) kept their diagnosis a secret, 9.6% (n=27) felt embarrassed, and 10.3% (n=29) stigma.

The HADS-A, HAD-D, and PHQ-9 scores were not associated with the agreement of all perceived risk and stigma items (all P>0.05; Table 3).

DISCUSSION

The present study investigated the psychological impact, perceived risk, stigma, and emotional regulation strategies of patients with psychiatric problems during the COVID-19 pandemic and the association between the characteristics, emotion regulation strategies, and psychiatric symptoms.

Anxiety and depression

Regarding the first objective, the results showed that about a quarter of participants reported having depressive or anxiety symptoms using the HADS-A or HADS-D, and about half reported having depression using the PHQ-9. There was a significant difference in the median age of patients with anxiety and depression. While the other features were similar, the participants with anxiety or depression were younger. Although the number of participants with anxiety differed between the two questionnaires, HADS-D and PHQ-9, we discovered a moderately significant correlation (r=0.767, P<0.01) between both questionnaires (Table 4). The results of the two questionnaires differ because the HADS-D concentrates on emotions and interests, whereas the PHQ-9 assesses neurovegetative symptoms. Other than age, we could not find an association between mood states (depression and anxiety), sex, and employment.

The current study’s participants were comparable to that of a previous study, which enrolled outpatients with a mental health condition from university hospitals.9 However, the objectives of this study were different. The earlier study concentrated on the participants’ COVID-19 knowledge, preventive behaviors, and anxiety levels, whereas the current study included emotion control, perceived risk, and stigma.

Perceived risk and stigma

According to the perceived risk in the three statements, the majority of the participants (87.5%) agreed with

the perceived risk of COVID-19 infection in others. Approximately half (58.2%) of the participants reported a risk of infection. This could imply that believing others are afflicted is simpler than believing oneself. Participants may have been confident in their preventive behaviors toward COVID-19, which is consistent with a recent study that found that almost half of the participants had a low-risk perception of COVID-19.

Regarding stigma, around a quarter of the participants agreed with the statement of avoidance, while “keeping a secret” received the least agreement. This could be because most participants did not see the illness as an issue that needed to be concealed. Avoidance is one of the defense mechanisms used by neurotic patients to deal with worry. We hypothesized that anxiety and sadness are related to perceived danger or stigma. However, no association was found between perceived risk and stigma in participants with high anxiety (HADS-A ≥ 11), depression (HADS-D ≥ 11), or PHQ-9 ≥ 9. There are several possible explanations for this, including 1) because the data were collected during the country’s second wave of the pandemic (around April-May 2020), which did not have a high prevalence rate compared to Europe, most people felt they were not at a high risk of being infected; and 2) at that time, most people may have known about COVID-19 and how to deal with it. As a result, the perceived threat or stigma may have been lower.

Emotional regulation strategies

According to the results, participants preferred cognitive reappraisal over expressive suppression. Anxiety and depression were significantly related to emotional regulation style, and participants who were anxious or depressed preferred expressive suppression. Emotional regulation is a complex mental process controlled by various elements such as mental health, mood state, and life satisfaction.21 Effective emotion regulation is crucial for effective functioning in a dynamic context.

                        Previous studies have shown that greater use of cognitive

reappraisal and less use of expressive suppression are

TABLE 4. Correlation between HADS and PHQ-9

**. Correlation is significant at the 0.01 level (2-tailed).

associated with more positive outcomes in the domains of mood, well-being, and social functioning.22 A previous meta-analysis showed a significant positive correlation between cognitive reappraisal and a positive indicator of mental health, while expressive suppression showed a negative correlation with a positive indicator of mental health. In addition, this meta-analysis found that cultural values may influence individual emotion regulation strategies.23 A previous study revealed that regulating emotions using cognitive reappraisal was associated

with greater resilience (i.e., the ability to seek enjoyable activities and social support and feelings of hope and resourcefulness).24 Cognitive reappraisal is a technique that may be learned and utilized to mitigate the impact of stress on an individual’s well-being, including stress induced by a pandemic.

The current study revealed that patients with psychological problems are especially susceptible and in need of care, particularly during the current COVID-19 pandemic. Psychological therapy focusing on emotional regulation skills could be used to support them cope with the COVID-19 pandemic. Even though face-to- face interventions are not possible during the pandemic, psychological support via the Internet and phone can ensure that patients with a psychiatric condition receive the care they require. It appears that people are now better equipped to deal with COVID-19. The current study sought to better understand the perceptions and emotional control styles of people suffering from psychiatric diseases. This increases preparedness to deal with mental health issues in the event of future pandemics.

Strengths and limitations

The present study has several strengths and limitations. To the best of our knowledge, this is the first study in Thailand to investigate emotion regulation styles and the relationship between these factors and anxiety and depression in people with mental health problems. This study also included a large number of patients with mood and anxiety disorders, which could be investigated using an emotional regulation lens. However, this study has significant drawbacks. First, this study was conducted at a single site and included only outpatients with mood and anxiety disorders, which may have resulted in a selection bias, and the representativeness of the population was limited. Inpatients may have more active symptoms, and their reactions may differ. Second, not all psychiatric disorders were included. Therefore, our findings may not be representative of other psychotic problems. Furthermore, the symptoms were established through self-report rather than face-to-face questioning, which may have resulted in a reporting bias. However, during the pandemic, these methods proved useful for data collection. Next, because the data were collected only once during the second wave of the pandemic, which was around April-May 2020, we could not examine the change or trend of the response during the pandemic. Therefore, the data should be carefully interpreted because it may only reflect the perception and behavior toward COVID-19 of the population at that specific point in time. Although we observed an increase in negative feelings

and emotional regulation among mental patients, as noted previously in the introduction and results of the manuscript, it is difficult to regulate external factors that influence each participant’s perception of COVID-19. Each participant has a unique viewpoint on COVID-19 based on how they feel about various aspects, such as health, costs, employment, etc. Furthermore, we could not conclude that patients with depression and anxiety disorders tended to use expressive suppression during the COVID-19 pandemic any more than normal because only one time point of data was collected.

Future recommendations and implications

In contrast to other examples, such as HIV infection, it can have a direct impact on specific groups of people, such as minorities or vulnerable groups, which may not be socially acceptable. COVID-19, on the other hand, can have an individual influence in cluster outbreaks like those in pubs and clubs, or on groups that can easily be identified as spreaders. The participants in this study were not infected with COVID-19 at the time of the study. As a result, this could assist to rule out strong thoughts about being infected, such as being labeled, feeling guilty, and so on. However, in the current study, external influences and one’s own views about the individual perspective were difficult to control. In order to address this challenge and obtain a clearer answer to the study, there may be several ways to design for additional research, such as:

1) inquiring into psychosocial issues that directly affect individual subjects for a better understanding of individual details, or 2) incorporating psychosocial variables into the analysis.

Further research across different sites and over time is necessary to understand the impact of the emerging disease pandemic on psychiatric problems and emotional control. Further research should cover additional mental illnesses, particularly in psychotic patients and inpatients who are more prone to stress. Furthermore, comparing people with and without psychiatric diagnoses should be considered in future studies. Nevertheless, the authors believe that the findings of this study contribute significantly to the literature on the experience of patients with psychiatric illnesses during the emerging disease pandemic. Furthermore, the association between emotion regulation strategies and mental health was investigated, which provides a foundation for future research and may lead to the development of useful interventions.

CONCLUSION

The results of the current study revealed that about a quarter to a half of the participants reported depressive

or anxiety symptoms. There was a significant difference in the median age of patients with anxiety and depression; participants with anxiety or depression were younger. Anxiety and depression were found to be associated with emotion regulation strategies. Patients with anxiety or depression tended to use expressive suppression more often than cognitive reappraisal. Understanding mood states and cognitive emotion regulation strategies will aid the future promotion of psychosocial interventions during pandemics.

ACKNOWLEDGEMENTS

The authors would like to thank the staff of the psychiatric outpatient unit, Siriraj Hospital for participants recruitment and Ms. Lakkhana Thongchot and Ms. Narathip Saguanpanich for their coordination and data analysis.

Potential conflicts of interest

None.

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Clinical Characteristics and Treatment Outcomes of Patients with Primary Ocular Adnexal Lymphoma at Siriraj Hospital

Sumalee Vangveeravong, M.D.*, Sanya Sukpanichnant, M.D.**, Mongkol Uiprasertkul, M.D.**, Archrob Khuhapinant, M.D., Ph.D.***, Tassapol Singalavanija, M.D.****

*Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, **Department of Pathology, Faculty of Medicine Siriraj Hospital, ***Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand, ****Department of Ophthalmology, Chulabhorn Hospital, HRH Princess Chulabhorn College of Medical Science, Chulabhorn Royal Academy, Bangkok, Thailand.

ABSTRACT

Objective: Malignant lymphoma represents the most common primary orbital malignant neoplasm.

Materials and Methods: We retrospectively reviewed data and analyzed clinical characteristics and treatment outcomes of patients with primary ocular adnexal lymphoma at Siriraj Hospital, a tertiary health-care and teaching center in Bangkok, Thailand between January 2004 and June 2017.

Results: From the total of 94 patients with primary ocular adnexal lymphoma, 77 cases (81.9%) were indolent, of which extranodal marginal zone lymphoma (EMZL) was the most common (76.6%). Aggressive subtype accounted for 17 cases (18.1%), consisting of diffuse large B-cell lymphoma (9.6%), mantle cell lymphoma (4.3%), and extranodal NK/T cell lymphoma (3.2%). Plasmacytic differentiation was found in half of EMZL and 4 cases of EMZL had association with IgG4-related disease. Most of the patients presented with proptosis (54.3%), followed by a palpable mass (42.6%). Most patients had Ann Arbor stage I (66.3%) with zero ECOG performance status (91.1%). Chemotherapy was the main treatment for both indolent and aggressive lymphomas. The overall response rate and complete response rate were 88.6% and 68.7%, respectively. The 5-year progression-free survival (PFS) and overall survival (OS) rates were 60.1% and 84.2%, respectively. The indolent group had better overall (92.4% vs 69.2%) (P=0.01) and complete response rates (73.1% vs 50%) (P=0.01) than the aggressive group.

Conclusion: Histopathological subtypes and clinical stages of lymphoma are the best indicators of prognosis and treatment outcomes. Chemotherapy was an effective treatment modality for both indolent and aggressive lymphoma subtypes with better treatment outcomes in the indolent group.

Keywords: Primary ocular adnexal lymphoma; extranodal marginal zone lymphoma with plasmacytic differentiation; IgG4-related disease; clinical characteristics; treatment outcomes (Siriraj Med J 2022; 74: 865-873)

INTRODUCTION

LymphomasaremalignanttumorsofBorTlymphocytes and, in rare cases, of natural killer (NK) cells. They are divided into Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Ocular adnexal lymphoma refers to a

malignant lymphoproliferative disease that involves the conjunctiva, lacrimal gland, eyelid, or orbit.1 Despite the uncommon orbital involvement by NHL, it represents the most common primary orbital and ocular adnexal malignant neoplasm.1–3 Lymphomas are clinically divided

Corresponding author: Tassapol Singalavanija E-mail: tassapol.sin@cra.ac.th

Received 13 August 2022 Revised 23 October 2022 Accepted 27 October 2022 ORCID ID:http://orcid.org/0000-0001-6651-7158 http://dx.doi.org/10.33192/Smj.2022.101

All material is licensed under terms of the Creative Commons Attribution 4.0 International (CC-BY-NC-ND 4.0) license unless otherwise stated.

into two groups. The first group, low-grade lymphoma, includes extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissue, follicular lymphoma, small lymphocytic lymphoma, and lymphoplasmacytic lymphoma. The second group, high-grade lymphoma, is composed of diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, T-cell lymphoma, and extranodal NK/T cell lymphoma. Most type of ocular adnexal lymphoma is low-grade B-cell NHL. EMZL is the most common subtype, followed by follicular lymphoma, DLBCL, and mantle cell lymphoma.4–10 Uncommon ocular adnexal lymphoma variants include plasmablastic lymphoma, extrannodal NK/T cell lymphoma, small lymphocyte lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, plasmacytomas, and peripheral T-cell lymphoma, NOS.4–10 There are many clinical manifestations of ocular adnexal lymphoma. Disease within the lacrimal glands and eye lids usually presents as palpable masses. In case that the disease extends behind the globe, then pain, proptosis, ophthalmoplegia, and globe displacement may be present. Localized plaque (termed a “salmon patch”) on the conjunctiva could be presented as an early sign of a conjunctival lymphoma. Orbital imaging typically shows a poorly defined mass that molds to surrounding structures without direct invasion or bony erosion.11 Suspicion of the diagnosis-either as a result of clinical or radiological findings—should lead to a confirmatory biopsy. Once a definite diagnosis is established, patients are referred for treatment by a multidisciplinary team composed of an ophthalmologist, a hemato-oncologist, a pathologist, and a radiation therapy specialist.

The purpose of this study was to evaluate the clinical characteristics and treatment outcomes of patients with primary ocular adnexal lymphoma at Siriraj Hospital. Deeper understandings gained through this research may contribute to improvements in multidisciplinary therapeutic planning.

MATERIALS AND METHODS

The protocol for this study was approved by the Institutional Review Board, Faculty of Medicine Siriraj Hospital, Mahidol University (Si 212/2018). Medical records were reviewed to collect data on all patients diagnosed with primary ocular adnexal lymphoma between 1 January 2004 and 30 June 2017 (13 years) at Siriraj Hospital, a tertiary health-care and teaching center in Bangkok, Thailand. The collected data related to the patients’ demographics, clinical and pathological assessments, disease stage, treatment modalities, and outcomes. Demographic data consisted of age, sex, provincial address, and underlying diseases. As to the clinical assessment data, they detailed

the clinical presentations, affected anatomical locations, laterality, time to diagnosis, and serological findings for lactate dehydrogenase levels and the statuses of the human immunodeficiency, hepatitis B, and hepatitis C viruses. The pathological assessment data comprised the biopsy site, date, and histological subtypes of the lymphomas which were independently reviewed by an experienced ocular pathologist (MU) and hematopathologist (SS). The disease stages were based on the Ann Arbor stage at the time of diagnosis, the Eastern Cooperative Oncology Group (ECOG) performance status, the number of extranodal sites involved, and the International Prognostic Index. In terms of treatment-modality, the details comprised type (chemotherapy, radiotherapy, surgery, immune- modulating therapy, or a combination of such treatment), the number of cycles of each treatment method, the kind of chemotherapy, and the salvage therapies employed for patients with recurrence. The treatment outcome assessments evaluated the responses to treatment, disease status, and current patient-status, all of which were based on WHO criteria. The outcome categories used were “complete response” (disappearance of the lesion), “partial response” (a decrease of ≥ 50% of the longest diameter of the tumor), “stable disease” (a decrease of

< 50% or an increase of < 25%), and “progressive disease” (an increase of ≥ 25%). All treatment outcomes were evaluated at the end of treatment, whereas the disease status was appraised at the final follow-up.

Progression-free survival (PFS) was defined as the length of time following the completion of the primary cancer treatment during which a patient remained free of the complications or events that the treatment was intended to prevent or delay. In comparison, overall survival (OS) was defined as the length of time between the date of diagnosis and death from any cause.

Quantitative data were analyzed with descriptive statistics. Univariable analyses were performed with SPSS for Windows, version 16.0 (SPSS Inc., Chicago, Il, USA). A p-value of < 0.05 was considered statistically significant. OS was calculated from the date of diagnosis to the date of the last follow-up or death from any cause. PFS was measured from the date of diagnosis to the date of the last follow-up, relapse/progression, or death from any causes. The probabilities of OS and PFS were estimated using the Kaplan–Meier method and the log-rank test for survival comparison.

RESULTS

From 99 patients diagnosed with lymphoma, after pathological reviews by an experienced ocular pathologist (MU) and hematopathologist (SS), 5 cases were excluded

with 3 cases diagnosed with IgG4-related disease, one case of plasmacytoma and one case of B-cell acute lymphoblastic leukemia. A total of 94 patients with primary ocular adnexal lymphoma were collected which comprised 56 males (59.6%) and 38 females (40.4%). Their ages ranged from 39 to 99 years, with a median of 61.5 years. Median time to diagnosis was approximately 3 months, while the median follow-up duration was 33 months. Most patients presented with proptosis (56%), followed by a palpable mass (42%) and cellulitis (2%). The most commonly involved anatomical locations were the orbit (62%), conjunctiva (17%), lacrimal gland (15%), and eyelid (5%) (Table 1). For the immunophenotype, 90 cases (95.7%) were of B-cell and the remaining 4 cases (4.3%) were of T-cell or NK cell. In terms of subtypes, 77 cases (82%) were indolent, of which extranodal marginal zone lymphoma (EMZL) was the most common (76.6%), followed by follicular lymphoma (2.1%), small lymphocytic lymphoma (1.1%) and unclassifiable small B-cell lymphoma (1.1%). Of the 72 cases of EMZL, there were 37 cases (51.4%) with plasmacytic differentiation. Interestingly, there were 4 cases (5.5%) of EMZL with accompanying IgG4-related disease and 2 of them also showed plasmacytic differentiation. While the aggressive subtype accounted for 17 cases (18.1%), consisting of DLBCL (9.6%), mantle cell lymphoma (4.3%), extranodal NK/T cell lymphoma (3.2%), plasmablastic lymphoma (1.1%) and peripheral T-cell lymphoma, NOS (1.1%). We reviewed the one case diagnosed with unclassifiable small B-cell lymphoma; the tissue biopsy was too small and the patient lost to follow-up so that further clinical examination and tissue biopsy could not be performed. Regarding the other laboratory findings, most patients had normal lactate dehydrogenase levels (92%). Three patients (4%) were found positivity with the human immunodeficiency virus, two (2.7%) tested positive for hepatitis B, while three (4%) were positive for hepatitis C.

After definitive tissue diagnosis and disease staging, the treatments were planned. Of the 94 cases, 64 (68%) were treated at Siriraj Hospital, while the remainder (30; 32%) were referred to other hospitals to meet patients’ preference or insurance-coverage requirements. Most patients had Ann Arbor stage I (66.3%) with zero ECOG performance status (91%). Of those, the International Prognostic Index of low risk (82.4%) was the most common (Table 1).

Regarding treatment modalities, 42 (67.2%) cases received chemotherapy and 11 cases (17.2%) received radiotherapy (Table 2). CVP regimen (cyclophosphamide, vincristine, and prednisolone; 59.5%) was the most common chemotherapy regimen, followed by CHOP

regimen (cyclophosphamide, doxorubicin, vincristine, and prednisolone; 16.7%) and others. Seven cases (10.9%) received combination chemotherapy and radiotherapy, while a small number of patients (3.1%) underwent surgery only or were administered with an immune modulating agent (1.6%).

Analysis of the treatment outcomes revealed that the overall response rate and complete response rate were 88.6% and 68.7%, respectively. Most cases of indolent subtype were from the first Ann Arbor staging (76.4%) following by 11.1% of stage II, 6.9% of stage III and 5.6% of stage IV. In contrast to aggressive subtype, the staging was varied with 23.5% in stage I, 41.1% in stage II, 5.8% in stage III, and 29.4% in stage IV. The 5-year PFS and OS rates were 60.1% and 84.2%, respectively (Table 3). Subgroup analysis found that the indolent lymphoma group had a 92.4% overall response rate and a complete response rate of 73.1%. 5-year PFS and OS rates for the indolent group were 62.8% and 92.5%, respectively. In aggressive lymphoma types, the overall response rate was 69.2%, with a complete response rate of 50%. The 5-year PFS and OS rates were 50% and 65.9%, respectively. Fig 1A illustrates the Kaplan-Meier PFS rates for various lymphoma subtypes, while Fig 1B presents the Kaplan- Meier OS rates.

DISCUSSION

Results of the current study were compared with those of the study by Yen et al.,12 which involved a review of the level 3 evidence of 27 studies on ocular adnexal lymphoma treatments. We grouped each of those 27 studies into regions to identify the characteristics of primary ocular adnexal lymphoma specific to each continent (Table 4). Of the 1,938 patients, females predominated slightly (54%)12 but males predominated in our study (59.6%). The median age at diagnosis (60.1 years) was nearly the same as our study (61.5 years). Their study and our study found that orbit was the most commonly affected anatomical location. However, some other reports found that conjunctiva was the most common site of involvement, followed by orbit.12 In general practice, orbital lesions are difficult for general ophthalmologists to biopsy so that many patients with proptosis and orbital lesions are referred from primary or secondary care hospitals to our hospital that is a tertiary care center and medical school. This may explain why orbit was the most commonly affected site for primary ocular adnexal lymphoma.

Distribution of the lymphoma subtypes in our study revealed that extranodal marginal zone lymphoma (EMZL) was the most frequent subtype (76.6%), followed by

TABLE 1. Demographic data, clinical assessment, pathological assessment, and stage of lymphoma.

Abbreviations: ECOG, Eastern Cooperative Oncology Group; HBV, Hepatitis B virus; HCV, Hepatitis C virus; HIV, Human immunodeficiency virus; LDH, Lactate dehydrogenase; NK, natural killer; SD, Standard deviation

TABLE 2. Treatment modalities.

Abbreviations: CHOP, Cyclophosphamide, doxorubicin, vincristine, and prednisolone; CP, Chlorambucil hydrochloride and prednisone; CVP, Cyclophosphamide, vincristine, and prednisolone; R-BAC, Rituximab, bendamustine, and cytarabine; R-CVP, Rituximab, cyclophosphamide, vincristine, and prednisolone; RT, Radiotherapy

TABLE 3. Treatment-outcome assessments.

Abbreviations: CMT, chemotherapy; CR, complete response rate; ORR, overall response rate; OS, overall survival; PFS, progression-free survival; RT, radiotherapy

A

B

Fig 1. A) Kaplan-Meier progression-free survival (PFS) for the lymphoma subtypes; B) Kaplan-Meier overall survival (OS) for the lymphoma subtypes.

DLBCL (9.6%) and others. This distribution corresponds with other studies. Seresirikachorn et al. from northern Thailand reported EMZL as the most common (85.2%), followed by DLBCL (5.6%) and others.13 Similarly, Yen et al. reported EMZL as the most common (75.2%), but followed by follicular lymphoma (7.9%) and DLBCL (6.1%).12 A distinctive finding in our study is the recognition of plasmacytic differentiation in half of EMZL cases (51.4%). This phenomenon is in fact quite common in marginal zone lymphoma and it is expected to see in lymphoplasmacytic lymphoma while it is rare in mantle cell lymphoma.14 Moreover, in our study, there were 4 cases (5.5%) of EMZL with accompanying IgG4- related disease and 2 of them also showed plasmacytic differentiation. As IgG4-related disease is quite common to produce ocular mass and, recently, a number of cases with ocular adnexal lymphoma of EMZL type have been reported to have accompanying IgG4-related disease.15 It is possible that a subset of EMZL may arise in IgG4-related disease.16 The benefit from the detection of plasmacytic differentiation in EMZL and other B-cell lymphomas is that the immunophenotype of restriction to cytoplasmic immunoglobulin light chain (light chain restriction) in the plasma cell component can be used to monitor the disease. For example, if EMZL with plasmacytic differentiation has lambda light chain restriction, evaluation of marrow staging or any subsequent tissue sample can determine whether any lambda light chain restriction exists in the plasma cells. The same light chain restriction in plasma cells as that found in the primary lesion is supportive for involvement by lymphoma.

The treatment of ocular adnexal lymphoma usually depends on its stage and the clinical judgement of the

attending physician’s. Although irradiation is the treatment of choice for stage I and II localized diseases, it is challenging because of the nearby radiosensitive structures, such as the lens, lacrimal gland, and retina, all of which may be adversely affected by radiation.17 The majority of cases use radiotherapy either as a stand-alone treatment or in combination with chemotherapy or surgery; however, the present study found that chemotherapy, rather than radiation, was the primary treatment in 67.2% of cases. There are several reasons for this situation. Firstly, as Siriraj Hospital is a tertiary-care hospital, most of the patients were above stage I (33.7%), and many cases had the aggressive lymphoma. Furthermore, the data were collected in a 13-year retrospective period; during that time the radiation therapy was not as effective or safe as at present. Moreover, a long radiation-therapy waiting list coupled with the negative side effects on the local ocular structures would also have influenced physicians to select chemotherapy over radiotherapy. Even though nowadays radiotherapy universally gives very good outcomes for extranodal marginal zone lymphomas12, the authors still performed the subgroup analysis to compare the outcomes of the extranodal marginal zone lymphoma patients who received chemotherapy versus radiotherapy. Both groups responded well to their treatment modalities, with no statistically significant difference between the outcomes of the two groups (p = 0.311).

In this study, the characteristic of lymphoma in terms of its presentation, anatomical location, and subtypes are similar to those found by other studies in Thailand, but with some small differences compared to Western countries (Table 4). When a comparison is made with various studies from Northern Thailand, Asian countries,

TABLE 4. Characteristic and treatment outcome of primary adnexal ocular lymphoma.

Abbreviations: B-LBL: B lymphoblastic lymphoma, BL: Burkitt lymphoma, CLL/SLL: chronic lymphocytic leukemia/small lymphocytic lymphoma, CMT: chemotherapy, CR: complete response rate, DLBCL: diffuse large B-cell lymphoma, EMZL: extranodal marginal zone lymphoma, EFS: event-free survival, FL: follicular lymphoma, HG: high grade, LG: low grade, LPL: lymphoplasmacytic lymphoma, MCL: mantle cell lymphoma, NK/T: NK/T-cell lymphoma, OS: overall survival, PBL: plasmablastic lymphoma, PFS: progression free survival, PTCL: peripheral T-cell lymphoma, RT: radiotherapy, SLL: small lymphocytic lymphoma

and Western countries where radiation has been used as the primary treatment, the PFS and OS results of the current investigation are not markedly different (Table 4). Analysis of the low-grade or indolent lymphoma group found a better prognosis than the high-grade or aggressive lymphoma subtype (5-year OS, 92.5% vs 65.9%; and 5-year PFS, 62.8% vs 50%, respectively). Yen et al. reported that the 5- and 10-year disease-free survival rates for EMZL (884 patients) were 86.4% and 78.7%, and that the 5- and 10-year OS rates were 93.8% and 84.9%, respectively.12 In lymphoma other than EMZL (988 patients), the 5- and 10-year disease-free survival rates were 75.7% and 71.0%, and the 5- and 10-year OS rates were 78.9% and 73.5%, respectively.12 EMZL is the most common type found in the orbit, and it generally shows the best prognosis. Follicular lymphoma patients usually have good long-term survival prospect. DLBCL shows an aggressive natural history but responds well to chemotherapy, whereas mantle cell lymphoma is usually very aggressive and often fatal.23

There were a number of limitations in our study. Firstly, it was a retrospective study, and many patients had been referred back for treatment by other hospitals; thus, complete data could not be elicited. Moreover, there were few cases of radiation therapy compared to the other treatment modalities, and only a small number of aggressive lymphomas. In order to evaluate the potential etiologic factors, a systematic, multicenter effort would therefore be needed to collect a large enough sample size. Further studies are needed to better understand each type of lymphoma, especially the aggressive type, which shows inferior survival rate.

CONCLUSION

Primary ocular adnexal lymphoma patients at Siriraj Hospital mostly presented with proptosis with an orbital lesion. Extranodal marginal zone lymphoma with an early stage and low ECOG was the most common subtype. Chemotherapy was still the main treatment utilized for both the indolent and aggressive lymphomas, in which the indolent group demonstrates better overall and complete response rates than the aggressive group. The histopathological subtype and clinical stage are the two best indicators of prognosis and treatment outcomes.

ACKNOWLEDGMENTS

The authors Sanya Sukpanichnant and Mongkol Uiprasertkul are supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University.

REFERENCES

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14. Swerdlow SH, Kuzu I, Dogan A, Dirnhofer S, Chan JK, Sander B, et al. The many faces of small B cell lymphomas with plasmacytic differentiation and the contribution of MYD88 testing. Virchows Arch. 2016;468:259-75.

15. Cheuk W, Yuen HK, Chan AC, Shih LY, Kuo TT, Ma MW, et al. Ocular adnexal lymphoma associated with IgG4+ chronic sclerosing dacryoadenitis: a previously undescribed complication of IgG4-related sclerosing disease. Am J Surg Pathol. 2008;32: 1159-67.

    
    

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17. Rootman DB, Mavrikakis I, Connors JM, Rootman J. Primary, unilateral ocular adnexal lymphoma: disease progression and long-term survival. Ophthal Plast Reconstr Surg. 2011;27:405-9.

18. Rosado MF, Byrne Jr GE, Ding F, Fields KA, Ruiz P, Dubovy SR, et al. Ocular adnexal lymphoma: a clinicopathologic study of a large cohort of patients with no evidence for an association with Chlamydia psittaci. Blood. 2006;107:467-72.

19. Tanimoto K, Kaneko A, Suzuki S, Sekiguchi N, Watanabe T, Kobatashi Y, et al. Primary ocular adnexal MALT lymphoma: a long-term follow-up study of 114 patients. Jpn J Clin Oncol. 2007;37:337-44.

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23. Coupland SE, Hellmich M, Auw-Haedrich C, Lee WR, Anagnostopoulos I, Stein H. Plasmacellular differentiation in extranodal marginal zone B cell lymphomas of the ocular adnexa: an analysis of the neoplastic plasma cell phenotype and its prognostic significance in 136 cases. Br J Ophthalmol. 2005; 89:352-9.

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Peripheral Atherosclerotic Profile in Type 1

Diabetic Patients: Lipid Ratios as a Predictive Marker of Asymptomatic Patients

Walid Hassene HAMRI*,**, Ph.D., Mustapha DIAF*, AP., Noria HARIR*,**

*Department of Biology, Faculty of Natural and Life Sciences, Djillali Liabes University, Sidi-Bel-Abbes, Algeria, **Laboratory of Molecular Microbiology Proteomics and Health, Djillali Liabes University, Sidi-Bel-Abbes, Algeria.

ABSTRACT

Objective: To investigate the relationship between Lipid ratios and asymptomatic peripheral artery disease (aPAD) in type 1 diabetic patients.

Materials and Methods: This cross-sectional study was performed among 223 diabetics. Patients were segregated with aPAD when their ankle-brachial index (ABI) was abnormal (ABI ≤ 0.90, or ABI > 1.20). Patients were segregated into the following groups (patients with normal ABI vs. patients with arterial stiffness). The association between lipid ratios and aPAD was analyzed using multivariate logistic regression analysis and the receiver operator characteristic curve.

Results: Our study reported a slight preponderance of females (108 males vs. 115 females), with a mean age of 30.70±9.69 years and a diabetes duration of 11.13±8.95 years. The prevalence of arterial stiffness was 38.11%. TC/ HDL-C ratio was a significant predictor for atherosclerosis with a sensitivity of 77.3%, specificity of 62.5%, and diagnostic accuracy of 0.758%. The results revealed that the 4th quartile (odds ratio [OR]=12.52 [5.06-31.00], p=<0.001) of TC/HDL-C ratio was statistically higher in patients with arterial stiffness. Similarly, the last quartiles of LDL-C/ HDL-C and TG/HDL-C ratio were higher in the arterial stiffness group (OR=3.70 [1.68-8.11], p=0.001; OR=4.74 [2.12-10.59], p=<0.001; respectively). In the arterial stiffness group, non-traditional lipid values were significantly higher in males compared to females.

Conclusion: Lipid ratios are correlated with aPAD in type 1 diabetic patients, and should thus be assessed in clinical decision-making and risk stratification on atherosclerotic cardiovascular disease.

Keywords: Ankle-brachial index; asymptomatic peripheral artery disease; atherosclerosis; lipid ratios. (Siriraj Med J 2022; 74: 874-882)

INTRODUCTION

Patients with type 1 diabetes (T1D) are highly predisposed to develop early atherosclerosis.1 Asymptomatic peripheral artery disease (aPAD) is frequent in diabetics.2 It is usually caused by atherosclerosis, in which an atherosclerotic plaque produces arterial stenosis or occlusion. As a consequence, blood flow to the affected

limb is reduced. The majority of subjects are asymptomatic, although many suffer from occasional claudicating.3 Diabetes-related peripheral neuropathy has a reported prevalence ranging from 15% to 86%,4 with painful diabetic neuropathy described in around 26% of diabetic patients.5 People with aPAD have a higher risk of diabetic foot amputation, myocardial ischaemia, and stroke, as well as

Corresponding author: Mustapha DIAF E-mail: diafmustapha@gmail.com

Received 7 July 2022 Revised 13 November 2022 Accepted 13 November 2022 ORCID ID:http://orcid.org/0000-0001-6065-6659 http://dx.doi.org/10.33192/Smj.2022.102

All material is licensed under terms of the Creative Commons Attribution 4.0 International (CC-BY-NC-ND 4.0) license unless otherwise stated.

mortality.6 According to recent data, aPAD affects more than 200 million individuals globally.7 Although current guidelines for screening of aPAD vary significantly,8 the ankle-brachial index (ABI) as a routine examination to identify aPAD is highly recommended. Type 1 diabetic patients aged over forty have an aPAD prevalence of 20%, which rises to 29% over the age of fifty.9

Additionally, the clinical characteristics of aPAD in diabetic people differ from those reported in the general population, resulting in a worse prognosis.10 Thus, an early detection is essential for diabetic patients, since aPAD is a significant risk factor for amputation, and is directly linked to cardiovascular diseases (CVDs).11 Arterial stiffness, which may be regarded as a sign of subclinical atherosclerotic disease,12 is an important parameter to study since it reflects CVD and mortality in diabetics and may serve as a valuable marker to prevent future vascular complications.13

Dyslipidemia is considered a risk factor for aPAD.14 Conventional lipid measures, such as abnormal values of low-density lipoprotein cholesterol (LDL-C) and high- density lipoprotein cholesterol (HDL-C), are prevalent risk factors for aPAD.15 Specifically, numerous studies have highlighted that atherosclerosis develops and progresses under conditions of abnormal lipid values in diabetes. A high triglyceride (TG)/HDL-C ratio may be a favorable indicator of CVD.17,18 Similarly, the total cholesterol (TC)/ HDL-C ratio has also been linked to an increased risk of CVD.19,20 The connection between the LDL-C/HDL-C ratio and aPAD has been well-established,21 indicating that the LDL-C/HDL-C ratio was also a stronger marker for major CVD in a prospective observational study.22 As a result, identifying atherosclerotic risk factors in patients with T1D will ameliorate diabetes-based screening and preventive strategies in diabetics at higher risk for vascular complications. To that end, in this study, we sought to investigate the prevalence of arterial stiffness among diabetics in order to determine whether there is a link between aPAD and CVD, mainly subclinical atherosclerosis by evaluating lipid ratios in patients with

T1D, as similar studies are currently scarce.

MATERIALS AND METHODS

Study design and sampling

The research was designed as a cross-sectional retrospective study that collected data from March 1, 2016, to February 2, 2020, on type 1 diabetic patients admitted to the Endocrinology Unit from an Academic Hospital in Sidi-Bel-Abbes, northwestern Algeria. The study was performed among 370 patients with T1D diagnosed in their pubertal phase (according to the WHO

criteria). All the participants’ files were revised for the following: medical history, other associated diseases, and complications such as diabetic retinopathy, neuropathy, and nephropathy.

The study included 223 patients with T1D who were above the age of 18, admitted to the hospital, and had no history of CVD. A total of 147 patients were excluded because they were younger than 18, lacked medical data, with missed informed consent (Fig 1).

Fig 1. Flow chart of the study population

Data collection

All patients had a full anthropometric evaluation, which comprised height, weight, body mass index (BMI), and waist circumference measures. A sphygmomanometer was used to measure blood pressure in the supine position, followed by a second measurement (after a few minutes) in a standing position. Hypertension was defined as systolic blood pressure (SBP) of 140 mmHg or higher and diastolic blood pressure (DBP) of 90 mmHg or higher. Fasting blood glucose (FBG), glycated hemoglobin (HbA1c), urea, serum creatinine, urinary albumin excretion rate (UAER), and serum lipids (TC, HDL-C, LDL-C, and TG), as well as thyroid-stimulating hormone (TSH), were all collected from patients’ medical records. Two noninvasive tests were used to assess the prevalence of aPAD (pulse examination, and ABI). Lower limbs were assessed for symptoms of aPAD, and peripheral pulses were measured at both dorsalis pedis and tibialis posterior arteries.23 ABI was calculated using the lowest ankle SBP for each leg divided by the maximum brachial SBP to increase the positive test rate in a screening population.24 ABI measurements were conducted using the Minidop ES 8 Mhz sonographic device.25

Normal (ABI = 0.91 to 1.20), decreased (ABI 0.90), and increased (ABI > 1.20) were the categories for ABI

results. This was considered to be comparable to arterial stiffness, using the following lipid ratios (TC/HDL-C, LDL-C/HDL-C, and TG/HDL-C) as markers of atherogenic risk. The presence of subclinical athérosclerosis was later evaluated in every patient.

Data management and analysis

Qualitative data were reported as percentages (%) and relative frequencies, and continuous variables were presented as mean ± standard deviation (SD) with associated 95 percent confidence intervals (95% CI). The Chi-square test was used for qualitative categorical factors and the Student t-test for quantitative data.

The association between ABI status and atherosclerosis, odds ratios (OR) and its 95% CI for lipid values were calculated utilizing the multivariate logistic regression analysis after adjusting across quartiles of lipid ratios. To establish the appropriate cut-off value and validity of lipid ratios, the receiver operator characteristic (ROC) curve was employed, and the area under the ROC curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were computed. When the p-value was less than or equal to 0.05 (p ≤0.05), statistically significant differences were maintained. SPSS software was used to compute and analyze all data (SPSS 22, IBM Corporation; Chicago, IL. August 2013).

Ethical considerations

Written informed consent was maintained from all patients. This retrospective study was approved by the Ethics Committee of Academic Hospital with approval number AAU-5/2022.

RESULTS

The general features of the participants are summerized in Table 1. Two hundred and twenty three type 1 diabetic patients (108 males and 115 females) were admitted to the Diabetes Unit. Patients were arbitrarily segregated into the following groups based on their ABI whether

<0.91 or >1.20 for arterial stiffness. Of the 223 diabetic patients, 138 (61.88%) patients had normal ABI while 85 (38.12%) patients had arterial stiffness. The mean age was 30.70±9.69 years, and the mean of diabetes duration was 11.13±8.95 years. The average age of diabetics with arterial stiffness was statistically higher than those with normal ABI (38.76±8.85 years vs. 25.73±6.24 years, p<0.001). The mean of duration diabetes was statistically increased in the arterial stiffness group compared to normal ABI group (16.05±9.67 years vs. 8.10±6.94 years, p <0.001) (Table 2).

Clinical features of the diabetics are presented in

Table 2. In terms of anthropometric measurements, there was a higher statistical difference in body weight between the two groups (60.08 ± 11.82 kg and 63.60 ±

8.56 kg for the patients with and without normal ABI, respectively, p=0.01). Similarly, significant differences were noted in BMI (p=0.02), and waist circumference (p<0.001), while there was no significant difference in body height. Furthermore, statistically higher SBP and DBP values were reported in the arterial stiffness group compared to the normal ABI type 1 diabetic patients (p<0.001) (Table 2).

Remarkably, unlike FBG values, there was a statistical difference in HbA1c values (normal ABI: 10.06±2.51%, arterial stiffness: 10.79±2.25 %, p =0.03) between both groups on admission. Concerning lipid values, as shown in Table 2 when the t-test was applied to the TC, triglycerides, and HDL-C for the two groups, p-value was statistically significant (p<0.001, p<0.001, p=0.01; respectively). Moreover, all lipid ratios were statistically higher in diabetics with arterial stiffness (p <0.001).

To determine the optimal cut-off lipid ratios for detecting atherosclerosis, the ROC curve was used. The TC/HDL-C ratio was a reliable predictor of atherosclerosis. The best cut-off value was ≥ 4.0, with a sensitivity of 77.3%, specificity of 62.5%, a PPV of 71.2%, and a NPV of 53.6% with a diagnostic accuracy of 0.758 (Fig 2).

The multivariate regression between lipid ratio quartiles, as potent marker of atherosclerosis, revealed that the 4th quartile (OR=12.52[5.06-31.00]; p<0.001) of TC/HDL-C ratio was greater in the arterial stiffness group as indicated in Table 3. Likewise, in patients with arterial stiffness, the last quartile (4th) of the LDL-C/ HDL-C, and the TG/HDL-C ratios were statistically higher (OR=3.70 [1.68-8.11], p=0.001; OR=4.74 [2.12-

10.59], p<0.001; respectively).

As shown in Fig 3, all lipid ratios were significantly greater in men than females with arterial stiffness. Males with arterial stiffness had greater TC, LDL-C, and TG values as compared to females

DISCUSSION

Our study examined the clinical and biological characteristics in long-term patients with T1D with a broad range of ABI to discover the variables linked with the existence of aPAD in these patients and to assess whether the link of these findings with more established atherosclerosis could be validated. The gender distribution of diabetics revealed a slight preponderance of females (51.60%) over males (48.40%) with a sex-ratio of 0.93. In patients with arterial stiffness, the percentage of men was statistically higher than that of women (70.60 % vs.

TABLE 1. General characteristics of the type 1 diabetic patients.