|Year : 2019 | Volume
| Issue : 2 | Page : 98-104
A study of various investigative modalities in establishing the cause of chronic urticaria
N Naseerudeen1, Sandeep Khuraiya2, Vinod Kumar Jain1, Rajeev Khullar1, Dilip Kachhawa1
1 Department of Skin and VD, Dr. S. N. Medical College, Jodhpur, Rajasthan, India
2 Department of Skin and VD, Gandhi Medical College, Bhopal, Madhya Pradesh, India
|Date of Submission||03-Oct-2018|
|Date of Acceptance||17-Sep-2019|
|Date of Web Publication||28-Jan-2020|
Dr. Sandeep Khuraiya
21-A, Naveen Nagar, Near Bhopal Academy School, Aishbagh, Bhopal, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
INTRODUCTION: Urticaria is a transient eruption of erythematous or edematous swellings of the dermis and is usually associated with itching. Angioedema consists of transient swellings in the deeper dermal, subcutaneous, and submucosal tissues. Urticaria and angioedema occur as clinical manifestations of various immunological and inflammatory mechanisms, or they may be idiopathic.
MATERIALS AND METHODS: The study included 500 patients of chronic urticaria who were recruited from the outpatient department. The detailed history, cutaneous, and systemic examination was done. All patients should be subjected to a complete blood count, absolute eosinophil count, ESR, urine analysis, stool examination, liver function test, blood sugar, and renal function test. In the patients where history will be suggestive of any specific disease, screening for hepatitis B and C, antinuclear antibody test, and rheumatoid factor. Autologous serum skin test (ASST) will be performed in all the patients with chronic urticaria.
RESULTS: Out of 500 patients, 211 were female and 289 were male. Most common age group was 21–40 years (58%). Most cases (60%) of chronic urticaria were of 2–6 months duration. Most patients (47%) were worse in the evening and night time. A personal history of atopy was present in 6.6% patients. A total of 38 (7.6%) had chronic urticaria with evidence of a focus of infection. In the present study, food items were observed to be aggravating chronic urticaria in 10 (2%) patients. 19 patients of chronic urticaria were found to have concomitant systemic disease, which included 16 with thyroid disease and 3 with rheumatoid arthritis. ASST was found to be positive in 172 patients with raised serum IgE levels in 120 patients.
CONCLUSION: The present study demonstrates that an extended diagnostic workup may be helpful in patients with chronic urticaria in addition to thorough history taking and physical examination for proper treatment.
Keywords: Atopy, Autologous serum skin test, chronic idiopathic urticaria
|How to cite this article:|
Naseerudeen N, Khuraiya S, Jain VK, Khullar R, Kachhawa D. A study of various investigative modalities in establishing the cause of chronic urticaria. Indian J Allergy Asthma Immunol 2019;33:98-104
|How to cite this URL:|
Naseerudeen N, Khuraiya S, Jain VK, Khullar R, Kachhawa D. A study of various investigative modalities in establishing the cause of chronic urticaria. Indian J Allergy Asthma Immunol [serial online] 2019 [cited 2021 Apr 21];33:98-104. Available from: https://www.ijaai.in/text.asp?2019/33/2/98/276954
| Introduction|| |
Urticaria is a transient eruption of erythematous or edematous swellings of the dermis and is usually associated with itching. Angioedema consists of transient swellings in the deeper dermal, subcutaneous, and submucosal tissues.
Acute urticaria is the presence of wheal for <6 weeks duration. Chronic urticaria is defined as wheals present on most days for longer than 6 weeks. Urticaria and angioedema may occur together or individually. The individual wheal of urticaria lasts for <24 h. Urticaria and angioedema occur as clinical manifestations of various immunological and inflammatory mechanisms, or they may be idiopathic. They may develop after an IgE or IgE receptor-dependent reaction, in association with abnormalities of the complement system and other plasma effecter systems, in relation to activation of cellular arachidonic acid metabolic pathway, and after direct mast cell degranulation.
Urticaria is not uncommon, and on the basis of published data has an incidence of 15%. Chronic urticaria is likely to be present in about a quarter of the patients with urticaria., Many acute urticarias are allergic reactions, but in most of the chronic urticarias, no causative factor, allergic, or otherwise can be reliably incriminated., Only 20% of chronic urticaria has clearly identifiable allergic cause. Instead, most frequent eliciting factors are physical factors or certain chemicals in food and drugs. Drugs, especially salicylates and other nonsteroidal anti-inflammatory drugs, preservatives, dyes, and natural pseudoallergens, can also aggravate chronic urticaria. Chronic urticaria is frequently flared by foci of infections in the body including Helicobacter pylori, Candida, Protozoa, and helminths., Thyroid autoimmunity has been reported to be associated with chronic urticaria. Urticaria occurring premenstrually has been attributed to “progesterone” sensitivity, and more recently, “estrogen” sensitivity. Psychological stress has also play a contributory role in urticaria. Implants such as femur pin and dental amalgams are also provocating factors.
Various types of physical urticaria occur commonly with ordinary urticaria, for example, delayed pressure urticaria occurs in 37% patients with ordinary chronic urticaria. Despite being one of the common skin diseases, the possible etiology of chronic urticaria remains obscure.
Aims and objective
The objective is to study different investigative modalities of chronic urticaria with identify any causal or aggravating factors the management of which can augment response to standard therapy and prevent relapse.
| Materials and Methods|| |
The study was conducted at the Department of Dermatology, Venereology, and Leprosy, MDM Hospital, Jodhpur.
Number of patients
A total of 500 patients participated in this study.
The study duration was 12 months.
Patient selection criteria
- Almost daily appearance of wheals for ≥6 weeks.
- Urticarial lesion <6 weeks duration.
All patients should be subjected to a complete blood count including hemoglobin, total leukocyte count, differential leukocyte count, absolute eosinophil count (AEC), erythrocyte sedimentation rate (ESR), urine analysis, stool examination on 3 consecutive days, liver function test (LFT), blood sugar (both fasting and post prandial) and renal function test.
In the patients where history will be suggestive of any specific disease, screening for hepatitis B and C (hepatitis B virus surface antigen [HbsAg], anti-hepatitis C virus [HCV]), antinuclear antibody test, and rheumatoid factor. Given the association of thyroid disease with chronic urticaria, thyroid function test including thyroid antibodies should be done.
Autologous serum skin test (ASST) will be performed in all the patients with chronic urticaria because a positive test suggests that an autoimmune mechanism underlies the disease.
- All antihistamines are withdrawn 2 days prior to the skin test
- No steroid intake 3 weeks before testing
- Venous blood is taken into sterile glass tubes without clotting accelerators (vacationers) and allowed to clot at room temperature for 10 min
- Serum was separated by centrifugation at 2000 rpm for 10 min
- 0.1 cc of autologous serum and sterile saline are injected by 31-gauge needle into volar aspects of forearm leaving 5 cm between each injection sites
- Areas known to have been involved in spontaneous wheals in the last 24 h avoided.
- Wheal and flare responses are measured at 30 min after injection
- ASST will be considered positive when the average of two perpendicular diameters of the autologous serum wheal will be ≥1.5 mm more than the normal saline wheal.
Serum IgE levels should be done where ASST will be positive which may be elevated due to overexpression of IgE receptor FcεR1α which will bind to IgG autoantibodies.
A skin biopsy should be carried out whenever urticarial vasculitis will be suspected.
The diagnosis of chronic idiopathic urticarial (CIU) will be considered when there will be no evident cause even after detailed history taking and necessary investigations.
| Results|| |
A total of 500 cases of chronic urticaria attending the Outpatient Department of Dermatology of MDM Hospital attached to Dr. S. N. Medical College, Jodhpur, from October 2014 to November 2015 were included in the present study.
Age and sex distribution of the patients [Table 1]
Out of 500 cases, 211 were female and 289 were male. Maximum cases were in the age group of 21–40 years (58%), followed by age group 11–20 years. The age of the youngest patient was 8 years and that of the oldest was 76 years.
Duration of urticaria
The duration of urticaria was 2–6 months in maximum number of patients (60%), followed by 1–2 year in 20% patients. Only 3 (0.6%) patients continued to be symptomatic more than 10 years.
Time of appearance of wheals
The time of appearance of wheals was variable in different patients. Usually, patients were better during the day, as only 13.4% of them were symptomatic during the daytime. Most patients (47%) were worse in the evening and night time.
Investigations in chronic urticaria [Table 2]
Complete hemogram-49 patients were detected to have eosinophilia. A personal history of atopy was observed among 33 of these patients.
ESR – It was found to be raised in 7 patients.
Stool examination – Detected parasitic worm infestation in 20 patients. Ova and cyst of ascariasis, hookworm, and Entamoeba histolytica were seen.
LFT and serology for hepatitis B and C – Abnormality of LFT was seen in 8 patients and 12 were HbsAg positive and 2 showed Anti-HCV antibody.
Screening for H. pylori – Four patients who gave history of gastritis had IgG for H. pylori.
Thyroid function test was abnormal in 16 patients.
ASST – A wheal of more than 1.5 mm on intradermal injection of 0.1 ml of autologus serum as compared to normal saline control was found in 172 patients.
Rheumatoid factor – Rheumatoid factor was positive in three patients.
Serum IgE levels – It was done in patients who were ASST positive and had history of atopy. It was found to be elevated in 120 patients.
Skin biopsy – It was done in three patients who had persistence of wheals for more than 24 h. Histopathology was suggestive of urticarial vasculitis.
Other routine investigations such as urine routine examination, blood sugar levels, and renal function test were normal.
Based on the history, clinical examination, and investigations following etiologies were considered.
Association of atopy
A personal history of atopy was observed in 33 (6.6%) case. These patients also had raised levels of AEC.
Chronic urticaria with focus of infection
Focus of infection was observed in a total number of 38 (7.6%) cases, which included bacterial infection (4), viral (14), and parasitic infestations (20). Four of these patients had H. pylori infection, 12 patients were positive for hepatitis B antigen, 2 had antibodies to HCV, and 20 had parasitic worm infestation.
Food items causing chronic urticaria
Food items were observed to cause chronic urticaria in 10 (2%) cases [Table 3].
|Table 3: Common food items causing chronic urticaria (on basis of patient's history)|
Click here to view
Systemic diseases associated with chronic urticaria
A total of 19 (3.8%) patients of chronic urticaria were found to have concomitant systemic disease, which included 16 with thyroid disease and 3 with rheumatoid arthritis.
A total of 172 (34.4%) patients had autoimmune urticaria based on ASST positivity and 120 of these patients had raised IgE levels.
Three (0.6%) patients had wheals persisting for more than 24 h and skin biopsy of this patient was suggestive of vasculitis.
Association of chronic urticaria and angioedema
Out of the 500 patients of chronic urticaria, in the present study, urticaria occurred alone in 87.6% of the patients. Urticaria and angioedema were found to occur together in 12.4% of the cases.
Chronic urticaria associated with mental stress/psychiatric illness, menstrual cycle, drug intake
Mental stress or psychiatric illness was observed in none of the patients in the present study. No female patient gave a history of exacerbation of urticarial symptoms during menstruation. No drug was found to exacerbate chronic urticaria.
Chronic idiopathic urticaria
In 225 (45%) patients, no causative factor was found, and they were grouped as CIU [Table 4].
| Discussion|| |
Urticaria in all of its manifestations is a common affliction. Although rarely life-threatening, widespread urticaria and its associated angioedema can be both debilitating and frightening. Approximately 15%–20% of the population may experience at least one episode of urticaria in their lifetime, and about one-quarter of these patients are likely to develop chronic urticaria. The rational therapy for urticaria is the identification and avoidance of causative agents that directly or indirectly precipitate the eruption. In chronic urticaria, the search for a cause is much more difficult 25. Patients often seek medical attention with the hope that a reversible cause can be identified. Being able to efficiently apply a cost-effective workup for urticaria is challenging. Therefore, the challenge for the clinician is to try to identify a cause that could lead to a specific treatment or avoidance strategy.
Age and sex distribution of the patients
In the present study, the incidence of chronic urticaria was found to be more in males than females [Table 1]. Sarojini et al. and Jacobson et al. also observed a male preponderance (56% and 52%, respectively) in their studies. However, Metzger, Singh et al., Trachsel et al., and Liutu et al. observed a female preponderance. The difference in the sex distribution in the later studies may be due to the difference in the study population.
Maximum number of patients in the present study was in the age group of 21–30 years (34.4%) [Table 1]. Juhlin, Metzger, and Liutu et al. made similar observations in their studies. Urticaria was more common in male in all age group.
Duration of urticaria
In the present study, the duration of the urticaria ranged from 2 months to 21 years. Most of these cases (60%) had urticaria of 2–6 months duration. Similar observations were made by Sarojini et al., Juhlin, and Kennard.
Time of appearance of wheals
In the present study, it is observed that most patients were better during the day. Most patients (47%) in the present study were worse in the evening and night probably due to low cortisol levels in blood at night. This compares well with the study by Juhlin, in which there was a marked diurnal variation of appearance of wheals with a larger number of patients being symptomatic in the evening and at night (70%).
Association of atopy
In the present study, 33 patients (6.6%) gave a personal history of atopy. These patients also had raised AEC. Juhlin  and Thune and Granholt  found a personal history of atopy in 9% and 8% in their study group of patients of chronic urticaria. The discrepancy in the incidence of atopy in the above-mentioned study could have arisen from differing population and test methods used.
Chronic urticaria with focus of infection
In the present study, a total of 36 (7.2%) patients had chronic urticaria with evidence of a focus of infection somewhere in the body [Table 5].
In the present study, parasitic infestation was observed in a total number of 20 (4%) cases. Sarojini et al. found E. histolytica in 1% and ascariasis in 10% of their study population of 100 patients. Kozel et al. in their study of 220 patients of chronic urticaria, found evidence of parasitic infestation in 10 (4.5%) cases which is similar to the present study.
Serology for hepatitis B (HbsAg) was found to be positive in 12 (2.4%) patients in the present study group. Varda et al. screened 114 patients of chronic urticaria and angioedema for serological markers of hepatitis B virus infection. In another study conducted by Chung et al. HBV was tested in 150 patients of chronic urticaria and 100 healthy controls from Guangdong, China. 19.3% of patients with chronic urticaria were positive for HbsAg, while it was only 10% in the control group.
In the present study, four patients were found to have IgG antibodies to H. pylori infection. Wedi et al. assessed 100 patients with chronic urticaria, detecting positive H. pylori serology in 47%. Thus, H. pylori can be considered to be associated with chronic urticaria and patients should be screened for H. pylori infection with a history of associated dyspepsia. Bonamigo et al. studied 12 patients with chronic urticaria and H. pylori infection, observing remission of the urticaria in six and improvement in four.
Association of food items with chronic urticaria
Patients frequently suspect allergy to food, but this is rarely found in chronic urticaria. The responsible agent in food-induced urticaria can be either proteins or substances added to food for color, preservative, or taste. Common urticogenic foods are shellfish, fish, eggs, fresh berries, milk, and other dairy products including cheese and chocolates. The incidence of food causing chronic urticaria is <3.5% according to Black and Grattan. In the study conducted by Kozel et al., in 220 patients of chronic urticaria 15 patients (6.8%) had adverse reactions to food. Young et al, in a population study of food intolerance found a prevalence of 1.4% in the general population.
Systemic diseases associated with chronic urticaria
In the present study, 19 (3.8%) patients were found to have associated systemic disease.
In the present study, 16 patients were found to have raised levels of thyroid-stimulating hormone which was similar to findings of Turktas et al. and Kandeel et al., Similar observation was made by Kozel et al, Leznoff et al, Rumbyrt and Schocket , Leznoff et al. and Kozel et al. found evidence of systemic disease in 3 out of 220 (1.4%) patients of chronic urticaria.
Autoimmune chronic urticaria
In the present study, 172 (34.4%) patients were found to be ASST positive. 120 (24%) of these patients had raised IgE levels [Table 6]. There is now growing evidence that 30%–50% of CIU results from an autoimmune process involving functional histamine-releasing anti-FcεRI, autoantibodies or less commonly, and anti-IgE autoantibodies. Various studies have reported that the prevalence of autoimmune urticaria ranged 30%–40%.
As patients with autoimmune antibodies have no distinctive diagnostic clinical features, current clinical diagnosis depends on ASST. A positive test is suggestive but not diagnostic of an autoimmune basis. Although Western blot analysis or an enzyme-linked immunosorbent assay test was developed to detect autoantibodies, these tests do not distinguish functional histamine-releasing antibodies from nonfunctional antibodies. These time-consuming tests are only performed in a few specialist laboratories. In our setting, ASST is the only available test for diagnosis of autoimmune urticaria. It is simple, inexpensive, semi-invasive, and an easy to perform test which can be done and recorded by the dermatologist.
The high level of IgE may play a role in the pathogenesis of chronic autoimmune urticarial (CAU) due to overexpression of IgE receptor FcεR1α which will bind to IgG autoantibodies. Another proof for the role of the high-level IgE in CAU is improvement of patients with CAU by anti-IgE, omalizumab which selectively binds to IgE. Kaplan et al. postulated that omalizumab by decreasing circulating IgE level, will secondarily decrease IgE receptor density on basophils and cutaneous mast cells, preventing activation by autoantibodies.
Patients who were ASST positive were treated with autohemotherapy which consisted of weekly intramuscular injections of 5 ml of autologus serum for 9 weeks. Many of them showed good response at the end of 9 weeks of therapy with respect to decrease in severity of urticaria and reduction in dose of antihistaminics.
Three patients (0.6%) who had presented with urticarial wheals persisting for more than 24 h were diagnosed as urticarial vasculitis based on clinical and histopathological features [Table 3]. Tharp  observed histological findings of vasculitis in lesional skin in 3 of 24 (12.5%) patients of chronic urticaria. However, according to Kaplan, the incidence of urticarial vasculitis accounts for less than 1% of all cases of chronic urticaria.
Aggravating or provoking factors in chronic urticaria
Aggravating or provoking factors of chronic urticaria such as drugs, psychiatric illness, mental stress, and menstruation were not found in this study. This was not in concordance with the study conducted by Trachsel et al. and Mekkes et al.
| Conclusion|| |
The present study demonstrates that an extended diagnostic workup may be helpful in patients with chronic urticaria in addition to thorough history taking and physical examination.
On medical and economic grounds, it appears prudent to perform comprehensive laboratory diagnostics only to exclude a particular; individually suspected causative factor.
CAU was diagnosed in 34.4% of patients with help of ASST which proved to be simple, easy, and cost-effective method of diagnosing CAU. IgE levels were also raised in 24% of patients. Patients with CAU responded well to autohemotherapy.
Thus, investigations helped in establishing the various causes of chronic urticaria in 55% of cases. In only 45% of cases, no causative factor could be found, and they were termed as CIU.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Black AK, Grattan CE. Urticaria. In: Burns DA, Breathnach SM, Cox NH, Griffiths CE. editors. Rooks Textbook of Dermatology. 8th
ed. Blackwell Publishing Ltd.; 2010. p. 22.1-22.39.
Grattan CE, Black AK. Urticaria and angioedema. In: Bolognia JL, Jorizzo JL, Rapini RP, editors. Dermatology. 2nd
ed. London: Elsevier Ltd.; 2008. p. 263-4.
Sheldon JM, Mathews KP, Lovell RG. The vexing urticaria problem: Present concepts of etiology and management. J Allergy 1954;25:525-60.
Greaves MW. Chronic urticaria. N Engl J Med 1995;332:1767-72.
Sarojini PA, Gopinathan T, Mohandas PP. Studies on 100 cases of urticaria with particular reference to the etiology. Indian J Dermatol Venereol 1972;38:132-6.
Champion RH, Roberts SO, Carpenter RG, Roger JH. Urticaria and angio-oedema. A review of 554 patients. Br J Dermatol 1969;81:588-97.
Kozel MM, Mekkes JR, Bossuyt PM, Bos JD. The effectiveness of a history-based diagnostic approach in chronic urticaria and angioedema. Arch Dermatol 1998;134:1575-80.
Czarnetzki BM. The history of urticaria. Int J Dermatol 1989;28:52-7.
Henz BM, Zuberbier T. Urticaria. New developments and perspectives. Hautarzt 2000;51:302-8.
Tebbe B, Geilen CC, Schulzke JD, Bojarski C, Radenhausen M, Orfanos CE. Helicobacter pylori
infection in chronic urticaria. J Am Acad Dermatol 1996;34:685-6.
James J, Warin RP. An assessment of the role of Candida albicans
and food yeasts in chronic urticaria. Br J Dermatol 1971;84:227-37.
Heymann WR. Chronic urticaria and angioedema associated with thyroid autoimmunity: Review and therapeutic implications. J Am Acad Dermatol 1999;40:229-32.
Kennard CD. Evaluation and treatment of urticaria. Immunol Ailergy Clin North Am 1995;15:785-801.
van Arsdel PP. Classification and risk factors for drug allergy. Immunol Allergy Clin North Am 1991;11:475-92.
Jacobson KW, Branch LB, Nelson HS. Laboratory tests in chronic urticaria. JAMA 1980;243:1644-6.
Metzger WJ. Urticaria, angioedema and hereditary angioedema. In: Patterson R, Grammer LC, Greenberger PA, editors. Allergic Diseases. 5th
ed. Philadelphia: Lippincott-Raven; 1977. p. 265-83.
Singh G, Minocha YC, Sood VK. Aetiological spectrum of urticaria. Indian J Dermatol Venereol Leprol 1989;55:173-6.
] [Full text]
Trachsel C, Pichler WJ, Helbling A. Importance of laboratory investigations and trigger factors in chronic urticaria. Schweiz Med Wochenschr 1999;129:1271-9.
Liutu M, Kalimo K, Uksila J, Kalimo H. Etiologic aspects of chronic urticaria. Int J Dermatol 1998;37:515-9.
Juhlin L. Recurrent urticaria: Clinical investigation of 330 patients. Br J Dermatol 1981;104:369-81.
Thune O, Granholt A. Provocation test with antiphlogistica and food additives in recurrent urticaria. Dermatologica 1975;151:287.
Varda GA, Goldman MA, Bloch KJ. Testing for hepatitis B Virus in patients with chronic urticaria and angioedema. J Allergy Clin Immunol 1983;72:193-8.
Chen MC; Huang Y; Zou YN. The relationship between chronic urticaria and hepatitis B virus infection. J China Trop Med 2009;9:1717-938.
Wedi B, Wagner S, Werfel T, Manns MP, Kapp A. Prevalence of Helicobacter pylori
-associated gastritis in chronic urticaria. Int Arch Allergy Immunol 1998;116:288-94.
Bonamigo RR, Leite CS, Bakos L. Association of Helicobacter pylori
and chronic idiopathic urticaria. Rev Assoc Med Bras (1992) 1999;45:9-14.
Young E, Stoneham MD, Petruckevitch A, Barton J, Rona R. A population study of food intolerance. Lancet 1994;343:1127-30.
Turktas I, Gokcora N, Demirsoy S, Cakir N, Onal E. The association of chronic urticaria and angioedema with autoimmune thyroiditis. Int J Dermatol 1997;36:187-90.
Kandeel AA, Zeid M, Helm T, Lillie MA, Donahue E, Ambrus JL Jr, et al.
Evaluation of chronic urticaria in patients with Hashimoto thyroiditis. J Clin Immunol 2001;21:335-47.
Leznoff A, Sussman GL. Syndrome of idiopathic chronic urticaria and angioedema with thyroid autoimmunity: A study of 90 patients. J Allergy Clin Immunol 1989;84:66-71.
Rumbyrt JS, Schocket AL. Chronic urticaria and thyroid disease. Immunol Allergy Clin North Am 2004;24:215-23, vi.
Sabroe RA, Seed PT, Francis DM, Barr RM, Black AK, Greaves MW. Chronic idiopathic urticaria: Comparison of the clinical features of patients with and without anti-fcepsilonRI or anti-IgE autoantibodies. J Am Acad Dermatol 1999;40:443-50.
Greaves M. Chronic urticaria. J Allergy Clin Immunol 2000;105:664-72.
Kaplan AP, Joseph K, Maykut RJ, Geba GP, Zeldin RK. Treatment of chronic autoimmune urticaria with omalizumab. J Allergy Clin Immunol 2008;122:569-73.
Tharp MD. Chronic urticaria: Pathophysiology and treatment approaches. J Allergy Clin Immunol 1996;98:S325-30.
Kaplan K P. Chronic urticaria and angioedema. N Engl J Med 2002;346:175-9.
Mekkes JR, Kozel MM, Bossuyut PM, Bos JD. A questionnaire for patients with chronic urticaria: A diagnostic approach based on structured Anamnesis. Ned Trjdschr Geneeskd 1999;143:1748-9.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]