|Year : 2021 | Volume
| Issue : 2 | Page : 85-92
Filariasis: A major potential imported parasitic disease in Najran District of Saudi Arabia
Ahmed Saif1, Mohammed Abdulrahman Alshahrani1, Ahmad A Alshehri1, Basim A Othman2, Mosa M Bahnass3, Mutaib M Mashraqi1, Gaffar Sarwar Zaman4, Yasser Alraey4, Aymen M Madkhali5
1 Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Najran University, Najran, Saudi Arabia
2 Department of Public Health, Faculty of Applied Medical Sciences, Albaha University, Albaha, Saudi Arabia
3 Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Najran University, Najran, Saudi Arabia; Department of Animal Medicine (Infectious Diseases), Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
4 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia
5 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Jazan University, Gizan, Saudi Arabia
|Date of Submission||21-Oct-2021|
|Date of Acceptance||22-Nov-2021|
|Date of Web Publication||12-Jan-2022|
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Najran University, Najran
Source of Support: None, Conflict of Interest: None
Introduction: Expatriate workers usually contribute to the origination of some infectious diseases in the countries they reside. The aim of this study was to identify and analyze available information on filariasis imported into Najran, Saudi Arabia, by expatriate workers. Methodology: Serum (n = 497) were acquired from expatriate workers of both genders from different nationalities and various ages and also from randomly chosen parts and at different time periods of Najran. The serum samples were stored in a deep freezer at a temperature of − 65°C until utilized for the serological diagnosis of filariasis by using filariasis immunoglobulin G/immunoglobulin M (IgG/IgM) combo rapid test and human filariasis antibody (IgG4) enzyme-linked immunosorbent assay (ELISA). Results: Based on IgG/IgM rapid test, the prevalence of filariasis IgG was 3.8% (19 out of 497), whereas IgG4 ELISA showed that the prevalence of filariasis was 10.9% (10 out of 92). The highest prevalence of filariasis occurred in Indian workers, in adulthood (30–49 years), and in male workers. There was no significant agreement in results between IgG/IgM rapid test and ELISA. The percentage specificity of IgG/IgM rapid test, relative to filariasis IgG4 ELISA was 80.5%. Conclusions: The possibility of health hazard due to imported filariasis and preeminently the initiation of a novel carrier focal point of the disease are likely to relied upon the abundance of carriers of microfilaria and the density of vector mosquitoes who feed on the carriers. Furthermore, the results of this study underscore the need for screening and therapeutic strategies that guarantee the control and prevention of parasitic infectious diseases.
Keywords: Expatriate workers, filariasis, Najran, protozoa, risk factors, Saudi Arabia
|How to cite this article:|
Saif A, Alshahrani MA, Alshehri AA, Othman BA, Bahnass MM, Mashraqi MM, Zaman GS, Alraey Y, Madkhali AM. Filariasis: A major potential imported parasitic disease in Najran District of Saudi Arabia. King Khalid Univ J Health Sci 2021;6:85-92
|How to cite this URL:|
Saif A, Alshahrani MA, Alshehri AA, Othman BA, Bahnass MM, Mashraqi MM, Zaman GS, Alraey Y, Madkhali AM. Filariasis: A major potential imported parasitic disease in Najran District of Saudi Arabia. King Khalid Univ J Health Sci [serial online] 2021 [cited 2022 Jan 17];6:85-92. Available from: https://www.kkujhs.org/text.asp?2021/6/2/85/335632
| Introduction|| |
Parasitic diseases such as schistosomiasis, filariasis, onchocerciasis, and leishmaniasis are among the most neglected tropical diseases (NTDs) which can lead to morbidity and mortality worldwide. Similarly, malaria which is distributed widely, particularly in the tropics and sub-tropical regions, is life-threatening if left untreated. These diseases are related with prolonged sub-clinical course that can be diagnosed only by laboratory investigations. Saudi Arabia receives a large number of immigrants for Hajj (pilgrims) from Asian tropical countries, specifically from South and South-east regions. These regions have very high prevalence of Helminth infections, leprosy, kala-azar, and particularly lymphatic filariasis (LF). Indeed, LF imposes a socioeconomic burden on about 1.3 billion people who are at danger in 83 endemic countries. Approximately, it has been estimated that 120 million individuals have already been infected with parasitic LF. The organism responsible for about 90% of LF infections is Wuchereria bancrofti.
Earlier studies on LF in Saudi Arabia delineated 10 cases comprising of Saudi nationals who manifested with LF without microfilaremia, many of whom, in all probability, got it in the south-western part of the country. Nevertheless, subsequent night blood surveys did not reveal the presence of microfilariae (MF) in that region.
Efficient and advanced determination of W. bancrofti infection is a crucial step in monitoring, therapy, and succeeding eradication of LF. The identification of MF through microscopic investigation of the thick blood film or the nucleopore-filtered blood (NFB) has dearth of sensitivity and contributes only a superficial measure of the adult worm load. Furthermore, the contemporaneous filarial antibody detection tests are unable to distinguish between recent active and past infections.
LF confirmed to be found in Yemen as a parasitic infection which is caused by filarial worms transmitted by mosquitoes. The deer fly transmits another filarial parasite called Loa loa. However, not much is known about the prevalence of these parasites in the Kingdom.
Parasitic diseases imported by expatriates constitute global problems in many countries of the world. Due to the ease with which people travel across countries as a result of improved aviation facilities, these diseases are no more restricted to the tropics and subtropics. Nowadays, there is a high tendency for the filarial parasites to form new foci whenever appropriate conditions are available.
The KSA is surrounded by many countries where many endemic parasitic diseases are prevalent. For example, Egypt and Sudan are the endemic areas for schistosomiasis., In Yemen, malaria, onchocerciasis and schistosomiasis are widely spread, constituting a real-life threat. India, Pakistan, and Afghanistan are countries where NTDs such as filariasis and leishmaniasis are prevalent. Expatriates coming from these countries to work in KSA could be potential sources for the spread of parasitic infections. Although KSA has an effective health monitoring system, some of the NTDs harbored by expatriates may escape detection. This could be due to inadequate experience in the surveillance and reporting of infections. Recent studies showed that most cases of tropical and NTDs in KSA were diagnosed and proven to be imported by expatriates. These accumulated studies were focused mainly on gastrointestinal parasitic diseases, with little or no coverage of other systemic parasitic diseases.,
Therefore, there is a need for studies on these systemic parasitic diseases to enhance current strategies for controlling the importation of parasitic diseases filariasis into KSA.
| Methodology|| |
Study participants and settings
This cross-sectional research was undertaken in the Department of Clinical Laboratory Sciences, Applied Medical Sciences College, Najran University. Najran is located in-between 17° 30' 20” North and 44° 11' 3” East, about 1260 km away from Riyadh, South of Saudi Arabia. The average range of temperature in Najran varies from 14.6°C to 30.9°C, whereas the average annual rainfall is 83 mm. The study subjects were selected through proportional random serum sampling from Najran hospitals and clinical laboratories, with approval from relevant authorities. Proportionate sampling is a sampling strategy (a method utilized for gathering participants for a research) utilized when the population is composed of several subgroups that are vastly different in number. Proportionate random sampling is done by first defining the population, and then choosing the relevant stratification, listing the population according to the chosen stratification, choosing our sample size, and then calculate a proportionate stratification, and finally, using a simple random or systematic sample to select our sample. The sample size was calculated with Epi Info version 2000 at a level of confidence of 95% and level of precision of 5% utilizing the following formula:
Where n is the required sample size, P is prevalence, Q is 100-P, and D is the level of precision.
Collection of human serum samples
Four hundred and ninety-seven human blood samples were collected. Expatriate workers of both genders from different nationalities and various ages were chosen. Known filariasis cases were excluded from the study. All volunteers from almost all Najran hospitals who agreed to participate in this study were given a predesigned questionnaire on sociodemographic data such as age, sex, and residence. Blood specimen was taken from each participant. Within 1 h of collection, the blood samples were shifted to the research laboratory situated inside the Department of Clinical Laboratory Sciences, Applied Medical Sciences College, Najran University. The blood samples were obtained from male and female individuals of different ages from different nationalities and from various parts and in the different environmental seasons of Najran. Obtained samples were from apparently healthy human subjects, in clean, dry tubes and left to clot in slanting positions at the room temperature for about 3 h. Then, the samples were centrifuged at 600 g for 10 min. Only clear and non-hemolyzed sera were transferred carefully with automatic pipette into clean and dry Eppendorf tubes. All the sera were saved in a deep freezer at a temperature of −65°C until used for serological diagnosis.
Tests for anti-filarial antibodies
Filariasis immunoglobulin G/immunoglobulin M combo rapid test
The Filariasis immunoglobulin G/immunoglobulin M (IgG/IgM) Combo Rapid Test (Zhuhai Encode Medical Engineering Co. Ltd) is defined as a lateral flow immunoassay for the concurrent identification and demarcation of anti-lymphatic filarial parasites (mainly W. bancrofti and Brugia malayi), IgG and IgM in human plasma, serumor whole blood. Its main intention is to be utilized as a screening test, and it gives preliminary results that helps to diagnose lymphatic filarial infection. IgM (to parasite antigens) presence reveals present-day infection, whereas detection of IgG indicates a later stage or earlier infection. For IgM test, relative sensitivity is 95.8%, relative specificity is 100%, and overall agreement is 99.6%, while for IgG test, relative sensitivity is 92.3%; relative specificity is 100%, and overall agreement is 99.1%.
Human filariasis antibody enzyme-linked immunosorbent assay test
The filariasis enzyme-linked immunosorbent assay (ELISA) (Abbkine, Inc, China) test kit was used to determine human filariasis antibody (IgG4) concentration in serum, in line with the manufacturer's protocol.
Data set for individual variables was coded and entered into Excel 16.0. IBM SPSS Statistics for Windows, Version 23.0. (IBM Corp. Released 2015. Armonk, NY: IBM Corp.) was used for the analysis of data with the Chi-square test. Values of P < 0.001 were taken as indicative of statistical significance of differences. Cohen's Kappa agreement was obtained regarding the sensitivity, specificity, and negative and positive predictive values (PPVs). Quantitative data are presented as mean ± standard deviation, whereas qualitative data are presented as frequency distribution. Logistic regression analysis was used to determine risk ratio (odds ratio, [OR]) of factors that affect filariasis. Statistical significance was set at P < 0.05. Descriptive statistics and categorical data presented as proportions were analyzed at 95% confidence interval.
| Results|| |
The prevalence of filariasis, based on IgG/IgM rapid test, was 3.8% (19 out of 497), while IgG4 ELISA showed that the prevalence of filariasis was 10.9% (10 out of 92). Indian workers formed the highest number of expatriate workers that participated in the study, with 22.5% (112 out of 497), followed by Egyptians, Pakistani, and Filipinos, accounting for 17.7, 14.5, and 12.5%, respectively. Food workers comprised 6.2% of the study participants (31 out of 497), whereas nonfood workers among the study population of expatriates were 56.3% (280 out of 497). Female domestic workers constituted 34.8% (173 out of 497), as shown in [Figure 1]. Moreover, the majority of workers were aged between 30 and 50 years, as shown in [Figure 2]. Male workers comprised 71.4% of the study population (355 out of 497). The mean of age of expatriate workers was 39.8 ± 6.7 years, with majority being in the adult age group (30–49 years) which comprised 92.2% (458 out of 497) of the population [Table 1].
|Figure 1: Descriptive chart for filariasis based on nationality and expatriate workers in Najran, Saudi Arabia|
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|Figure 2: Descriptive chart for expatriate workers based on age group in Najran, Saudi Arabia|
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|Table 1: Descriptive statistics for filariasis among expatriate workers in Najran, Saudi Arabia (n=497)|
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The IgG/IgM rapid test revealed that nonfood workers had 2.0% prevalence of filariasis (10 out of 497; χ2 = 1.384a, P = 0.84 and Cramer V small effect size value = 0.05, P = 0.84). On the other hand, the results of the ELISA test showed a 7.7% prevalence of filariasis in nonfood workers (7 out of 92; χ2 = 12.289a, P = 0.683 and Cramer V small effect size value = 0.159, P = 0.683), as shown in [Table 2].
|Table 2: Relationships of study variables with filariasis among expatriate workers in Najran, Saudi Arabia|
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The IgG/IgM rapid test showed 0.6% prevalence of filariasis in Indian workers (3 out of 497; χ2 = 9.9 a, P = 0.76, and Cramer V small effect size value = 0.14, P = 0.76). Based on results of ELISA test, the prevalence of filariasis in Indian workers was 4.3% (4 out of 92; χ2 = 10.915a, P = 0.536 and Cramer V small effect size value = 0.344, P = 0.536).
The IgG/IgM rapid test showed that the prevalence of filariasis in adulthood (30–49 years) was 3.6% (18 out of 497), χ2 = 0.24a, P = 0.88 and Cramer V small effect size value small effect size = 0.02, P = 0.88). In contrast, results from ELISA test showed that the prevalence of filariasis in adulthood (30–49 years) was 9.8% (9 out of 92; χ2 = 0.971a, P = 0.615 and Cramer V small effect size value = 0.103, P = 0.615).
The IgG/IgM rapid test showed a 2.6% morbidity from filariasis in male workers (13 out of 497; χ2 = 0.096a, P = 0.756; Cramer V small effect size value = 0.014, P = 0.756). With the ELISA test, there was 7.6% filariasis-associated mortality in male workers (7 out of 92; χ2 = 0.001a, P = 0.975; Cramer V small effect size value = 0.003, P = 0.975), as illustrated on [Table 2].
The IgG/IgM rapid test showed that positive cases of filariasis, relative to negative case fit model for nationality of expatriate workers, were not significant (χ2 = 9.906, P > 0.05), which indicated that the null model was significantly worse, or less accurately predictive than the final or full model [Table 3]. A similar result was obtained in the diagnosis with ELISA test (χ2 = 14.008, P < 0.05).
|Table 3: Contrasting persisting group variables with each of the other group variables, and its predictors' unique contributions using multinomial logistic regression|
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The IgG/IgM rapid test showed that positive cases of filariasis, relative to negative case model fit for expatriate workers was not significant χ2 (5) = 2.220, P > 0.05, which indicated that null model was significantly worse, or less accurately predictive than the final or full model [Table 3]. A similar result was seen in diagnosis with ELISA test (χ2 = 3.335, P < 0.05).
The IgG/IgM rapid test revealed that positive cases of filariasis, relative to negative case fit model for age of expatriate workers, was not significant (χ2 = 1.901, P > 0.05), indicating that the null model was significantly worse, or less predictively accurate than the final or full model [Table 3]. A similar result was obtained in the diagnosis of filariasis with ELISA test (χ2 = 0.914, P < 0.05).
The IgG/IgM rapid test revealed that positive cases of filariasis, relative to negative case fit model for age of expatriate workers were not significant (χ2 = 1.503, P > 0.05), which indicates the null model was significantly worse, or predictively less accurate than the final or full model [Table 3]. A similar result was obtained in the diagnosis of filariasis with ELISA test (χ2 = 0.960, P < 0.05).
Multinomial logistic regression was used to analyze the predictors of positive cases of filariasis based on IgG/IgM rapid test and ELISA. The reference category for the outcome variable was “negative cases.” Each of the other categories was compared with this reference group. The main interest of current analysis was focused on the relationship among the prevalence of filariasis and type of expatriate workers, nationality of workers, age, and sex [Table 3] and [Table 4].
|Table 4: Degree of agreement between filariasis immunoglobulin G/immunoglobulin M rapid test and filariasis immunoglobulin G 4 ELISA test|
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[Table 3] shows the outcomes of “positive cases of filariasis based on IgG/IgM rapid test and ELISA, when compared to “negative cases” (reference category). The IgG/IgM rapid test showed that nationality of the worker had no significant effect on prevalence of filariasis. With respect to nationality, participants from Saudi Arabia (OR = 3.59) were more likely to be quarantined less than once, when compared to Vietnamese workers. Furthermore, “type, sex and age of expatriate workers” were not significant predictors of filariasis, based on IgG/IgM rapid test and ELISA.
There was no significant agreement between IgG/IgM rapid test and ELISA with respect to the prevalence of filariasis [K = −0.073, PV = 0.464; [Table 4]]. The sensitivity of IgG/IgM rapid test, when compared with results from filariasis IgG4 ELISA, was 10%. Thus, if positive cases of filariasis were present, there was only a 10% (0.1) chance of the IgG/IgM rapid test picking them up. The percentage specificity of IgG/IgM rapid test, relative to that of filariasis IgG4 ELISA was 80.5%. Thus, if there were no positive cases of filariasis, there was 80.5% (0.805) chance of the test being negative, and 19.5% chance of a false-positive result with the IgG/IgM rapid test. PPV was 5.9%. This implies that if the IgG/IgM rapid test was positive, there was a 5.9% (0.059) chance that the patient actually had filariasis. Negative predictive value was 88%, implying that if the test was negative, there was 88% (0.88) chance that the patient did not have filariasis. However, there was still 10% chance of a false negative, i.e., that the patient did have filariasis. The likelihood ratio was 0.5. This means that if the IgG/IgM rapid test was positive in a patient, that patient had equal chance of having or not having filariasis, as presented on [Table 5].
|Table 5: Sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratio of filariasis immunoglobulin G/immunoglobulin M rapid test, relative to filariasis immunoglobulin G 4 ELISA|
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The cconcentrations of IgG4 in the samples were determined by extrapolation from a standard calibration curve. The concentrations ranged from 27.447 to 186.114 ng/L [Table 6].
|Table 6: Concentrations of human filariasis antibody (immunoglobulin G 4) in positive samples, on filariasis immunoglobulin G 4 ELISA|
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| Discussion|| |
One of the major reasons of morbidity and a stumbling block or hindrance to socioeconomic development in almost 80 countries of the world is LF. In the present research, the highest prevalence filariasis, based on IgG/IgM rapid test, was 0.6% in Indian workers (3 out of 497; χ2 = 9.9 a, P = 0.76; Cramer V small effect size value = 0.14, P = 0.76). Results from ELISA test showed that the highest prevalence of filariasis (4.3%) was also in Indian workers (4 out of 92; χ2 = 10.915a, P = 0.536; Cramer V small effect size value = 0.344, P = 0.536). A recent statistical report on the populations of nationals in Saudi Arabia showed that 1,228,652 Indian nationals (mostly males) reside in the Kingdom., If we assume that 3.5% of this population had microfilaremia, almost 43,000 Indians with bancroftian filariasis now living in this country. Studies by previous researchers have reported the presence of filariasis mainly among foreign workers. Other studies, for example, Sri Lanka revealed that these infected migrants have also started a new foci of filariasis in those areas where it was previously not found. Many countries have already started their eradication programs for prevention and eradication of filarial. The present peril posed by these MF carriers to the native population still remains to be ascertained. Nevertheless, continued influx of filariasis infected people from the endemic regions into Saudi Arabia is going on, especially from the regions of South-East Asia, where they usually reside in large congregations among the native residential communities for long periods. Moreover, there is the peril of exposure of these people to bites from mosquitoes, which are potential vectors of MF. In view of these factors, there is a need for the establishment of novel and self-sustained endemic cycles of bancroftian filariasis in the guest country. Population migration has also been implicated as a danger by many studies. A retrospective study of three cases of filariasis during a period of 20 years (1981–2001) in the Riyadh Armed Forces Hospital has been reported. We have many studies which gives suggestion on how to eliminate this menace.,, These observations justify the need for confirmation, by drawing evidences from recent serological and entomological surveys which have been conducted in the study area. The inferences and results presented here support. An urgent need to focus on the research sites where the pockets of infections are presumed, to delineate the areas of disease transmission. Most observers have noted that the peak of elephantiasis occurs in patients aged between 30 and 50 years, since this is one of the most productive age, where these people are usually involved in busy professional activities. These results have also been aligned with past studies which have reported that LF, in most cases, initiates at the economically active age of the individuals.,, Even though the disease LF was well precepted by the people living in that area of endemicity, the causes are still poorly understood by many people who are strongly influenced by sociocultural norms and own beliefs. It is difficult to say that the majority of participants − both the patients and their healthy counterparts − rarely know the exact causes of filariasis, which ultimately confuses the patients while seeking appropriate care.,
One major limitation in the serological tests is cross-reactivity. Filariasis infections caused by STH (soil-transmitted helminths [STH]) cause cross-reactivity. These infections are prevalent mainly in children in areas where brugian filariasis is endemic. It has been reported that sera from people infected with nonfilarial helminths showed high cross-reactivity in filariasis rapid test.,,
However, another study in which 20.5% of the population were children from STH endemic area, showed 100% specificity in filariasis rapid test. STH are endemic in Bachok. Thus, examinations of serology and stool were not included in the study population of Bachok, Kelantan. Despite the study on STH infection reported by Mahendra and his group in 1997, the laboratory of University Sains Malaysia, Kelantan campus, often reports positive results for STH in stool specimens from the residents. In the present study, the specificity of IgG/IgM rapid test, relative to filariasis IgG4 ELISA was 80.5%. This result is consistent with the finding of previous studies. Complete elimination of filariasis requires high-specificity test such as the dipstick test. This is significant for mapping an area of filaria infection, and for ensuring that mass-treatment is restricted only to endemic areas. Another important criterion for certifying the elimination of filariasis is sampling of children from the endemic areas. Therefore, the use of specific diagnostic test is crucial for the certification phase of the program.
The rapid test has been accepted by field staff due to the ease of testing and interpretation of results. One of the advantages of the test is that when villagers in endemic areas visualize the positive dipstick outcome of the test, it makes them to increase their drug compliance. However, one limitation of the dipstick test is that when it is used outdoors, it may be affected by the wind. Furthermore, staff have reported a cutoff in the blood filter stub which could lead to inconvenience.
LF infections differ between males and females. Reports from different areas on brugian filariasis showed that the prevalence of lymphedema and infection cases are more in males than in females. In contrast, a study by previous researchers demonstrated that there was no variation between the genders. This study has also demonstrated the effect of diagnostic tool on the epidemiological profile of a population. Rapid test showed that the population of infected females was less than that of infected males. However, results from ELISA showed that infection in males was thrice more than that in females. This disparity might be due to the fact that the sample size in males was larger than that in females. In the near future we can apply newer techniques for fast detection of filariasis.,, The use of high-resolution melting real-time polymerase chain reaction, in the detection of filariasis will be a real boon in future studies. BLF Rapid™, a prototype immunochromatographic IgG4-based test utilizing BmSXP recombinant protein, for the detection of bancroftian filariasis is also found successful.
The inferences drawn from the present study recommend that ELISA would be a much more useful diagnostic tool for filariasis infection than rapid test. This is in accordance with the results of a past study in an endemic filariasis area near the border of Malaysia–Thailand, in which ELISA test was used, along with an identical recombinant protein. Approximately 0.35% (18 out of 5138 cases) of infections were seen in school children. On the other hand, the health department in this area reported that only 0.05% (3 out of 5601) infections were detected using thick blood smears test.,,
| Conclusions|| |
In this study, the prevalence of filariasis (based on IgG/IgM rapid test) was 3.8% (19 out of 497), while the prevalence of filariasis based on IgG4 ELISA was 10.9% (10 out of 92). Based on IgG/IgM rapid test, the highest prevalence of filariasis (0.6%) was in Indian workers (3 out of 497), and results of ELISA test showed that the highest prevalence of filariasis (4.3%) was also in Indian workers (4 out of 92). It is on this account obligatory to conduct further epidemiological research to recognize and pin-point the magnitude of imported bancroftian filariasis in Saudi Arabia, in view of the fact that new South-East Asian carriers enter the country regularly in search of employment. In the meantime, screening of expatriate workers for filarial parasites in their countries of origin should be mandatory before employment. For workers from endemic areas living in the Kingdom, selective treatment with diethylcarbamazine citrate for persons with MF and/or identification of clinical signs in night blood examination surveys should be commenced.
NFB should be used to screen migrant workers for filarial infection using daytime blood specimens. This type of blood (NFB) requires minimal laboratory and technical support, and it is very suitable for the diagnosis of filarial infection in clinical laboratories and for surveillance studies.
Financial support and sponsorship
This study was financially supported by Deanship of Scientific Research, Najran University-Kingdom of Saudi Arabia for their financial and technical support under code number (NU/MID/16/083).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]