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    Images of some dipsticks/cassettes tested with sera from Groups 1, 2, and 4 before and after adsorption. SsRapid, BLF Rapid, and XEh Rapid are rapid tests for strongyloidiasis, bancroftian filariasis, and amoebic liver abscess, respectively. rNIE, rBmSXP, rPPDK are recombinant proteins for Strongyloides stercoralis, Wuchereria bancrofti, and Entamoeba histolytica, respectively. Group 1: Strongyloides sera with various test line intensity scores; Group 2: Strongyloides seropositive, positive for W. bancrofti and E. histolytica; Group 3: Strongyloides seropositive, positive for other infections/allergies. Arrows show the positions of the test lines, the line above it is the control line (goat anti-mouse IgG). The reddish-pink stain at the bottom ends of some dipsticks is from excess gold conjugate. Enlarging the images will allow better visualization of the test lines. This figure appears in color at www.ajtmh.org.

  • 1.

    Buonfrate D, Bisanzio D, Giorli G, Odermatt P, Fürst T, Greenaway C, French M, Reithinger R, Gobbi F, Montresor A, Bisoffi Z, 2020. The global prevalence of Strongyloides stercoralis infection. Pathogens 9: 468.

    • Search Google Scholar
    • Export Citation
  • 2.

    Montresor A et al., 2020. The global progress of soil-transmitted helminthiases control in 2020 and World Health Organization targets for 2030. PLOS Negl Trop 14: e0008505.

    • Search Google Scholar
    • Export Citation
  • 3.

    Yunus MH, Arifin N, Balachandra D, Anuar NS, Noordin R, 2019. Lateral flow dipstick test for serodiagnosis of strongyloidiasis. Am J Trop Med Hyg 101: 432435.

    • Search Google Scholar
    • Export Citation
  • 4.

    Noordin R, Anuar NS, Juri NM, Wongphutorn P, Sirowan Ruantip S, Kulthida Y, Kopolrat KY, Worasith C, Sithithaworn J, Sithithaworn P, 2021. Evaluation of a rapid IgG4 lateral flow dipstick test to detect Strongyloides stercoralis infection in northeast Thailand. Am J Trop Med Hyg 105: 688691.

    • Search Google Scholar
    • Export Citation
  • 5.

    Noordin R, Yunus MH, Robinson K, Won KY, Babu S, Fischer PU, Hisam S, Mahmud R, 2018. Laboratory evaluation of a rapid IgG4 antibody test (BLF Rapid™) for bancroftian filariasis. Am J Trop Med Hyg 99: 15871590.

    • Search Google Scholar
    • Export Citation
  • 6.

    Noordin R, Mohd Zain SN, Yunus MH, Sahimin N, 2017. Seroprevalence of lymphatic filariasis among migrant workers in Peninsular Malaysia. Trans R Soc Trop Med Hyg 111: 370372.

    • Search Google Scholar
    • Export Citation
  • 7.

    Lammie PJ, Weil G, Noordin R, Kaliraj P, Steel C, Goodman D, Lakshmikanthan VB, Ottesen E, 2004. Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis-a multicenter trial. Filaria J 3: 9.

    • Search Google Scholar
    • Export Citation
  • 8.

    Supali T, Djuardi Y, Bradley M, Noordin R, Rückert P, Fischer PU, 2013. Impact of six rounds of mass drug administration on brugian filariasis and soil-transmitted helminth infections in eastern Indonesia. PLoS Negl Trop Dis 7: e2586.

    • Search Google Scholar
    • Export Citation
  • 9.

    Saidin S, Yunus MH, Zakaria ND, Razak KA, Huat LB, Othman N, Noordin R, 2014. Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess. BMC Infect Dis 14: 182.

    • Search Google Scholar
    • Export Citation
  • 10.

    Noordin R, Yunus MH, Saidin S, Mohamed Z, Fuentes Corripio I, Rubio JM, Golkar M, Hisam S, Lee R, Mahmud R, 2020. Multi-laboratory evaluation of a lateral flow rapid test for detection of amoebic liver abscess. Am J Trop Med Hyg 103: 22332238.

    • Search Google Scholar
    • Export Citation
  • 11.

    Norsyahida A, Riazi M, Sadjjadi SM, Muhammad Hafiznur Y, Low HC, Zeehaida M, Noordin R, 2013. Laboratory detection of strongyloidiasis: IgG-, IgG4 - and IgE-ELISAs and cross-reactivity with lymphatic filariasis. Parasite Immunol 35: 174179.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 

 

 

 

 

Serum Adsorption Study to Validate the Specificity of a Rapid Test to Detect Strongyloides stercoralis Infection

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  • 1 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia;
  • | 2 Department of Parasitology and Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

ABSTRACT.

A lateral flow rapid test for strongyloidiasis will greatly facilitate the control and elimination of the disease. Previously SsRapid prototype rapid test showed high diagnostic specificity to detect Strongyloides infection, determined using non-Strongyloides sera negative by IgG-ELISAs. Since high specificity is crucial before a test is used for public health control activities, further validation of its specificity is needed. Also, it needs to be ascertained whether non-Strongyloides sera positive by IgG-ELISAs and SsRapid are truly positive for Strongyloides or are cases of cross-reactivity. We performed 84 rapid tests (two types of dipsticks and cassettes) using 34 serum samples. They were divided into four groups based on Strongyloides infection and coinfection with other parasites and the availability of recombinant proteins and rapid tests for the latter. Sera was adsorbed using polystyrene microspheres beads separately coated with four recombinant parasite proteins. The small sample size is a limitation of this study; however, the overall results showed that the sera adsorption procedure was successful, and the SsRapid test is specific.

Strongyloides stercoralis is estimated to infect 613.9 million worldwide.1 The autoinfection ability of this parasite causes almost lifelong infection in many people. When they are immunosuppressed, fatal hyperinfection may ensue. Due to the infection’s high and wide prevalence and its potential to cause serious outcomes, the WHO has added Strongyloides to the list of soil-transmitted helminth parasites targeted for control in its road map for neglected tropical diseases for 2021 − 2030.2 A rapid diagnostic test with high sensitivity and specificity will be important in assessing the disease’s public health burden and assisting its control efforts. Previously, the SsRapid prototype rapid test showed high diagnostic specificity, determined using non-Strongyloides sera negative by Strongyloides IgG-ELISAs.3,4 However, for use in public health control programs, the specificity of SsRapid needs to be further validated. Also, we would like to elucidate whether non-Strongyloides sera positive by the IgG-ELISA and SsRapid are truly positive for Strongyloides or are cases of cross-reactivity.

SsRapid is a prototype IgG4 rapid test for strongyloidiasis, with the test line mainly comprising Strongyloides NIE recombinant protein (rNIE). A diagnostic evaluation study in Malaysia reported 91.3% sensitivity and 100% specificity, while another study in Thailand demonstrated 82% sensitivity and 96% specificity.3,4 The different sensitivity results may be attributed to different sets of serum samples used in the evaluations and may also be contributed by some technical issues due to it being a prototype test with pipetting steps. Both studies reported high diagnostic specificity; nevertheless, confirmation of this parameter by performing a serum adsorption study would be desirable. If serum antibodies specifically react with the rNIE lined on the rapid test (thus giving a positive SsRapid result), the same serum adsorbed with rNIE-coated beads should give a negative SsRapid result. On the other hand, if the positive SsRapid is due to serum antibodies to another antigen that cross-reacts with the rNIE on SsRapid, the dipstick may remain positive with rNIE-adsorbed serum. Also, cross-reactivity may be suspected if a serum positive by SsRapid gives a negative result upon testing using the same serum adsorbed with another recombinant parasite protein.

Serum samples.

Serum samples used in the present study were anonymized stored specimens collected over the years. The use of the archived samples was approved by the Human Research Ethics Committee at Universiti Sains Malaysia (No. USM/JEPeM/20040230). All serum samples (N = 34) used in this study were previously positive by SsRapid and Strongyloides IgG ELISAs (Scimedx Corporation, Denville, NJ and/or Euroimmun Medical Laboratory Diagnostics AG, Lubeck, Germany). They were divided into four groups; all were tested again with SsRapid to recheck their test line intensity scores. Group 1 (N = 10) comprised Strongyloides-seropositive samples showing different test line intensity scores with SsRapid. Group 2 samples (N = 8) were from individuals positive with SsRapid and coinfected with other parasites with available specific recombinant proteins for serum adsorption and rapid tests to detect them. The coinfections were with Wuchereria bancrofti, diagnosed by microscopy and BLF Rapid (N = 3); Brugia malayi, diagnosed by microscopy and Brugia Rapid (N = 3); and Entamoeba histolytica (amoebic liver abscess) diagnosed by clinical presentation, a commercial IgG-ELISA (RIDASCREEN® Entamoeba IgG, R-Biopharma AG, Darmstadt, Germany), and XEhRapid (N = 2). BLF Rapid is a prototype IgG4 cassette rapid test for bancroftian filariasis lined with recombinant BmSXP5,6; Brugia Rapid is a commercial IgG4 cassette rapid test for brugian filariasis lined with recombinant BmR1 (Reszon Diagnostics International, Selangor, Malaysia);7,8 and XEh Rapid is a prototype IgG4 dipstick rapid test for extraintestinal amoebiasis lined with recombinant pyruvate phosphate dikinase (rPPDK).9,10 Group 3 samples (N = 11) were from individuals who were positive with SsRapid as well as coinfected with other parasitic infections, diagnosed via microscopy (malaria, ascariasis, trichuriasis, schistosomiasis), and DNA detection (microsporidium). However, we did not have recombinant proteins for serum adsorption or rapid tests to detect them. Two sera from individuals with high total IgE antibodies due to hyperallergic conditions were also included in this group; they were negative with the other available rapid tests, that is, BLF Rapid, Brugia Rapid, and XEh Rapid. Finally, Group 4 (N = 5) comprised samples from blood donors.

Sera in Groups 1, 3, and 4 were individually adsorbed with polystyrene microspheres beads coated with rNIE. Sera in Group 2 were adsorbed separately with rNIE and the respective recombinant parasite proteins (rBmSXP, rBmR1, or rPPDK). We then tested the adsorbed sera with both SsRapid and the rapid test for other parasitic infections (BLF Rapid, Brugia Rapid, or XEh Rapid, respectively).

Preparation of coated microspheres beads.

Polystyrene microspheres beads (Bangs Laboratories, Inc., Fishers, Indiana) with a mean diameter of 0.42 μm were used to coat the recombinant proteins. First, the polystyrene microspheres beads (130 µL) were washed with distilled water containing 0.01% (v/v) Tween-20, then centrifuged at 10,000 × g, at 4°C for 15 minutes. The washing was repeated two more times. Recombinant protein at 2 mg/mL was incubated with the polystyrene microspheres beads on a rotator for 8 hours at 4°C. It was then centrifuged at 10,000 × g, 4°C for 15 minutes, and the supernatant discarded. The excess recombinant protein was removed by a washing step using phosphate-buffered saline with 0.05% Tween 20 (PBST), then centrifuged at 10,000 × g, at 4°C for 15 minutes. The coated polystyrene microspheres beads were then blocked with 1% bovine serum albumin in PBST for 20 minutes on a rotator at 4°C, then centrifuged at 10,000 × g, 4°C for 15 minutes, followed by a washing step with PBST. The pellet containing the coated polystyrene microspheres beads were then used for serum adsorption.

Serum adsorption.

Serum adsorption using a recombinant protein involved two adsorption cycles. In the first cycle, 40 µL serum was added to the coated beads and incubated overnight at 4°C on a rotator. The next day, it was centrifuged for 15 minutes at 10,000 × g, 4°C. The supernatant was saved for a second serum adsorption cycle. It was added to a tube of polystyrene microspheres beads newly coated with the same recombinant protein and incubated for 8 hours on a rotator at 4°C. It was then centrifuged for 15 minutes at 10,000 × g, 4°C, and the serum in the supernatant was considered to be well-adsorbed.

Table 1 summarizes the results obtained before and after sera adsorption with the recombinant parasite proteins. A total of 84 runs of the four rapid tests (dipsticks and cassettes) were performed. Some results of the rapid tests with sera from Groups 1, 2, and 4 before and after adsorption are shown in Figure 1. Before adsorption, ten sera in Group 1 were positive with SsRapid with different test line intensity scores; however, after the adsorption process, all samples were negative with SsRapid. The scoring guide for line intensity (+1, +2, +3, +4) is a rough indicator of differences in the intensities; it is not to discern subtle differences of line intensities or quantitative comparison.

Figure 1.
Figure 1.

Images of some dipsticks/cassettes tested with sera from Groups 1, 2, and 4 before and after adsorption. SsRapid, BLF Rapid, and XEh Rapid are rapid tests for strongyloidiasis, bancroftian filariasis, and amoebic liver abscess, respectively. rNIE, rBmSXP, rPPDK are recombinant proteins for Strongyloides stercoralis, Wuchereria bancrofti, and Entamoeba histolytica, respectively. Group 1: Strongyloides sera with various test line intensity scores; Group 2: Strongyloides seropositive, positive for W. bancrofti and E. histolytica; Group 3: Strongyloides seropositive, positive for other infections/allergies. Arrows show the positions of the test lines, the line above it is the control line (goat anti-mouse IgG). The reddish-pink stain at the bottom ends of some dipsticks is from excess gold conjugate. Enlarging the images will allow better visualization of the test lines. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 5; 10.4269/ajtmh.21-0674

Table 1

Summary of SsRapid results before and after sera adsorption with recombinant parasite proteins

Serum typeRapid test results before serum adsorptionRecombinant protein coated on microsphere beadsRapid test results after serum adsorption
Group 1: Strongyloides-seropositive samples
1Strongy R60SsRapid +1rNIESsRapid Neg
2Strongy R95SsRapid +1rNIESsRapid Neg
3Strongy R3SsRapid +2rNIESsRapid Neg
4Strongy R36SsRapid +3rNIESsRapid Neg
5Strongy TS7SsRapid +3rNIESsRapid Neg
6Strongy R50SsRapid +1rNIESsRapid Neg
7Strongy N3SsRapid +2rNIESsRapid Neg
8Strongy A6SsRapid +2rNIESsRapid Neg
9Strongy R38SsRapid +3rNIESsRapid Neg
10Strongy R59SsRapid +4rNIESsRapid Neg
Group 2: Strongyloides seropositive, positive for Wuchereria bancrofti, Brugia malayi, and Entamoeba histolytica
1Wb BF 10SsRapid +1, BLF Rapid +4rNIESsRapid Neg, BLF Rapid +4
rBmSXPSsRapid +1, BLF Rapid Neg
2Wb BF 22SsRapid +2, BLF Rapid +4rNIESsRapid Neg, BLF Rapid +4
rBmSXPSsRapid +2, BLF Rapid Neg
3Wb BF17SsRapid +1, BLF Rapid +4rNIESsRapid Neg, BLF Rapid +4
rBmSXPSsRapid +1, BLF Rapid Neg
4Bm TS3SsRapid +4, Brugia Rapid +3rNIESsRapid Neg, Brugia Rapid +3
rBmR1SsRapid +3-+4, Brugia Rapid Neg
5Bm SB13SsRapid +4, Brugia Rapid +4rNIESsRapid Neg, Brugia Rapid +4
rBmR1SsRapid +3-+4, Brugia Rapid Neg
6Bm TU2SsRapid +4, Brugia Rapid +4rNIESsRapid Neg, Brugia Rapid +4
rBmR1SsRapid +4, Brugia Rapid Neg
7Eh CS027SsRapid +2, XEh Rapid +3rNIESsRapid Neg, XEh Rapid +3
rPPDKSsRapid +2, XEh Rapid Neg
8Eh SA19SsRapid +2, XEh Rapid +1rNIESsRapid Neg, XEh Rapid +1
rPPDKSsRapid +2, XEh Rapid Neg
Group 3: Strongyloides seropositive, positive for other infections/allergies
1Malaria S11PfSsRapid +2rNIESsRapid Neg
2Malaria S10 PvSsRapid +2rNIESsRapid Neg
3Malaria S6PfSsRapid +1rNIESsRapid Neg
4Ascariasis CS016SsRapid +2rNIESsRapid Neg
5Ascariasis P3SsRapid +1rNIESsRapid Neg
6Ascariasis CS018SsRapid +1rNIESsRapid Neg
7Trichuriasis Tri10SsRapid +1rNIESsRapid Neg
8Schistosomiasis SM59SsRapid +1rNIESsRapid Neg
9Microsporidium NMC010SsRapid +4rNIESsRapid Neg
10Hyperallergy (high IgE) CS003SsRapid +3rNIESsRapid Neg
11Hyperallergy (high IgE) CS004SsRapid +3rNIESsRapid Neg
Group 4: Strongyloides seropositive, blood donors from non-endemic area
1NH050SsRapid +2rNIESsRapid Neg
2NH058SsRapid +2rNIESsRapid Neg
3NH036SsRapid +2rNIESsRapid Neg
4NH030SsRapid +2rNIESsRapid Neg
5NH10SsRapid +1rNIESsRapid Neg

Strongy = Strongyloides; Ss = Strongyloides stercoralis; Wb = Wuchereria bancrofti; Bm = Brugia malayi; Eh = Entamoeba histolytica; NH = normal healthy; PPDK = pyruvate phosphate dikinase; BLF = bancroftian lymphatic filariasis.

We were concerned that the NIE bead adsorption could remove a significant proportion of cross-reactive IgG4 antibodies. Cross-reaction with antibodies to other parasites, especially lymphatic filarial, in Strongyloides serology tests is commonly reported;11 thus, we created Group 2 to include samples from filariasis and amoebiasis patients who were also positive by the Strongyloides dipstick. Thus, if the NIE beads removed antibodies to filarial and amoebic parasites, then after adsorption, the test line intensity on the rapid tests (Brugia Rapid, BLF Rapid, and XEh Rapid) should significantly decrease or become negative. However, as shown in Table 1 and Figure 1 (Group 2), the rapid tests remained positive with test line intensities similar to those before sera adsorption; thus, the NIE bead adsorption did not remove significant cross-reactive IgG4 antibodies. The same set of sera were then adsorbed with recombinant proteins to the other parasite infections, that is, rBmSXP, rBmR1, or rPPDK. The results showed that after adsorption, the respective rapid tests were negative. Meanwhile, SsRapid maintained a similar level of reactivity as before sera adsorption with those non-rNIE recombinant proteins.

The above results showed that the serum adsorption process successfully removed the antibodies to the recombinant protein coated on the beads, resulting in a negative result of the respective rapid test. SsRapid showed specific reactivity since polystyrene microspheres beads coated with rNIE did not significantly reduce sera reactivity to other recombinant parasite proteins. Similarly, the polystyrene microspheres beads coated with other recombinant parasite proteins did not significantly reduce the sera reactivity to rNIE.

Group 3 consists of sera from Strongyloides-seropositive individuals infected with other parasites (N = 9) and sera of hyperallergic cases (N = 2). We do not have specific non-Strongyloides proteins for adsorption of these sera, nor rapid tests to detect them; thus, they were only adsorbed with rNIE, which showed negative SsRapid post-adsorption. Based on the results of Groups 2 and 3, other infection sera positive with SsRapid can be considered coinfections of Strongyloides with other parasites and not cases of cross-reactions. Group 4 sera (N = 5) were from blood donors who were positive with SsRapid they also were negative with the rapid test after serum adsorption. It thus shows that blood donors may harbor the parasite via chronic asymptomatic infection.

We randomly selected pairs of pre- and post-adsorbed samples from several sera groups and retested/tested them with Euroimmun IgG-ELISA. The results were variable; some samples showed negative results post-adsorption, while others showed slight reductions in optical density values (data not shown). The results were not surprising since we did not adsorb the sera with Strongyloides larvae lysate antigen as used in the Euroimmun IgG-ELISA kit.

A limitation of this study is the small sample size of each sera group; however, the overall results suggest that the SsRapid is specific. In conclusion, this study showed that the sera adsorption procedure was successful, and SsRapid is specific in detecting anti-Strongyloides antibodies. It demonstrates that a serum positive with SsRapid is highly likely to be positive for Strongyloides infection. Thus, SsRapid has good potential to be used in diagnosing strongyloidiasis and for public health control activities.

REFERENCES

  • 1.

    Buonfrate D, Bisanzio D, Giorli G, Odermatt P, Fürst T, Greenaway C, French M, Reithinger R, Gobbi F, Montresor A, Bisoffi Z, 2020. The global prevalence of Strongyloides stercoralis infection. Pathogens 9: 468.

    • Search Google Scholar
    • Export Citation
  • 2.

    Montresor A et al., 2020. The global progress of soil-transmitted helminthiases control in 2020 and World Health Organization targets for 2030. PLOS Negl Trop 14: e0008505.

    • Search Google Scholar
    • Export Citation
  • 3.

    Yunus MH, Arifin N, Balachandra D, Anuar NS, Noordin R, 2019. Lateral flow dipstick test for serodiagnosis of strongyloidiasis. Am J Trop Med Hyg 101: 432435.

    • Search Google Scholar
    • Export Citation
  • 4.

    Noordin R, Anuar NS, Juri NM, Wongphutorn P, Sirowan Ruantip S, Kulthida Y, Kopolrat KY, Worasith C, Sithithaworn J, Sithithaworn P, 2021. Evaluation of a rapid IgG4 lateral flow dipstick test to detect Strongyloides stercoralis infection in northeast Thailand. Am J Trop Med Hyg 105: 688691.

    • Search Google Scholar
    • Export Citation
  • 5.

    Noordin R, Yunus MH, Robinson K, Won KY, Babu S, Fischer PU, Hisam S, Mahmud R, 2018. Laboratory evaluation of a rapid IgG4 antibody test (BLF Rapid™) for bancroftian filariasis. Am J Trop Med Hyg 99: 15871590.

    • Search Google Scholar
    • Export Citation
  • 6.

    Noordin R, Mohd Zain SN, Yunus MH, Sahimin N, 2017. Seroprevalence of lymphatic filariasis among migrant workers in Peninsular Malaysia. Trans R Soc Trop Med Hyg 111: 370372.

    • Search Google Scholar
    • Export Citation
  • 7.

    Lammie PJ, Weil G, Noordin R, Kaliraj P, Steel C, Goodman D, Lakshmikanthan VB, Ottesen E, 2004. Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis-a multicenter trial. Filaria J 3: 9.

    • Search Google Scholar
    • Export Citation
  • 8.

    Supali T, Djuardi Y, Bradley M, Noordin R, Rückert P, Fischer PU, 2013. Impact of six rounds of mass drug administration on brugian filariasis and soil-transmitted helminth infections in eastern Indonesia. PLoS Negl Trop Dis 7: e2586.

    • Search Google Scholar
    • Export Citation
  • 9.

    Saidin S, Yunus MH, Zakaria ND, Razak KA, Huat LB, Othman N, Noordin R, 2014. Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess. BMC Infect Dis 14: 182.

    • Search Google Scholar
    • Export Citation
  • 10.

    Noordin R, Yunus MH, Saidin S, Mohamed Z, Fuentes Corripio I, Rubio JM, Golkar M, Hisam S, Lee R, Mahmud R, 2020. Multi-laboratory evaluation of a lateral flow rapid test for detection of amoebic liver abscess. Am J Trop Med Hyg 103: 22332238.

    • Search Google Scholar
    • Export Citation
  • 11.

    Norsyahida A, Riazi M, Sadjjadi SM, Muhammad Hafiznur Y, Low HC, Zeehaida M, Noordin R, 2013. Laboratory detection of strongyloidiasis: IgG-, IgG4 - and IgE-ELISAs and cross-reactivity with lymphatic filariasis. Parasite Immunol 35: 174179.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Rahmah Noordin, Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia. E-mail: rahmah@usm.my

Financial support: This study was funded by a grant from the Malaysian Ministry of Higher Education (MOHE) MyLab grant 1/2018 (No 203.CIPPM.673014 2). We also acknowledge support from MOHE’s Institution Centre of Excellence (HICoE) program (No: 311/CIPPM/4401005).

Authors’ addresses: Rahmah Noordin, Nor Suhada Anuar, Nor Mustaiqazah Juri, Anizah Rahumatullah, and Nur Athirah Ahmad Hilmi, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Pulau Pinang, Malaysia, E-mails: rahmah8485@gmail.com, norsuhada@usm.my, mustaiqazah@yahoo.com, anizahrahumatullah@usm.my, and hilmi.nurathirah@gmail.com. Emelia Osman, Department of Parasitology and Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, E-mail: emelia.osman@ukm.edu.my.

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