Profile of loiasis infection through clinical and laboratory diagnostics: the importance of biomarkers

Abstract

Background: Detection of Loa loa microfilariae in peripheral blood is insensitive given only 30% of individuals are microfilaraemic while 70% are amicrofilaraemic with a variety of clinical signs. Biomarkers may improve the diagnosis of loiasis.

Methods: A total of 545 individuals exposed to L. loa were analysed using clinical data collected through a questionnaire (requesting information on eye worm, Calabar swelling, pruritis) and detection of microfilariae, immunoglobulin G4 (IgG4), DNA and antigens using microscopy, enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR) and Western blot, respectively.

Results: The results revealed that the rates of detection of L. loa microfilariae in the blood, of DNA by qPCR, of IgG4 by ELISA and of antigen by Western blot were 4.7%, 5.5%, 15.60% and 10.09%, respectively.

Conclusions: This study showed that clinical signs based on a questionnaire are highly subjective. Therefore it is imperative to use IgG4 and DNA biomarkers as well as antigens detected by Western blot to identify individuals infected with L. loa.

Keywords: L. loa DNA; L. loa adult antigen; IgG4 response; diagnosis; loiasis profile; qPCR.

Figure 1.

Representative western blot results for microfilaraemic probe with anti-IgG4 subclass: L. loa adult antigens were separated by 12% SDS-PAGE and separated antigens were transferred onto a membrane. Western blotting using diluted serum (1/100) was performed followed by mouse monoclonal antibodies against IgG4 (1/1000 dilution). Detection by diluted antibody against mouse IgG (1/1000) conjugated with alkaline phosphatase. (A) Serum reactivity: microfilaraemic individuals with ELISA IgG4-positive status (1–14), pool of positive sample (Pool+), pool of negative French (F), Turkish (T) samples (Pool−: F-T). Left, molecular weight standard (PM); right, antigen molecular size. (B) Microfilaria-positive serum reactivity: microfilaraemic individuals with IgG4-positive status (line 1); microfilaraemic individuals but with ELISA IgG4-negative status (lines 2–6); cryptic infection sample (lines 7–11 and 12–14), pool of positive samples (Pool+), pool of negative French (F) and Turkish (T) samples. Left, molecular weight standard; right, antigen molecular size.

Figure 2.

Representative Western blot results for endemic control probe with anti-IgG4 subclass: L. loa adult antigens were separated by 12% SDS-PAGE and separated antigens were transferred onto a membrane. Western blotting using diluted serum (1/100) was performed followed by mouse monoclonal antibodies against IgG4 (1/1000 dilution). Detection by diluted antibody against mouse IgG (1/1000) conjugated with alkaline phosphatase: endemic control sample (lines 1–15), pool of positive samples (Pool+), pool of negative French (F) and Turkish (T) samples (Pool−). Left, standard molecular weight (PM); right, antigen molecular size.

Figure 3.

Representative Western blot results from amicrofilaraemic ELISA IgG4-positive probe with anti-IgG4 subclass: L. loa adult antigens were separated by 12% SDS-PAGE and separated antigens were transferred onto a membrane. Western blotting using diluted serum (1/100) was performed followed by mouse monoclonal antibodies against IgG4 (1/1000 dilution). Detection was by diluted antibody against mouse IgG (1/1000) conjugated with alkaline phosphatase. (A) First set, individuals with ELISA IgG4-positive, ocular passage-negative (OP−), and Calabar oedema-positive (Ed+) status (lines 1–7). Second set, ELISA IgG4-positive, ocular passage-positive (OP+), Calabar oedema-negative (Ed−) status (lines 8–15); pool of positive samples (Pool+), pool of negative French (F) and Turkish (T) samples (Pool−). Left, standard molecular weight (PM); right, antigen molecular size. (B) First set, individuals with ELISA IgG4-positive, ocular passage-negative (OP+), Calabar oedema-positive (Ed+) status (lines 1–7). Second set, individuals with ELISA IgG4-positive, ocular passage-negative (OP−), Calabar oedema-negative (Ed−) status (lines 8–15); pool of positive samples (Pool+), pool of negative French (F) and Turkish (T) samples (Pool−). Left, standard molecular weight (PM); right, antigen molecular size.

Figure 4.

Representative Western blot results from amicrofilaraemic ELISA IgG4-negative probe with anti-IgG4 subclass. L. loa adult antigens were separated by 12% SDS-PAGE and separated antigens were transferred onto a membrane. Western blotting using diluted serum (1/100) was performed followed by mouse IgG (1/1000) conjugated with alkaline phosphatase. (A) First set, individuals with ELISA IgG4-negative, ocular passage-positive (OP+), Calabar oedema-positive (Ed+) status (lines 1–8). Second set, ELISA IgG4-negative, ocular passage-positive (PO+), Calabar oedema-negative (Ed−) status (lines 9–15); pool of positive samples (Pool+), pool of negative French (F) and Turkish (T) samples (Pool−). Left, standard molecular weight; right, antigen molecular size. (B) First set, individuals with ELISA IgG4-negative, ocular passage-negative (OP−), Calabar oedema-positive (Ed+) status (lines 1–15). Pool of positive samples (Pool+), pool of negative French (F) and Turkish (M) samples (Pool−). Left, molecular standard weight; right, antigen molecular size.