Diagnostic performance of capillary and venous blood samples in the detection of Loa loa and Mansonella perstans microfilaraemia using light microscopy

Background Loa loa and Mansonella perstans–the causative agents of loiasis and mansonellosis—are vector-borne filarial parasites co-endemic in sub-Saharan Africa. Diagnosis of both infections is usually established by microscopic analysis of blood samples. It was recently established that the odds for detecting Plasmodium spp. is higher in capillary (CAP) blood than in venous (VEN) blood. In analogy to this finding this analysis evaluates potential differences in microfilaraemia of L. loa and M. perstans in samples of CAP and VEN blood. Methods Recruitment took place between 2015 and 2019 at the CERMEL in Lambaréné, Gabon and its surrounding villages. Persons of all ages presenting to diagnostic services of the research center around noon were invited to participate in the study. A thick smear of each 10 microliters of CAP and VEN blood was prepared and analysed by a minimum of two independent microscopists. Differences of log2-transformed CAP and VEN microfilaraemia were computed and expressed as percentages. Furthermore, odds ratios for paired data were computed to quantify the odds to detect microfilariae in CAP blood versus in VEN blood. Results A total of 713 participants were recruited among whom 52% were below 30 years of age, 27% between 30–59 years of age and 21% above 60 years of age. Male-female ratio was 0.84. Among 152 participants with microscopically-confirmed L. loa infection median (IQR) microfilaraemia was 3,650 (275–11,100) per milliliter blood in CAP blood and 2,775 (200–8,875) in VEN blood (p<0.0001), while among 102 participants with M. perstans this was 100 (0–200) and 100 (0–200), respectively (p = 0.44). Differences in linear models amount up to an average of +34.5% (95% CI: +11.0 to +63.0) higher L. loa microfilaria quantity in CAP blood versus VEN blood and for M. perstans it was on average higher by +24.8% (95% CI: +0.0 to +60.5). Concordantly, the odds for detection of microfilaraemia in CAP samples versus VEN samples was 1.24 (95% CI: 0.65–2.34) and 1.65 (95% CI: 1.0–2.68) for infections with L. loa and M. perstans, respectively. Conclusion This analysis indicates that average levels of microfilaraemia of L. loa are higher in CAP blood samples than in VEN blood samples. This might have implications for treatment algorithms of onchocerciasis and loiasis, in which exact quantification of L. loa microfilaraemia is of importance. Furthermore, the odds for detection of M. perstans microfilariae was higher in CAP than in VEN blood which may pre-dispose CAP blood for detection of M. perstans infection in large epidemiological studies when sampling of large blood quantities is not feasible. No solid evidence for a higher odds of L. loa microfilariae detection in CAP blood was revealed, which might be explained by generally high levels of L. loa microfilaraemia in CAP and VEN blood above the limit of detection of 100 microfilariae/ml. Yet, it cannot be excluded that the study was underpowered to detect a moderate difference.Microfilaraemia of Loa loa and Mansonella perstans was investigated by light microscopy in paired thick smears of capillary and venous blood; each sample was prepared using a standardised quantity of 10 microliters of blood and analysed by a minimum of two independent microscopists. Microfilaraemia was on average +34.5% (95% CI: +11.0 to +63.0) higher in capillary than in venous blood samples for L. loa and +24.8% (95% CI: +0.0 to +60.5) for M. perstans. This might have implications for treatment algorithms of onchocerciasis and loiasis, in which exact quantification of L. loa microfilaraemia is of importance. Furthermore, the odds for detection of M. perstans microfilariae was 65% higher in capillary than in venous blood which may pre-dispose capillary blood for detection of M. perstans infection in large epidemiological studies when sampling of large blood quantities is not feasible. No solid evidence for a higher odds of L. loa microfilariae detection in capillary blood was revealed, which might be explained by generally high levels of L. loa microfilaraemia in capillary and venous blood.Peer Reviewe

Burden of disease in Gabon caused by loiasis: a cross-sectional survey

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A) An overview of ELISA seropositivity and arbitrary units (AU) as well as B) Ll-SXP-1-RDT positivity and absolute intensity of Reader Units (RU) by loiasis subgroups.

*Arbitrary Antibody Units, ** Interquartile range, *** Wilcoxon-rank sum test comparing median AU or RU. ‡Participants were defined by microfilaremia density as low (1–7,999 mf/mL), high (8,000–19,999 mf/mL) and hyper-microfilaremic (≥20,000 mf/mL).</p

Database of the study.

BackgroundThe parasitic disease loiasis is associated with significant morbidity and mortality. Individuals with hyper-microfilaremia (greater than 20,000 microfilariae per mL of blood) may suffer from serious treatment-related or spontaneous adverse events. Diagnosing loiasis remains complex and primarily relies on direct parasite detection. In this study, we analyzed the performance of various diagnostic tests and the influence of parasitological and clinical factors on test outcomes in samples from individuals living in an endemic region.MethodsData and samples were collected from rural Gabon. Loiasis was defined as either detectable microfilaremia, or a positive history of eyeworm as assessed by the RAPLOA questionnaire. Diagnostic testing included a quantitative PCR (qPCR) for detection of Loa loa DNA in blood samples, an in-house crude L. loa antigen IgG ELISA, and a rapid test for antibodies against the Ll-SXP-1 antigen (RDT). Sensitivity and specificity were determined for each test and factors potentially influencing outcomes were evaluated in an exploratory analysis.ResultsELISA, RDT and qPCR results were available for 99.8%, 78.5%, and 100% of the 1,232 participants, respectively. The ELISA and RDT had only modest diagnostic accuracy. qPCR was specific for L. loa microfilaremia and Cycle threshold values correlated with microfilarial density. Anti-L. loa IgG levels were highest in occult loiasis, and antibody levels correlated inversely with L. loa microfilarial density as did RDT line intensities. Only 84.6% and 16.7% of hyper-microfilaremic individuals tested positive by ELISA (11/13) and RDT (2/12), respectively.ConclusionNone of the tests demonstrated high sensitivity and specificity for loiasis. Indirect diagnostic assays were characterized by low specificity. Additionally, hyper-microfilaremic individuals often tested negative by RDT and ELISA, indicating that these tests are not suitable for individual case management in endemic populations.</div

Sensitivity analysis with a loiasis case definition of eyeworm history during the previous year and/or detectable microfilaremia.

Sensitivity analysis with a loiasis case definition of eyeworm history during the previous year and/or detectable microfilaremia.</p

The PCR settings for the <i>Loa loa</i> qPCR and <i>Mansonella</i> real-time FRET assay.

The PCR settings for the Loa loa qPCR and Mansonella real-time FRET assay.</p

Saponin lysis laboratory protocol.

BackgroundThe parasitic disease loiasis is associated with significant morbidity and mortality. Individuals with hyper-microfilaremia (greater than 20,000 microfilariae per mL of blood) may suffer from serious treatment-related or spontaneous adverse events. Diagnosing loiasis remains complex and primarily relies on direct parasite detection. In this study, we analyzed the performance of various diagnostic tests and the influence of parasitological and clinical factors on test outcomes in samples from individuals living in an endemic region.MethodsData and samples were collected from rural Gabon. Loiasis was defined as either detectable microfilaremia, or a positive history of eyeworm as assessed by the RAPLOA questionnaire. Diagnostic testing included a quantitative PCR (qPCR) for detection of Loa loa DNA in blood samples, an in-house crude L. loa antigen IgG ELISA, and a rapid test for antibodies against the Ll-SXP-1 antigen (RDT). Sensitivity and specificity were determined for each test and factors potentially influencing outcomes were evaluated in an exploratory analysis.ResultsELISA, RDT and qPCR results were available for 99.8%, 78.5%, and 100% of the 1,232 participants, respectively. The ELISA and RDT had only modest diagnostic accuracy. qPCR was specific for L. loa microfilaremia and Cycle threshold values correlated with microfilarial density. Anti-L. loa IgG levels were highest in occult loiasis, and antibody levels correlated inversely with L. loa microfilarial density as did RDT line intensities. Only 84.6% and 16.7% of hyper-microfilaremic individuals tested positive by ELISA (11/13) and RDT (2/12), respectively.ConclusionNone of the tests demonstrated high sensitivity and specificity for loiasis. Indirect diagnostic assays were characterized by low specificity. Additionally, hyper-microfilaremic individuals often tested negative by RDT and ELISA, indicating that these tests are not suitable for individual case management in endemic populations.</div

Sensitivity, specificity, positive and negative predictive values of the <i>Loa loa</i> qPCR, in-house ELISA and Ll-SXP-1-RDT to detect loiasis defined by either a positive life-time history of eyeworm assessed by the RAPLOA questionnaire and/or detectable microfilaremia.

Sensitivity, specificity, positive and negative predictive values of the Loa loa qPCR, in-house ELISA and Ll-SXP-1-RDT to detect loiasis defined by either a positive life-time history of eyeworm assessed by the RAPLOA questionnaire and/or detectable microfilaremia.</p

Results of exploratory test analysis of microfilaremic loiasis.

Linear regression models showing the association between A) Ct values of the qPCR, B) arbitrary antibody units (AU) of the IgG ELISA and C) reader units (RU) of the Ll-SXP-1-RDT, and L. loa microfilarial density assessed by microscopy. Percentages of test positivity are shown for microfilaremic subgroups in IgG ELISA (D) and the Ll-SXP-1-RDT (E). In A to D, blue dots represent microscopy results of thick blood smears and orange triangles represent results from leukocyte concentration. In Fig 2A, the negative qPCR Ct values were displayed as a value of 42, which is outside of the detection area. Cut-off values for test-positivity are marked by dashed lines.</p

Overview of employed diagnostic procedures.

(A) Diagnostic scheme. Procedures included history of eyeworm (B) assessed by the RAPLOA questionnaire, an in-house Loa loa IgG ELISA using crude adult worm antigen (C), a rapid diagnostic test detecting antibodies against the Ll-SXP-1 antigen (D), direct microfilaria detection including microscopy of Giemsa-stained thick blood smears (E) and saponin lysis (F), as well as a L. loa qPCR (G).</p