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Background: The control of lymphatic filariasis (LF) caused by Wuchereria bancrofti in the Central African Region has been hampered by the presence of Loa loa due to severe adverse events that arise in the treatment with ivermectin. The immunochromatographic test (ICT) cards used for mapping LF demonstrated cross-reactivity with L. loa and posed the problem of delineating the LF map. To verify LF endemicity in forest areas of Cameroon where mass drug administration (MDA) has not been ongoing, we used the recently developed strategy that combined serology, microscopy and molecular techniques., Methods: This study was carried out in 124 communities in 31 health districts (HDs) where L. loa is present. At least 125 persons per site were screened. Diurnal blood samples were investigated for circulating filarial antigen (CFA) by FTS and for L. loa microfilariae (mf) using TBF. FTS positive individuals were further subjected to night blood collection for detecting W. bancrofti. qPCR was used to detect DNA of the parasites., Results: Overall, 14,446 individuals took part in this study, 233 participants tested positive with FTS in 29 HDs, with positivity rates ranging from 0.0 to 8.2%. No W. bancrofti mf was found in the night blood of any individuals but L. loa mf were found in both day and night blood of participants who were FTS positive. Also, qPCR revealed that no W. bancrofti but L.loa DNA was found with dry bloodspot. Positive FTS results were strongly associated with high L. loa mf load. Similarly, a strong positive association was observed between FTS positivity and L loa prevalence., Conclusions: Using a combination of parasitological and molecular tools, we were unable to find evidence of W. bancrofti presence in the 31 HDs, but L. loa instead. Therefore, LF is not endemic and LF MDA is not required in these districts.