Detection of Wuchereria bancrofti L3 Larvae in Mosquitoes: A Reverse Transcriptase PCR Assay Evaluating Infection and Infectivity

Lymphatic filariasis is a disabling and disfiguring disease caused by a parasite that is transmitted by a mosquito. The life cycle of the parasite requires two hosts: the mosquito vector and the human host. Part of the developmental life cycle of the parasite occurs in the mosquito and the other part in the human host. The parasite develops through four stages in the mosquito, only the last of which is infectious to humans. The third larval stage (L3) is the infective stage that initiates human infections when infective mosquitoes bite humans. There is currently a global program attempting to eliminate this disease by administering drugs to affected communities with the goal of interrupting transmission of the parasite. The new diagnostic tool described in this paper uses molecular techniques to specifically detect the infective stage of the parasite in mosquitoes. Many mosquitoes can be tested at one time to assess the risk of ongoing transmission of filariasis in communities. In addition, this new L3-detection assay can simultaneously detect whether the mosquitoes contain ‘any-stage’ of the parasite. This provides information on infection rates in humans in the community. Both pieces of information can be used in assessing the progress of disease elimination efforts

National Mass Drug Administration Costs for Lymphatic Filariasis Elimination

Lymphatic filariasis (LF), commonly known as elephantiasis, is a profoundly disfiguring parasitic disease caused by thread-like nematode worms. This disease can often be disabling, thus reducing the potential productivity of the affected individuals. The WHO places the number of people at risk in 83 countries at 1.307 billion. This study was undertaken in seven countries—Burkina Faso, Ghana, Egypt, Tanzania, the Philippines, the Dominican Republic, and Haiti—using a common protocol to determine the costs of mass drug administration (MDA) programs to interrupt transmission of infection with LF, because there is lack of sufficient information about the costs of these programs. The results demonstrate that LF MDA is affordable and relatively inexpensive when compared to other public health programs. In the context of initiatives for integrating programs for the control and elimination of neglected tropical diseases, this study adds specifically to the relatively scarce body of information about the costs of MDA programs for LF. It also adds to the general knowledge about the application of methods that can be used to estimate the costs and cost-effectiveness of an integrated approach

A Reverse Transcriptase-PCR Assay for Detecting Filarial Infective Larvae in Mosquitoes

The Global Programme for the Elimination of Lymphatic Filariasis (GPELF) was launched in the year 1998 with the goal of eliminating lymphatic filariasis by 2020. As the success of mass drug administration (MDA) in the global program drives the rates of infection in endemic populations to very low levels, the development of new, highly sensitive methods are required for monitoring transmission by screening mosquitoes for the presence of L3 infective larvae. The current method of mosquito dissection to identify L3 larvae is laborious and insensitive and is not amenable to screening large numbers of mosquitoes. Existing molecular assays for the detection of filarial parasite DNA in mosquitoes are sensitive and can easily screen large numbers of vectors. However, current PCR-based methods cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. This paper reports the first development of a molecular L3-detection assay for a filarial parasite in mosquitoes based on RT-PCR detection of an L3-activated gene transcript. This strategy of detecting stage-specific messenger RNA from filarial parasites may also prove useful for detecting infective stages of other vector-borne pathogens

Detection of one <i>B. malayi</i> L3 added to pools of uninfected mosquitoes.

*<p>All samples were run in triplicate and average Ct values are shown.</p><p>Dashed lines indicate that the TC8100 RNA was not detected in the sample.</p

Specificity Testing of the <i>Brugia</i> L3 Detection Assay.

*<p>All samples were run in triplicate and average Ct values are shown.</p><p><i>BmL3</i> = <i>B. malayi L3</i>, <i>DiL3</i> = <i>D. immitis L3</i>, <i>WbL3</i> = <i>W. bancrofti L3</i>, <i>BpL3</i> = <i>B. pahangi L3 tph-1</i> is a constitutive target that is transcribed in all stages of <i>Brugia and Wuchereria</i>.</p><p>TC8100 is an L3-activated transcript that is only present in <i>Brugia</i> L3.</p><p>PBM = days post blood meal.</p><p>Dashed lines indicate that the RNA target was not detected in the sample.</p