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

Background: Existing molecular assays for filarial parasite DNA in mosquitoes 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. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript. Methodology/Principal Findings: Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayilife cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen) was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes. Conclusions/Significance: This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971) that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens) may also be useful for detecting infective stages of other vector-borne parasites

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

My Body and Me: Self-Injurious Behaviors and Body Modifications in Eating Disorders\u2014Preliminary Results

We investigated self-injurious behaviors and body modification practices in eating disorder patients, considering different ED diagnoses and illness severities. Of the total sample, 50.9% showed at least one form of self-injury and 50.9% reported at least one tattoo, piercing, or both. Patients reporting only body modifications showed more positive feelings toward their bodies, higher levels of self-esteem, less impulsivity, depression, and anxiety, and lower levels of social dysfunction than those reporting only self-injury or both self-injury and body modifications. Self-injury was influenced by both diagnosis and severity of disorders

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

Detection of a single infective mosquito in mosquito pools.

*<p>All samples were run in triplicate and average Ct values are shown.</p><p>Dashed lines indicate samples where no <i>B. malayi</i> RNA was detected.</p

cDNA Library PCR Screening of Gene Candidates.

<p>Gene Identifier = TIGR Cluster Number (TC) available at <a href="http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.plgudbb_malayi" target="_blank">http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.plgudbb_malayi</a>.</p><p>+ = PCR product detected, blank = no PCR product detected, and NT = not tested.</p><p><i>B. malayi</i> cDNA library abbreviations: BmMf = microfilarial stage, BmL2 = L2 stage, BmL3 = vector-derived L3 stage, BmL3d6 = L3 larvae collected 6 days post mammalian infection, BmL4 = mammalian derived L4 stage.</p