Diagnosis of feline filariasis assisted by a novel semi-automated microfluidic device in combination with high resolution melting real-time PCR

Abstract

Background The diagnosis of filariasis traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears. This approach has several limitations. We developed a semi-automated microfluidic device to improve and simplify the detection of filarial nematodes. Methods The efficiency and repeatability of the microfluidic device was evaluated. Human EDTA blood samples were ‘spiked’ with B. malayi mf at high, moderate, and low levels, and subsequently tested 10 times. The device was also used for a field survey of feline filariasis in 383 domesticated cats in an area of Narathiwat Province, Thailand, the endemic area of Brugia malayi infection. Results In the control blood arbitrarily spiked with mf, the high level, moderate level and low level mf-positive controls yielded coefficient variation (CV) values of 4.44, 4.16 and 4.66%, respectively, at the optimized flow rate of 6 µl/min. During the field survey of feline filariasis in Narathiwat Province, the device detected mf in the blood of 34 of 383 cats (8.9%) whereas mf were detected in 28 (7.3%) cats using the blood smear test. Genomic DNA was extracted from mf trapped in the device after which high-resolution melting (HRM) real-time PCR assay was carried out, which enabled the simultaneous diagnosis of filarial species. Among the 34 mf-positive samples, 12 were identified as B. malayi , 15 as Dirofilaria immitis and 7 as| D. repens . Conclusions We developed a semi-automated microfluidic device to detect mf of filarial parasites that could be used to diagnose lymphatic filariasis in human populations. This novel device facilitates rapid, higher-throughput detection and identification of infection with filariae in blood samples.

A miniPCR-duplex Lateral Flow Dipstick Platform in Combination with the Microfluidic Device for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

“…We have previously developed a semi-automated micro uidic device for rapid, higher-throughput detection of micro lariae in animal blood. We next ]performed a real time PCR with high resolution melting analysis (HRM-real time PCR) for species identi cation of the trapped mf [29]. The e ciency of the device was evaluated in laboratory and it was applied to study the prevalence of canine and feline micro laremia [29,37].…”

“…Micro uidic device coupled with miniPCR-DLFD Detection of micro lariae was undertaken in all study samples using the micro uidic device, as described [29]. Brie y, 50 μl of blood was suspended in 150 μl of lysis buffer A, mixed, and then incubated at room temperature for 10 min.…”

“…The present study proposed for the rst time to developed a miniPCR assay coupled with a duplex lateral ow dipstick (DLFD) to detect both B. malayi and W. bancrofti DNA in samples of human blood. We also applied this miniPCR-DLFD in combination with our recently developed semi-automated micro uidic device [29] to facilitate mass screening and rapid visual species identi cation of micro lariae of lymphatic lariae in human blood.…”

Lab-on-a-Chip Technologies for the Single Cell Level: Separation, Analysis, and Diagnostics

“…Some salient examples thereof are circulating tumor cells (CTC) of various types of cancer [1,[36][37][38], rare cells (e.g., sickle-cell variants of red blood cells) [39,40], parasites, like Plasmodium falciparum [1,7,10,11,[13][14][15][21][22][23]36,[41][42][43][44][45][46][47][48][49][50] and Trypanosoma spp. [8,44,[51][52][53][54][55][56][57] and even plant pathogens [26,58], as well as-after cells have been lysed-subcellular infection markers (e.g., DNA, RNA fragments) [10,11,22,43,[59][60][61][62]. Given the vast adaptability of microfluidics to any kind of single or multi-cellular assay [63], the ability to combine it with various light microscopy techniques…”

“…This can also be done with droplets that already contain the primers and nucleotides [210]. The application of PCR is especially widely applied for diagnostic chips for malaria detection [11,13,211] and other parasites [43,212].…”