An accurate diagnosis of filarial infections in mobile populations can be challenging. Infected individuals may present with nonspecific symptoms or laboratory findings, and proper evaluation requires both a strong degree of clinical suspicion as well as specialized knowledge of filarial epidemiology and pathogenesis and expertise in the morphological classification of filarial parasites by microscopic examination. Further difficulties arise from the extremely limited commercial availability of filarial diagnostic assays that can distinguish not only between specific pathogens but also between active current infections and those that occurred in the past.
In this study, we assessed the performance and feasibility in a clinical diagnostic setting of a panel of real-time PCR assays designed to detect species-specific genomic DNA target sequences of the four most prevalent pathogenic filariae of humans: W. bancrofti, B. malayi, O. volvulus, and L. loa. Compared to conventional parasitology methods, our PCR assays were overall equal to or significantly more sensitive among blood and skin snip samples collected from a cohort of 200 patients undergoing evaluations for suspected filarial infections.
Differences in assay performance were most striking for the detection of O. volvulus
in skin snip samples, with diagnosis by a positive PCR assay for 11 patients compared to diagnosis by microscopy for only one patient. One possible interpretation of this discordance is that only one patient was truly infected with O. volvulus
, with the remaining diagnoses representing false-positive PCR assays. This situation is unlikely, however, since DNA extraction and assay setup were conducted under rigorous conditions to protect against cross-sample contamination, and all PCR runs included internal negative controls with verified negative assay results. Moreover, each of the positive patients was treated definitively with ivermectin, and each patient had a clinical response. Some patients also had a Mazzotti-type reaction following ivermectin treatment, which indicates a high likelihood of O. volvulus
infection. Each of the PCR-positive patients was also found retrospectively to be positive by highly O. volvulus
-specific serological assays (3
; data not shown).
More likely, discordances represent situations in which very low numbers of O. volvulus
microfilariae are present in skin snip samples such that microscopy is truly insensitive compared to PCR (23
). It is possible that PCR allows the detection of O. volvulus
DNA found within the entirety of the tissue, while microscopy detects only organisms that are capable of extruding themselves from submerged tissue samples. The increased sensitivity of our O. volvulus
PCR assay is notable in that for each patient evaluation, 4 to 6 skin snip samples were typically examined microscopically, while only 2 samples were processed for DNA extraction and PCR. Our PCR assay therefore achieved a higher rate of detection despite having to overcome a potential loss of sensitivity due to sampling error. Furthermore, there were no patients diagnosed with O. volvulus
infection by conventional microscopy but negative by PCR assay.
The performance of our PCR assay for L. loa infection was similar to that of blood filtration. Three paired assays were positive by blood filtration but negative by PCR, all of which were posttreatment samples collected from patients whose pretreatment evaluation included positive blood filtration and PCR assays on paired samples. Quantification of microfilaremia by blood filtration in each of these instances was only 1 or 2 organisms/ml, and there were several positive PCR assays for which the paired blood filtration identified microfilaremia at only 1 to 2 organisms/ml. Therefore, the false-negative PCR assays were most likely due to sampling error in the context of very low-level microfilaremia rather than any inherent inability of the assay to detect small quantities of parasite DNA. In further support of this interpretation, there were four paired blood samples (from four patients) for which PCR was positive but blood filtration was negative. Two of the patients had recently undergone medical therapy for loiasis, with pretreatment blood samples being positive by both PCR and blood filtration, while the other two patients likely had low-level microfilaremia upon initial evaluation. These four cases illustrate the utility of our L. loa PCR assay for the detection of low-level infection both at the time of diagnosis and during the course of monitoring the response to therapy.
An important limitation of our study is that our clinic evaluated on average only 20 new patients each year, reflecting the scarcity of opportunities to diagnose filarial infections in the United States, even at a national referral center. In particular, there were relatively few evaluations for suspected lymphatic filariasis. Due to the limited number of positive assays for W. bancrofti and B. malayi (either PCR or blood filtration), it is difficult to draw firm conclusions regarding the performance of our PCR diagnostics for these organisms, although these types of assays have been used successfully in research laboratories in countries where filarial disease is endemic.
In addition to observed gains in sensitivity, there are some distinct advantages of PCR in comparison to other available diagnostic methodologies. First, PCR directly detects filarial DNA, in contrast to serological and circulating-antigen assays, which measure indirect indicators of infection (e.g., antibodies) and which may be persistently positive long after all organisms have died (for both antibody and circulating-antigen assays). Second, our PCR assays achieve similar increased sensitivities compared to that of conventional microscopy while requiring smaller amounts of patient material as a starting point (200 μl of blood for PCR versus 1 ml or greater for filtration and 1 to 2 skin snip samples for PCR versus 6 skin snip samples for microscopy). Finally, PCR assays can be run by laboratory personnel with generalized training and do not require a specialized knowledge of parasite morphology and classification.
There are several disadvantages of PCR that must also be recognized. At this time, PCR assays require costly reagents such as kits for DNA extraction from blood or tissue, enzymes and primers that must be stored frozen, and thermal cycler machines with the ability to detect fluorescence emission (for real-time PCR assays). On a per-assay cost (between $10 and $12 [2
]), once equipment is in place, PCR is likely to be of equal or lower cost than antibody-based or parasitologic methods because of significantly lower labor costs than those of classical methods.
PCR may soon become more suitable for point-of-care use in resource-poor countries with ongoing advances in the development of hand-held, battery-operated devices using microfluidic methods for all-in-one DNA extraction, amplification, and detection (9
). Unlike the situation with blood filtration, our PCR assay results are currently not reported quantitatively. However, the generation of a standard curve using defined numbers of organisms would be a relatively easy adjustment to the real-time PCR format to allow a quantitative assessment of microfilaremia (methods which we have recently developed for L. loa
). Another avenue for improvement would be to multiplex the PCR assays using a different fluorescent reporter for each organism. Such an adjustment would allow the detection of microfilarial coinfections by a single assay, negating the advantage that blood filtration provides in this regard. The development and validation of an M. perstans
-specific PCR assay would also be necessary for this purpose.
In summary, we have demonstrated the utility and feasibility of a panel of real-time PCR assays for the diagnosis of filarial infections among immigrants and travelers which have been used clinically for more than a decade. Our data demonstrate that the PCR panel is exquisitely species specific and slightly more sensitive than blood filtration for the detection of microfilaremia. Additionally, our real-time PCR assay for O. volvulus is significantly more sensitive than conventional microscopy for the detection of skin microfilariae. Although not quite ready for widespread use in areas of endemicity, the successful performance of these molecular assays is an important step forward in making accurate filarial diagnostic tools more accessible to clinical parasitology programs that serve internationally mobile populations.