DIGFA Clinical diagnosis N
3.4 Discussion
Early diagnosis of human cystic echinococcosis (CE) and especially for alveolar echinococcosis (AE) could provide significant improvement in the quality of clinical management and treatment prognosis of both these zoonotic diseases (WHO/OIE, 2001; Craig et al 2003). Diagnostic laboratory methods that are cheap, rapid and easy to use would be very useful for basic clinics in endemic rural areas and in support of mass screening programmes since most persons in the early stages of CE or AE are asymptomatic (Rogan and Craig, 1997, 2002; Craig et al., 2000, 2003; WHO/OIE, 2001). Specific antibody detection appears most valuable for serodiagnosis of human CE or AE and has also shown promise in some post-treatment follow-up studies (Wen et al, 1995; Ito, 2002; Rogan and Craig, 2002). Gold standard laboratory tests for human echinococcosis are currently based on ELISA or immunoblots using E. granulosus hydatid cyst fluid antigen B
for CE, and E. multilocularis metacestode antigen Em2 or antigen Em18 for AE
(Gottstein et al, 1983, 1987; Zhang et al., 2000, 2001; Rogan and Craig, 2002; Ito, 2002; Craig et al., 2003; Carmena et al., 2006).
Dot-ELISA rapid format has been applied in a few community based studies for human CE but has limitations since enzyme-conjugates are difficult to store and apply in field conditions (Zheng et al., 1986; Rogan et al., 1991; Qiao et al., 1999). Colloidal gold labeling techniques were first used in the 1970s to locate specific antigens on the cell surface using electron microscopy (Faulk and Taylor, 1971; Horisberger et al., 1975). “One-step pregnancy test strip” type tests are a good example of a colloidal gold based rapid diagnostic test (May, 1991; Millipore Corp, 1996). A dot-immunogold infiltration assay (DIGFA) was first developed for serodiagnosis of HIV in 1989 (Chun and Chu, 1989; Spielberg et al., 1989). The procedure is similar to dot-ELISA but has the advantage of an infiltration system and use of colloidal gold conjugated IgG to give a more rapid, reliable and clear result. Colloidal gold antibody conjugate based DIGFA or similar immunochromatographic assays have also become an acceptable rapid clinical bed-side detection method for drug screening and diagnosis of several microbial and parasitic infections (Dar et al., 1994; Xiao et al., 1995; Dylan and Kevin, 1999; Feng et al., 2000; Garcia et al., 2000; Feng et al., 2002; Zhu et al., 2002; Sorell et al., 2002; Yang, 2003; Hujakka et al., 2003; Chen et al., 2005). Initial applications of DIGFA for human echinococcosis in China indicated good potential as a rapid
test (Fu et al., 2000; Feng et al., 2002; Zhang et al., 2001).
The current study reports the most comprehensive assessment and application of a rapid DIGFA for quick serodiagnosis of human echinococcosis. We confirm that DIGFA exhibited the following features: (1) the test could give a reliable diagnostic result within 2--3 minutes using only 20μL of serum or 40 μL heparinized blood; (2) the test was able to detect human echinococcosis in approximately 80-93% of cases and differentiate human CE and AE in about 80% of confirmed cases; (3) the DIGFA procedure is simple and no special training was required and therefore it had practical value for support of both community mass-screening in conjunction with ultrasound, and for hospital based diagnostic confirmation of echinococcosis (Fu et al., 2000; Zhang et al., 2001; Feng et al., 2002; Chen et al., 2005).
We developed this DIGFA test which acted as a qualitative immunological diagnostic method for detection antibody of human echinococcosis. Antigens used for DIGFA had been studied from 1980s and the preparation had been partially modified by our research group in Xinjiang Hydatid Clinical Research Institute from 1997 to 2000. Those antigens had been tested by ELISA and dog immunoblot assay (DIBA) and a sensitivity of 91% for EgCF in ELISA and DIBA, 93% in ELISA and 91% in DIBA for EgP had been reported (Zhang et al., 2000). I had tried to apply DIBA for the community study in 1998 but the storing and application of enzyme conjugate became the main problems since no fridge or freezer available. And also the DIBA procedure was similar with ELISA and asked for at least 1 hour test time at room temperature. But the stability of antigens on the NC membrane kept effective for longer time (at least 2 months without special package) indicated that we might find another stable conjugate to develop a test for using in the rural area. Colloidal gold conjugate could be stable at 4°C in working dilution and had been successfully applied in DIGFA and other tests for some infection disease (e.g. HAV IgM for hepatitis, Feng et al., 2000). The four antigens were combined together in one test for obtain better sensitivity and also partially specific for differentiation of CE and AE. Optimization the concentration of four antigens could supply stable and reproductive results and commercial DIGFA kit had been allowed to apply in many hospitals in hydatid endemic area of northwest China for clinical diagnosis and community study. DIGFA kit with 4 antigens was accepted since no special device needed, easy to perform and effective for both CE and AE. Other antigens had been tried to apply as well.
Native E. multilocularis crude abstract showed a higher false positive in normal
controls in initial trial (not exactly calculated) and was given up. A recombined antigen B (r-AgB) was also regarded as a potential candidate antigen but need more work for that since initial poor sensitivity was observed due to lower concentration and poor stability. Em18 or recombined Em18 might also be optional antigens for future research and application in DIGFA for human AE. Based on a panel of 1601 serum samples from advanced CE or AE patients confirmed by imaging, pathology and/or surgery in Xinjiang Medical University Hospital (XMUH, Urumqi, China), and control sera, overall DIGFA sensitivity was 80.7% (692/857) for human CE and 92.9% (39/42) for human AE. Specificity for echinococcosis (both CE and AE) was 89.6% (629/702); while E. granulosus
antigen B specificity for CE was 93.4% (695/744), and E. multilocularis Em2
antigen specificity for AE was 90.3%(1408/1559).
When applied to community mass screening studies in western China (i.e. sites in Xinjiang, Ningxia, Sichuan, Tibet, see also Chapter 6), the Echinococcus DIGFA
showed slightly lower sensitivity (71.8% for CE and 90.7% for AE) and specificity (74.4% for echinococcosis in general, 94.6% with antigen B for CE, 97.1% with Em2 for AE) compared with the hospital based DIGFA assessment. The DIGFA test was nevertheless extremely useful in these resource-poor settings as a combined diagnostic tool with ultrasound. Sera could be tested within 1 hour of ultrasound scan and up to 200 sera tested in one day. Diagnosis of CE or AE was confirmed in more than 80% of community detected cases and therefore facilitated efficient clinical treatment and assisted follow-up recommendations. Reasons for lower sensitivity of DIGFA in community (vs hospital settings) may be due to exposure without a detectable abdominal cyst lesion, involvement of sites not ultrasound detectable, and / or presence of small cysts or lesions, or degenerate, calcified, or necrotic cysts/lesions. The false negative rate for hospitalized CE cases was 19.3% (165/857) compared to 7.1% (3/42) for AE, while false positives occurred in 6.6% of CE (49/744), and in 9.7% (151/1559) of AE cases in XMUH. The DIGFA test could reliably differentiate CE and AE cases from each other around 80% of the time and an Em2 positive reaction appeared in 17.1% (146/857) of CE case sera. The DIGFA results were comparable to those obtained with the standard ELISA (false negative for CE 25.0%, for AE 2.4%, false positive for both 10.3%), and in general the ELISA was less sensitive (p<0.01) but
exhibited comparable specificity with DIGFA for human CE. The AgB antigen
preparation from E. granulosus and Em2 metacestode extract from E.
multilocularis, showed reliable specificity (90.3% --97.1%) in DIGFA for CE or AE
disease, and were comparable to other studies using traditional ELISA formats (Gottstein et al., 1983, 1987; Liu and Zhao, 1993; Poretti et al., 1999; Carmena et al., 2006).
3.5 Summary
In conclusion, a rapid 3 minute eye-read dot immunogold filtration assay (DIGFA) for serodiagnosis of human cystic (CE) and alveolar (AE) echinococcosis was developed in which 4 crude or semi-purified native antigens from E. granulosus
(EgCF, EgP, AgB) and E. multilocularis (Em2) were utilized simultaneously. The
overall sensitivity of DIGFA in a hospital diagnostic setting was 80.7% for human cystic echinococcosis (CE) (n=857) and 92.9% for human alveolar echinococcosis (AE) (n=42). The E. granulosus protoscoleces (EgP) and crude cyst fluid (EgCF)
extracts provided high sensitivity for the test; while E. granulosus partially purified
antigen B (AgB) and E. multilocularis antigen (Em2) ensured specificity
comparable to standard ELISA. Highest specificity was 93.4% with AgB extract for CE, and 90.3% with Em2 antigen for AE when CE vs AE cross-reactivity was excluded. Anti-AgB antibodies were present in 35.5% of AE cases and anti-Em2 in 7.4% of CE cases. In endemic communities in northwest China screened for echinococcosis, the sensitivity of DIGFA ranged from 71.8% to 90.7% in comparison to abdominal ultrasound; specificity for CE using AgB was 94.6% and for AE using Em2 was 97.1%. The DIGFA format was used successfully in conjunction with ultrasound for mass screenings to identify or confirm asymptomatic CE and AE cases in co-endemic communities in western China.