The online version of this article (doi:10.1186/s12879-017-2362-4) contains supplementary material, which is available to authorized users.
Schistosomiasis, one of the neglected tropical diseases, is endemic in more than 70 countries. However, the clinical diagnosis of patients with a low degree of infection is an unsolved technical problem. In areas endemic for schistosomiasis japonica, proctoscopy detection of eggs has been one method used for clinical diagnosis. However, it is often a challenge to find typical live eggs and it is difficult to distinguish live eggs from large numbers of partially degraded and/or completely degraded eggs within colon biopsy tissue. To address this problem, we tested six different morphological and biochemical/molecular markers (ALP; morphological characteristics of egg; CalS (calcified substance); AOS (antioxidase); SDHG (succinic dehydrogenase) and SjR2 mRNA (retrotransposons 2 of S.japonicum genome mRNA)), including four new markers (CalS; AOS; SDHG and SjR2 mRNA.), to determine the viability of S. japonicum eggs deposited in human and mouse colon tissues. Our ultimate aim is to obtain a new method that is more sensitive, practical and accurate to clinically diagnose schistosomiasis.
Tissue samples were collected from mice at six different time points during S. japonicum infection with or without treatment with praziquantel (PZQ). Four new biochemical or molecular markers were used for the detection of egg viability from mouse liver and intestinal samples: CalS; AOS; SDHG and SjR2 mRNA. Subsequently, all markers were employed for the detection and analysis of eggs deposited in biopsy materials from patients with suspected schistosomiasis japonica for clinical evaluation. Microscopic examination of the egg morphology, worm burden in vivo and ALP (alkaline phosphatase) levels were used as a reference standard to evaluate the sensitivity and reliability of four new markers detecting egg viability.
The results of the study showed that the morphology of S. japonicum eggs deposited in tissues of hosts with schistosomiasis, especially cases with chronic schistosomiasis, is complex and egg viability is difficult to judge morphologically, particularly eggs with a fuzzy structure or partially modified eggs. We found that the majority of the viable schistosome eggs determined by four new markers (CalS, AOS, SDHG and SjR2 mRNA) were morphologically difficult to identify.
Among the markers, the most sensitive and specific method was the detection of SjR2 mRNA and the most simple, rapid and practical method was the detection of SDHG. Therefore, the detection of SDHG is the most practical for clinical application and its use could improve the accuracy in diagnosing active schistosome infection.
Additional file 1: Figure S1. Categories of eggs in colon tissue from infected mice. A: Immature eggs (smaller with embryonic cells present); B, D, I: Unknown viability eggs; C and E: Mature eggs (larger in size with miracidium present); F: Partially degraded eggs (miracidium with disordered structure with the the appearance of black particles); G and H: Completely degraded eggs (black particles present in eggs or whole egg appears black). Magnification is 100× for all images. (JPEG 52 kb)12879_2017_2362_MOESM1_ESM.jpg
Additional file 2: Figure S2. Types of eggs in biopsied colonic mucosa from schistosomiasis patients. Viable eggs with an intact miracidium are present in panels A, B and H (red arrows). Panels C, D and L contain partially degraded eggs that are light in color with disordered structure (green arrows). Panels B, G, I, J, K, M and N reveal completely degraded black eggs (black arrows). Unknown viability eggs containing miracidia with unclear structure are present in panels A, E, F and O (yellow arrows). Images are all at 100× magnification. (JPEG 88 kb)12879_2017_2362_MOESM2_ESM.jpg
Additional file 3: Table S1. Clinical data and egg viability in proctoscopic tissue samples collected from 76 schistosomiasis patients. (DOCX 12 kb)12879_2017_2362_MOESM3_ESM.docx
Additional file 4: Figure S3. ALP staining results for S. japonicum eggs in mouse colonic tissue. A: 45dPI group; B: 90dPI group; C: 180dPI group; D:30dPT group; E: 90dPT group; F: 180dPT group. The arrows in Fig. A point to positive eggs, which were stained blue/black by NBT. The staining results show that: all eggs in the 45dPI group were positive and negative eggs began to appear in the 90dPI group; Positive eggs decreased after treatment of mice with PQZ and, notably, eggs in the180dPI group were all negative. The eggs that had no color development were mostly empty or had a fuzzy structure (400× magnification). (JPEG 60 kb)12879_2017_2362_MOESM4_ESM.jpg
Additional file 5: Figure S4. CalS staining results for S. japonicum eggs in mouse colonic tissue. A: 45dPI group; B: 90dPI group; C: 120dPI group; D: 30dPT group; E: 90dPT group; F: 180dPT group. Eggs indicated by the blue arrows were negative (live) and stained blue with the von Kossa stain. Calcified eggs, were positively stained black? with the von Kossa stain and are highlighted (red arrows). In the Figure, as the tissue background was stained with eosin, the miracidium in the egg was also stained red (400× magnification). (JPEG 62 kb)12879_2017_2362_MOESM5_ESM.jpg
Additional file 6: Figure S5. AOS staining results for S. japonicum eggs in mouse colonic tissue. A, B and C indicate free eggs isolated from either tissues of infected mice with S. japonicum at 45 days and 180 days post infection or tissues of treated mice at 90 days post treatment. After staining with DAB, eggs in Fig. A developed a positive deep yellow or brown color. Some eggs in Fig. B were positive response and are indicated by blue arrows. In Fig. C, no eggs were positive. Figs 1, 2, 3, 4, 5 and 6 (1: 45 dPI group; 2: 90 dPI group; 3: 120 dPI group; 4: 30 dPT group; 5: 90 dPT group; 6: 180 dPT group) show DAB stained eggs in colon tissue from mice. Among them, all eggs in Figs 1 and 2 developed a positive deep yellow color. Some eggs in Figs 3 and 4 were positive indicated by blue arrows. None of the eggs in Figs 5 and 6 were positive. Eggs that were negative were morphologically empty and had a fuzzy structure or appeared black (400× magnification). (JPEG 82 kb)
Additional file 7: Figure S6. Amplified, standard and melt curve lines of S. japonicum-specific RNA real-time qPCR. The first 6 standards of the real-time qPCR (copy number was 105 Copies ~ 1 Copy) presented as a complete amplified curve, but the last standard (0.1 copy) did not have a t completely amplified curve. The linear relationship of the standard curve line was optimum, and the amplifying effectiveness was 98.05%; the dissolved melt curve line presented as single peak. (JPEG 50 kb)12879_2017_2362_MOESM7_ESM.jpg
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- Clinical diagnostic value of viable Schistosoma japonicum eggs detected in host tissues
Donald P. McManus
- BioMed Central
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