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Differentiating Taenia eggs found in human stools: does Ziehl-Neelsen staining help?

2010; Wiley; Linguagem: Inglês

10.1111/j.1365-3156.2010.02579.x

1365-3156

Juan A. Jiménez, Silvia Rodríguez, Luz M. Moyano, Yesenia Castillo, Héctor H. Garcı́a,

Congenital Anomalies and Fetal Surgery

Objective To determine whether Ziehl-Neelsen staining can differentiate Taenia solium from Taenia saginata eggs. Methods Tapeworm proglottids (33 specimens, 23 T. solium and 10 T. saginata) and eggs (31 specimens, 13 T. solium and 18 T. saginata) were stained. Four eggs from each sample were measured and average diameters were recorded. Results Taenia saginata eggs stained entirely magenta in seven of 13 cases. Taenia solium eggs stained entirely blue/purple in 4/18 cases and entirely magenta in one. Eggs of T. saginata were slightly larger and always ovoid, while T. solium eggs were smaller and mostly spheric. Conclusions Ziehl-Neelsen staining can occasionally distinguish fully mature T. solium from T. saginata eggs, but this distinction is neither very sensitive nor completely specific. Differential staining suggests differences in embryophore components between species which become evident with egg maturation. In this small series, egg morphology (shape, maximal diameter) provided appropriate differentiation between T. solium and T. saginata eggs. Différenciation des œufs de Taenia retrouvés dans les selles humaines: La coloration Ziehl-Neelsen aide t-elle? Objectif: Déterminer si la coloration Ziehl-Neelsen permet de différencier les œufs de T. solium de ceux de T. saginata. Méthodes: Des proglottis de ténia (33 spécimens: 23 de T. solium et 10 de T. saginata) et des œufs (31 spécimens: 13 de T. solium et 10 de T. saginata) ont été colorés. Quatre œufs de chaque échantillon ont été mesurés et les diamètres moyens ont été enregistrés. Résultats: Les œufs de T. saginata étaient entièrement colorés en magenta dans 7/13 cas. Les œufs de T. solium étaient entièrement colorés en bleu/violet dans 4/18 cas et entièrement en magenta pour un cas. Les œufs de T. saginataétaient légèrement plus grands et toujours ovoïde, tandis que ceux de T. solium étaient plus petits et surtout sphériques. Conclusions: La coloration Ziehl-Neelsen peut parfois distinguer les œufs à pleine maturité de T. solium et de T. saginata, mais cette distinction n'est ni très sensible ni tout à fait spécifique. Une coloration différentielle suggère des différences dans les composants embryophores entre les espèces, qui deviennent évidentes avec la maturation des œufs. Dans cette petite série, la morphologie des œufs (forme, diamètre maximal) a procuré une différenciation appropriée entre les œufs de T. solium et ceux de T. saginata. Diferenciando los huevos de Taenia encontrados en heces humanas: es útil la tinción de Ziehl-Neelsen? Objetivo: Determinar si la tinción de Ziehl-Neelsen puede diferenciar los huevos de T. solium y T. saginata. Métodos: Se tiñeron proglótidos (33 especies, 23 T. solium y 10 T. saginata) y huevos (31 especies, 13 T. solium y 10 T. saginata) de Tenia. Se midieron cuatro huevos de cada muestra y se anotaron los diámetros promedio. Resultados: Los huevos de T. saginata se tiñeron completamente de color magenta en siete de los 13 casos. Los huevos de T. solium se tiñeron completamente de azul/violeta en 4/18 casos y completamente de magenta en uno. Los huevos de T. saginata eran un poco más grandes y siempre ovoides, mientras que los huevos de T. solium eran más pequeños y casi esféricos. Conclusiones: La tinción de Ziehl-Neelsen puede, ocasionalmente, servir para distinguir huevos maduros de T. solium de los de T. saginata, pero dicha distinción no es ni muy sensible ni completamente específica. La tinción diferencial sugiere diferencias en los componentes embrióforos entre especies que podrían ser evidentes con la maduración del huevo. En esta pequeña serie, la morfología del huevo (forma, diámetro máximo) proveía una diferenciación apropiada entre huevos de T. solium y T. saginata. Three big tapeworms lodge in the human intestine: Diphyllobothrium sp, Taenia solium and Taenia saginata. It is a matter of debate whether a fourth, Taenia asiatica, is a new species or a T. saginata subspecies (Flisser et al. 2004; Garcia et al. 2007). Of these, only Taenia solium can lead to severe disease because of its capacity to infect the human brain with its larval form and cause neurocysticercosis, the major cause of acquired epilepsy in most of the world (Garcia & Del Brutto 2005). Differential diagnosis between these tapeworms is based on the morphology scolex or proglottids of adult worms. In most cases, however, only tapeworm eggs are found in stool samples, and no parasite tissue is available. While Diphyllobothrium eggs are easily distinguishable, T. solium and T. saginata eggs cannot be differentiated by microscopy. Only DNA-based probes enable differentiation between these eggs; but this type of assay is rarely available in endemic areas (Gonzalez et al. 2000; Mayta et al. 2000; Flisser et al. 2004; Garcia et al. 2007). Fifty years ago, Capron and Rose described the use of the acid-fast (Ziehl-Neelsen) staining to distinguish T. solium from T. saginata (Capron & Brygoo 1959; Capron & Rose 1962). While their work is occasionally quoted, it has neither been replicated nor refuted. Thus, we examined fresh and preserved material from both parasite species to determine whether this method can differentiate between these two tapeworm species. Expelled parasite material of human origin was used for this study. Cases were defined as T. solium or T. saginata on the basis of carmine staining and counting of main uterine branches (Mayta et al. 2000; Flisser et al. 2004; Garcia et al. 2007) as well as PCR of tapeworm material (Mayta et al. 2000). There were no discordances between results with either method. Preserved parasite material was used for this part of the study, comprising mature and gravid proglottids obtained from 33 patients (23 T. solium carriers, 10 T. saginata carriers) after antiparasitic treatment (Jeri et al. 2004). In some cases, multiple gravid and pre-gravid proglottids were available (14 and 9 proglottids from two T. solium tapeworms, and seven and four proglottids from two T. saginata tapeworms) and were processed to assess changes in staining according to proglottid maturation. As per our standard routine, proglottids were washed with distilled water, fixed in 10% formalin-phosphate-buffered saline (PBS) and stored at room temperature until histological processing. Eggs from 8 pre- or post-treatment stool samples (2 T. saginata and 6 T. solium) were examined and stained afresh before any fixation. We also stained archived stool samples from 23 patients which had been preserved in 5% formalin-PBS (11 T. saginata, 12 T. solium). Stool samples were concentrated by tube sedimentation. Sediments were placed in microscopy slides with polylysine and left to dry for staining. Four eggs from each sample were measured, and the average maximal and transversal diameters recorded (Table 1). Proglottids were washed to eliminate formalin and passed through increasing ethanol concentrations (70°, 80°, 90° and 100°) and then xilol three times. Proglottid samples were placed in paraffin blocks, sliced into 6-μm sections, freed from paraffin and placed on microscopy slides with polylysine for staining (Luna 1968). Samples were stained with carbol-fuchsin 3% for 15', washed with tap water and then decoloured with 70% ethanol 1% HCl for 2'. After a second washing, the slide was contrasted with 3% methylene blue for 5', washed again and left to dry at room temperature (Clavel et al. 1999; Chapin & Lauderdale 2007). Staining of more proximal and more distal gravid T. solium and T. saginata proglottids showed that the oncospheres always stain blue in both species, with magenta hooks. As the eggs mature, a blue oncospheral membrane is clearly defined, around which magenta blocks begin to form the embryophore. A substance apparently secreted from the oncosphere then begins to fill the space between blocks. This substance is initially blue in both species. As the embryophore matures and becomes thicker, colouration gets more intense, gradually changing from blue to mixed magenta tones in T. solium and to a more marked magenta colour in T. saginata (Figure 1). Histological sections showing stages of maturation of eggs in proglottids of Taenia saginata (left) and Taenia solium (right) showing oncospheres surrounded by an oncospheral membrane, small, magenta embryophoric blocks, and interstitial substance. There was no difference in staining of eggs from fresh vs. preserved stool samples. In T. saginata eggs, the external cover or embryophore was coloured entirely magenta on Ziehl-Neelsen in 7/13 cases (Figure 2a), and magenta with dark blue (some close to dark purple) areas in the remaining six. In T. solium eggs, the embryophore was mostly stained in a mix of magenta and blue and was entirely blue in 4 of the 18 cases (Figure 2b) and entirely magenta in one case (Table 1). Mature eggs of Taenia solium (a) and Taenia saginata (b) as seen in stool samples, showing differences in staining tonalities. Some morphological differences were also apparent. The eggs of T. saginata were slightly larger, with a maximal diameter of 35.58 ± 0.91 μm vs. 32.08 ± 1.45 μm for T. solium eggs (n = 13 for T. saginata, n = 18 for T. solium; mean ± SD; P < 0.001, Mann–Whitney test). T. saginata eggs were always ovoid (ratio between larger diameter and its transverse diameter was 1.14 ± 0.07), while most T. solium eggs were spheric (ratio was 1.03 ± 0.03; P < 0.001 compared to T. saginata, Mann–Whitney test). In 3 of 18 cases, however, T. solium eggs looked ovoid (ratios 1.11, 1.08, and 1.07) (Table 1). In direct comparison of sensitivity and specificity, egg size and form were better predictors for species differentiation between T. saginata and T. solium eggs than Ziehl-Neelsen staining (Table 2). Taenia spp. eggs are covered by a thick embryophore composed of prismatic keratin blocks (which give it its typical radial appearance) and kept together by a cement substance. By the time they reach the environment, the embryophore is still surrounded by a colloid vitellum layer. The vitelogen glands in the oncosphere produce an acid-fast resistant substance which occupies the spaces between embryophoric blocks, likely caused by the changes in colouration through egg maturation (Capron & Brygoo 1959; Capron & Rose 1962). This series showed that Ziehl-Neelsen staining can occasionally distinguish fully mature T. solium from T. saginata eggs, in cases where the cover is entirely magenta (7/13 T. saginata), or entirely blue/purple (4/18 T. solium). Staining was mixed in 19/31 cases, and equivocal (magenta) in one case of T. solium. While this distinction provided a species diagnosis in 35% of cases, it is by no means absolute and does not seem useful in practice. Application of Ziehl-Neelsen staining assumes that eggs are fully mature (which is not possible to determine in a proglottid) and carries some degree of subjectivity because colour differences may be subtle and thus require highly trained personnel. In most centres, the numbers of specimens to be tested for species differentiation will be small, and operators will have little experience with this. Oncospheres stained blue in both species, which did not help differentiation. Morphological criteria (diameter and shape) seemed more consistent in this series. In this small series, an arbitrary cut-off of 35 μm had 100% predictive value for differentiation. This had been reported before (Verster 1969) but not replicated. However, morphological differences are minor and would need to be replicated with T. saginata and T. solium material from other parts of the world. Interestingly, our data suggest differences in specific components of the embryophore between these two species which become evident through egg maturation, compatible with previous data on the different chemical composition of embryophoric blocks (Morseth 1966). Given that the ability of the oncosphere of T. solium to infect the human host and cause cysticercosis is not shared by T. saginata, further understanding and characterization of the enzymes and other active molecules present in one species but not the other may provide species-specific diagnostics and potential vaccine targets. This work was partially supported by the Fogarty International Center, National Institutes of Health, Bethesda, USA and by the Bill and Melinda Gates Foundation.