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Histopathological and ultrastructural analysis of the liver.. (a) Liver of a non-dengue case stained with HE and presenting normal aspect. (b-e) Liver sections of dengue cases, stained with HE, showing hepatic injuries, including micro (Mi) and macrovesicular (Ma) steatosis, necrosis (N), edema (E) and hemorrhage (He) near central vein (CV). (f) Semi-thin section of a non-dengue case presenting hepatocytes and sinusoidal capillaries with normal structures and (g) one dengue case presenting micro (Mi) and macrosteatosis (Ma), nuclear degeneration (black star) and numerous macrophage cells (Mo). (h) Ultrathin section of a non-dengue case exhibiting normal hepatocytes (H) and regular sinusoidal capillaries (SC) with the presence of monocytes (Mo) and Kupffer cells (KC) and (i and j) dengue cases showing large lipid droplets (LD) in the cytoplasm of hepatocytes, swollen mitochondria (red stars) and presence of platelet (Pt) inside sinusoidal capillaries (SC) with loss of endothelium. Semi-thin and ultrathin sections of liver were stained with methylene blue/azure II solution and uranyl acetate/lead citrate, respectively. Quantitative studies of histological damages were made individually in dengue (cases 1-4) and non-dengue patients (cont. 1-4), and statistical analysis were performed comparing the mean values of each group (dengue patients vs non-dengue patients). Damages were quantified by the percentage of affected area for (k) hemorrhage and (l) edema or (m) by steatosis degree using a scale ranging from 0 to 4. (n-o) Steatosis was also evaluated in each hepatic acini area (periportal, midzonal and central vein) by plotting different damage degrees (ten fields for each case). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05) and (***) (P<0.0001).
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As expected, in the liver of non-dengue patients we observed a regular structure of hepatic parenchyma, with hepatocytes around the central veins and sinusoid capillaries exhibiting normal endothelial cells (Figs.1a, 1f and 1h). The most prominent parenchyma lesion was the presence of abnormal retention of lipids inside hepatocytes with either small fat vacuoles around the nucleus (microsteatosis) or large vacuoles with displacement of the nucleus to the periphery of the cell (macrosteatosis) (Figs. 1b, 1c and 1g). Ultrastructural analysis also revealed numerous large lipid droplets inclusions inside hepatocytes, typical of macrosteatosis (Fig. 1i) and absent in the control (Fig. 1h). Quantification of steatosis revealed that three dengue cases, mainly case 2 who was obese, showed extensive areas with this damage, while case 4 presented a basal steatosis degree similar to those observed in non-dengue patients (Fig. 1m). Additionally, we observed that steatosis was present in all the three hepatic zones in the dengue cases, although it was more prominent around the portal space (Figs. 1n, 1o and 1p). Besides steatosis, the hepatic parenchyma of dengue cases also presented focal areas of necrosis with the presence of mononuclear infiltrates, mainly in case 2 (Fig. 1b). Cell death was also evidenced by detection of nuclear vacuolar degeneration in semi-thin analysis (Fig. 1g) and the presence of swollen mitochondria in ultra-thin investigations (Figs. 1i and 1j), indicating probably apoptotic processes, which were absent in the control of non-dengue patient (Figs. 1f and 1h).
Histopathological and ultrastructural analysis of the lung.. (a) Lung of a non-dengue case stained with HE and presenting normal aspect of alveoli (A) and alveolar septa (AS). (b-g) Lung sections of dengue cases, stained with HE, showing pulmonary alterations, including septal thickening (St), edema (E), hemorrhage (He), presence of mononuclear infiltrate (Inf), hyaline membrane formation (HM) and hypertrophy of alveolar macrophages (AM) and type II pneumocytes (PcyII). (h) Semi-thin section of a non-dengue case showing alveoli (A), alveolar septa (AS), endothelial cells (EC) and type I (PcyI) and II pneumocytes (PcyII) with normal aspects. (i) Semi-thin section of one dengue case presenting numerous platelets (Pt) and megakaryocytes (MK) inside alveolar septa. (j) Ultrathin section of one non-dengue case exhibiting regular alveoli, alveolar septum, type I and II pneumocytes and endothelial cell. (j-l) Ultrathin sections of dengue cases exhibited type II pneumocytes located in alveolar space in contact with numerous platelets, the appearance of hyaline membrane and the presence of virus particles (VP) in endothelium. Quantitative analysis of hemorrhage (m) and edema (n) observed in dengue (cases 1-4) and non-dengue patients (cont. 1-4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05). Semi-thin and ultrathin sections were stained as described in figure 1, as well as damage quantifications.
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As expected, we observed a regular structure of alveoli, alveolar septa and normal endothelial cells in the lung of non-dengue patients (Figs. 3a, 3h and 3j). Dengue cases presented septum thickening with an increase of cellularity (Fig. 3b), the presence of mononuclear inflammatory infiltrates (Fig. 3c) and hyperplasia of alveolar macrophages (Fig. 3g). Cases 1 and 2, who had co-morbidities (diabetes and obesity), also showed hyaline membrane formation (Fig. 3e and 3f), probably due to dengue shock syndrome, with the concomitant hypertrophy of type II pneumocytes (Fig. 3d). Alterations observed in cases 1 and 2 were evidenced with more detail in the ultrastructural analysis (Fig. 3k). Virus-like particles were also detected in the endothelium of the lung in case 1 (Fig. 3l).
Histopathological and ultrastructural analysis of the heart.. (a) Heart of a non-dengue case stained with H.E. and presenting normal aspect. (b and c) Heart sections of dengue cases, stained with HE, showing cardiac injuries, including hemorrhage (He), edema (E), presence of mononuclear infiltrate (Inf) and degeneration of muscle fibers (black star). (d and f) Semi-thin and ultrathin sections of a non-dengue case presenting cardiac fibers (CF) with normal nucleus (N), mitochondria (M), capillaries (Cap) and intercalated discs (ID). (e) Semi-thin section of one dengue case presenting degeneration of cardiac fibers (black star) characterized by absence of nucleus and a diffuse interstitial edema (E) and (g and h) ultrathin sections showing nuclear (white stars) and mitochondria alterations (M) in cardiomyocytes and interstitial edema. Quantitative analysis of hemorrhage (i) and edema (j) observed in dengue (cases 1-4) and non-dengue patients (cont. 1-4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05). Semi-thin and ultrathin sections were stained as described in figure 1, as well as damage quantifications. (Pt) platelets; (EC) endothelial cells.
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As expected, non-dengue patients presented a normal cardiac structure with branching fibers, central nuclei and intercalated discs (Figs. 5a, 5d and 5f). On the other hand, all dengue cases, except case 2, presented myocarditis with cardiac fibers degradation and the loss of striations and nucleus (Figs. 5b and 5c). Besides, focal areas of mononuclear infiltrated were detected in all dengue cases (Figs. 5b and 5c). Semi-thin sections revealed degeneration of cardiac fibers with the absence of nucleus and a diffuse interstitial edema, characteristic of myocarditis (Fig. 5e). Ultrastructural analysis suggested an apoptosis process in the cardiac fiber with picnotic nucleus and mitochondria alterations (Fig. 5g), which was not detected in the non-dengue patient (Fig. 5f).
Histopathological/ultrastructural analysis and dengue detection in the kidney.. (a) Kidney of a non-dengue case stained with HE and presenting normal aspect. (b and c) Kidney sections of dengue cases, stained with HE, showing injuries, including: hemorrhage (He), edema (E), sloughing of necrotic cells with loss of the basement membrane (black star), mainly in proximal convoluted tubule (PCT) but also detected in distal convoluted tubule (DCT), and areas of cellular regeneration (blue star) near renal glomerulus (RG). (d and e) Semi-thin sections of non-dengue cases showing Bowman's capsule (BC) and podocytes (Pdc) around glomerular capillaries (GC), mensagial cells (MC) and endothelial cell (EC) with regular structures and preserved capillaries (Cap), epithelial cells (Ep) and distal and proximal convolutes tubules (DCT and PCT, respectively). (f and g) Dengue cases with the presence of thrombus (T) in capillaries of renal glomerulus and necrotic cells (NC) in the lumen of proximal convoluted tubules. (h) Ultrathin of a non-dengue case exhibiting conserved glomerular capillaries. (i) Ultrathin of one dengue case showing necrotic cell with picnotic nucleus (white star) and dilatation of rough endoplasmic reticulum (ER). Quantitative analysis of hemorrhage (j) and edema (k) observed in dengue (cases 1-4) and non-dengue patients (cont. 1-4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05). Semi-thin and ultrathin sections were stained as described in figure 1, as well as damage quantifications. (l) Detection of DENV-3 antigens by immunochemistry in macrophages (M ) and monocytes (Mo) and (m) quantification of cells presenting these antigens.
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As expected, in non-dengue patients we observed a normal structure with preserved distal and proximal convoluted tubules and intact renal glomerulus (Fig. 7a). In contrast, three dengue cases (1, 2 and 4) presented acute tubular necrosis, characterized by sloughing of necrotic cells and loss of the basement membrane mainly in proximal convoluted tubules, but also to a lesser extend in distal tubules, with formation of casts of cellular debris (Figs. 7b and 7g). Ultra-structural analysis revealed pyknotic nucleus and dilatation of endoplasmic reticulum in these necrotic cells (Fig. 7i). On the other hand, cellular regeneration was also found in the cortical region, indicating thus an initial recovery of the organ (Fig. 7b).
Histopathological and ultrastructural analysis of the spleen.. (a) Spleen of a non-dengue case stained with HE and presenting normal aspect. (b and c) Spleen sections of dengue cases, stained with HE, showing vascular congestion (VC), edema (E) and an atrophy of lymphoid follicles. Red pulp (RP); white pulp (WP). (d) Semi-thin section of a non-dengue case revealing red pulp with regular aspect and normal splenocytes (S). (e) Semi-thin section of a dengue case showing vacuolization (V) and degenerated splenocytes (DS). (f) Ultra-thin section of a non-dengue case with regular splenocytes (S) and (g and h) dengue cases exhibiting vacuolization (V) around degenerated splenocytes and loss of the endothelium of splenic sinusoid (SS). Semi-thin and ultrathin sections were stained as described in figure 1. (i-k) Quantitative analysis of histological damages observed individually in dengue (cases 1-4) and non-dengue patients (cont. 1-4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). The media of lymphoid follicle areas were quantified (i), as well as the percentage areas with vascular congestion (j) and edema (k). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05) and (***) (P<0.0001).
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Spleen of non-dengue patients presented a regular structure, with normal splenocytes and well-defined regions of white and red pulp (Figs. 8a, 8d and 8f). On the other hand, dengue cases revealed prominent parenchyma lesion with remarkable atrophy of lymphoid follicles (Fig. 8b), disruption of the structural pattern and destruction of germinal centers (Fig. 8c). Quantitative analysis of lymphoid follicle areas revealed an approximately two fold reduction of these follicles in white pulp in dengue cases when compared to non-dengue patients (Fig. 8i). Additionally, analysis of semi and ultra-thin sections revealed areas with vacuolization around degenerated splenocytes (Figs. 8e and 8g) and loss of endothelium of sinusoids (Fig. 8h), which were not observed in non-dengue case (Figs. 8d and 8f).