The liver and splanchnic venous system contribute to the body's cardiovascular stability by acting as blood reservoirs for the circulation (1-3). Although its role in restoring effective circulatory blood volume in cases of hemorrhagic shock has been well documented (3), evidence of hepatosplanchnic venous dysfunction as a cause or contributor to shock has been sparse. Acute hepatic and/or splanchnic pathology exist in many diseases that potentially affect cardiovascular hemostasis via hepatosplanchnic circulatory dysfunction. One of these illnesses is dengue hemorrhagic fever (DHF), an acute illness caused by dengue virus infection in which shock and prominent hepatosplanchnic symptoms and/or dysfunction often occur concomitantly (4, 5). The purpose of this study was to investigate the potential role of the liver and splanchnic vein in the pathogenesis of shock in this disease.
Dengue virus infection is one of the most important emerging infectious diseases in tropical countries (5). The diseases commonly caused by the virus are undifferentiated fever (dengue fever, DF) and a clinical entity characterized by capillary leakage, decreased intravascular volume, and hemorrhagic diathesis (DHF) (4, 5). Shock is seen in patients with the most severe form of DHF (dengue shock syndrome, DSS). Thus far, the mechanism leading to shock in these patients has not yet been fully understood. Whereas capillary leakage is mostly recognized as the mechanism for hypovolemia and shock in DHF, a previous study failed to show any difference in capillary permeability in patients with and without shock (6). In this study, we looked at the possibility of splanchnic venous pooling as a contributor for circulatory dysfunction in these patients. Because the portal vein receives all venous blood from the splanchnic organs and is relatively easy to be studied by ultrasound, it was chosen as the representative for the splanchnic venous beds. Inferior vena cava (IVC) was chosen as the representative for the systemic venous beds.
MATERIALS AND METHODS
Previously healthy children (age 5-15 years) who were clinically diagnosed with dengue virus infection at King Chulalongkorn Memorial Hospital from July 2003 to December 2004 were prospectively enrolled. The patients who subsequently did not have serologic or PCR confirmation for dengue virus infection and patients who required blood transfusion were excluded. After written inform consent was obtained, blood samples were collected for serologic and/or PCR confirmation of dengue virus infection, and ultrasonographic studies of portal vein and IVC were done as follows: at deffervescence or clinical sign of shock/capillary leakage (toxic stage); 24 to 48 h after deffervescence (convalescent stage); and at follow-up, at least 1 week after deffervescence.
Sizes of portal vein and IVC-
Ultrasonographic studies of portal vein and IVC were done by one investigator (A.K.) using an Aloka Prosound SSD5500 (Aloka Inc., Tokyo, Japan) imaging system using a 3 to 5 MHz transducer with an electrocardiogram attached. The maximum anterior-posterior diameter of the IVC was measured in the sagittal subxiphoid view with the patient lying supine, during quiet respiration, at the point where it crossed with portal vein. The size of portal vein was measured in supine right subcostal position, with the vein seen longitudinally (7, 8). The maximal diameter of portal vein was obtained just distal to the site where the hepatic artery crossed the vein (8). All measurements were done from inner wall to inner wall, during expiration, and at/before QRS complex on the electrocardiogram. An average from three measurements, indexed to the patients' height (7), was used for further analysis.
To assess the reliability of the measurement of both veins, the images of IVC and portal vein were recorded on paper and a second set of measurement was done by another investigator (P.S.) who did not have any knowledge of the patients' clinical status.
Portal vein flow velocity and portal vein congestion index-
The velocity of blood flow in the portal vein was measured by Doppler interrogation of the right portal vein just before its first intrahepatic branching. A transducer was placed at right lateral aspect of the upper abdomen in a supine position such that the right portal vein was seen pointing toward the transducer during expiration. The mean velocity of portal vein flow was obtained during respiratory hold in expiration, and averaged for three cardiac cycles. In patients who could not hold respiration reliably (in small children), an averaged value from three respiratory cycles was used. Doppler measurements were done three times and the value was averaged. Only Doppler interrogation with less than a 45 degree angle between the ultrasound beam and the vessel was accepted. Angle correction was used for all Doppler measurements. Mean Doppler velocity of blood flow was calculated by the software provided with the ultrasound machine with automatic tracing of the Doppler spectral signal.
Portal vein congestion index was calculated by dividing the cross-sectional area of portal vein with the velocity of blood flow in the vein (9). We calculated the cross-sectional area of the portal vein by assuming that the portal vein cross-section was circular. The mean Doppler velocity of flow in the right portal vein was used in lieu of velocity of flow in the common portal vein because of its ease of measurement. Modified portal vein congestion index was calculated as 3.14 × (radius of common portal vein indexed to height in cm/m × 1.7)2/(mean Doppler flow velocity of right portal vein in cm/s). The radius of portal vein was multiplied by 1.7 to standardize with the previous study (9), which was done in adults. The portal vein blood flow velocity was not adjusted because a previous study had demonstrated a relatively constant portal vein blood flow velocity in normal children of different ages (10).
Confirmation of dengue virus infection
Serologic study of dengue virus infection was done at The Arm Force Research Institute of Medical Sciences (AFRIMS, Bangkok, Thailand) using the an enzyme-linked immunoabsorbant assay (ELISA) method. The laboratory procedures and interpretations were previously reported (11). A positive PCR test for dengue virus was accepted as an evidence of dengue virus infection in patients who did not have ELISA results because of a missing specimen or a lack of paired specimens. Nonserotype-specific reverse transcription (RT)-nested PCR was performed at the Division of Infectious Disease, Department of Medicine, Chulalongkorn University School of Medicine using consensus primers targeting the 3′-untranslated region of all dengue viruses. The sensitivity and specificity of the test has been determined to be 86.8% and 100% using plasma or serum specimens, respectively (12).
Diagnosis and grading of DHF was done according to the criteria published by the World Health Organization (4). Diagnosis of DHF required at least one evidence of capillary leakage (rising of hematocrit > 20% or presence of pleural effusion and/or ascites). DSS was diagnosed when the patient with DHF had clinical findings of shock as hypotension for age and cold clammy skin or restlessness or narrow pulse pressure (≤20 mmHg) and rapid and weak pulse (4). Ultrasonographic study of the right pleural cavity was used to detect the presence of pleural effusion during the convalescent stage in all cases. The classification of the patient's clinical severity was done by the agreement of two physicians who were blinded to the ultrasonographic data of the venous study. Treatments, including fluid administration of all patients, were given by house staff and attending staff of our department according to the guideline published by the World Health Organization (http://www.who.int/csr/resources/publications/dengue/Denguepublication/en/) (4).
Data are expressed as mean ± SD unless otherwise specified. One-way analysis of variance was used to compare the difference of continuous variables between groups (DF, DHF without shock, and DSS). A chi-square test was used to compare categorical variables between the three groups. Intraclass correlation was used to assess the agreement between the two sets of measurement. P < 0.05 was considered significant. Statistic analysis was done using a commercial statistic software package (SPSS Inc., Chicago, IL).
Ethic committee approval and patient's consent
The study was approved by the Ethic Committee of the Faculty of Medicine, Chulalongkorn University. Written informed consent was obtained from each subject and/or appropriate guardian before enrollment.
Forty-five patients (20 DF, 14 DHF without shock, and 11 DSS) were recruited. Thirty-eight patients had positive ELISA for dengue virus infection. A positive PCR test was used as evidence of dengue virus infection in the other seven patients in whom the ELISA result was not available because of a missing specimen or a lack of paired specimens. The sensitivity and specificity of the PCR test in the patients enrolled by clinical criteria using ELISA as the gold standard was 95% (20/21) and 100% (3/3), respectively. The only one patient with conflicting ELISA and PCR results (positive ELISA and negative PCR) was included because of the longer establishment of ELISA as the gold standard for diagnosis of dengue virus infection at our institution (11) and possible decreased sensitivity of PCR during a later date of infection. However, exclusion of this patient would not change the results of the study. The demographic and clinical data of all patients are shown in Table 1.
Measurements of the portal vein and IVC showed a good correlation between measurements done at real time and retrospectively from recording paper (Intraclass correlation, 0.88 for portal vein and 0.97 for IVC). The data read retrospectively by an investigator who was blinded to the patient's clinical severity were reported. Except for minor differences in the P value, both sets of data yielded similar results.
The summary of all venous studies is shown in Figure 1. The portal vein was more dilated in shock cases (DSS) than DHF without shock than DF during the toxic and convalescent stages (Fig. 1, top left, P = 0.025 and = 0.011, respectively). At follow-up, the difference in the size of portal vein became nonsignificant. The difference in the size of the IVC followed the opposite trend, with smaller IVC in shock cases (DSS) than DHF without shock than DF during toxic and convalescent stages, but the difference failed to reach statistical significance (Fig. 1, top right). Portal vein blood flow velocity was lower in shock cases (DSS) than DHF without shock than DF during the toxic and convalescent stages (Fig. 1, lower left, P = 0.001 and = 0.002, respectively). Larger size of the portal vein along with lower velocity of blood flow in the vein resulted in higher portal vein congestion index in shock cases (DSS) than DHF without shock than DF during toxic and convalescent stages (Fig. 1, lower right, P = 0.001 and < 0.001, respectively). There was no difference in the portal vein blood flow velocity and portal vein congestion index at follow-up among the three groups.
Shock in patients with DHF is often classified as hypovolemic shock resulting from capillary leakage as evidenced by rising hematocrit and presence of pleural effusion and/or ascites in most patients with a severe form of this disease (4). Increased capillary permeability in patients suffering from DHF was demonstrated by strain gauge plethysmography (6). Decreased left ventricular end-diastolic dimension additionally supported intravascular volume depletion as the main etiology of shock in this disease (13).
Whereas there is little doubt that capillary leakage causes decreased effective blood volume in patients with DHF, other mechanisms may coexist because the degrees of microvascular leakage were not different among patients with and without shock (6). Studies of cardiac function similarly failed to demonstrate any association between left ventricular ejection fraction and the clinical severity of DHF (14).
One of the human body's defense mechanisms against decreased intravascular volume is its ability to mobilize blood volume from capacitance veins, of which the most important is the splanchnic venous system (1-3). In experimental animals, 38% of the body's total blood volume is in this compartment. During acute hemorrhage, contraction of these splanchnic reservoir can transfer as much as 27% of the total blood volume into the central circulation (3). This “autotransfusion” occurs mainly from the contraction of venous space in the liver, spleen, and intestinal veins (1-3). Whether this mechanism is operative in dengue virus-induced shock is unknown. Hepatic pathology in these patients may impair this mechanism. Endothelial dysfunction in patients with DHF may affect the capability of splanchnic veins to constrict. The purpose of this study was to investigate the role of splanchnic veins in the maintenance of cardiovascular stability in patients with dengue virus infection. Because the portal vein receives all venous blood from splanchnic organs, and is easily seen by ultrasound, it was chosen for the study. IVC was similarly chosen to represent systemic capacitance veins. Congestion of the portal vein was assessed by its size and modified portal vein congestion index, which has been shown to correlate with the portal venous pressure and the severity of various liver diseases (9).
In this study, we demonstrated that the portal vein dilated, whereas the IVC shrunk during the toxic stage of DHF. The changes were more pronounced in shock cases and were minimal in patients without capillary leakage (DF). Whereas diminished size of the IVC concurs with decreased intravascular volume, dilatation of the portal vein is contradictory and signifies splanchnic venous pooling and impairment of the hepatosplanchnic venous defense mechanism. Lower portal vein blood flow velocity in patients with DHF, especially in shock cases even makes this finding more paradoxical because under normal circumstances, the portal vein dimension should decrease with lower portal blood flow as a result of passive elastic recoil of the vessel (2). In hypovolemic shock from hemorrhage, sympathoadrenal activation further decreases the size of the splanchnic vein by causing active venoconstriction (1, 2). In experimental animals with hypovolemic shock from acute hemorrhage, the portal vein was shown to decrease in size, with splanchnic pooling occurred only at the preterminal stage of shock (15, 16). Additional data that support splanchnic congestion in patients with DHF are from autopsy studies of patients who died of this disease that showed congestion of liver, spleen, and esophageal and intestinal mucosae (17, 18). Microscopic sections of liver generally showed hepatic sinusoidal congestion (18), and congestion and dilatation of blood vessels were seen in the mucosa and lamina propria of the intestine (17).
The reason(s) for dilatation of the portal vein (and probably other splanchnic veins) is unknown at this time. Splanchnic veins can be dilated passively from impediment of hepatic sinusoids caused by viral-induced hepatic pathology that results in increased hepatic venous resistance and higher distending pressure inside the vein (2). Alternatively, the vein may be dilated as a result of its own pathology, such as endothelial dysfunction or other nervous and/or humoral factors affecting its vascular smooth muscle, causing increased venous compliance. Whichever the cause is, the end result is the pooling of venous blood in the splanchnic circulation. If all splanchnic veins dilated similarly, and assuming that the length was constant, the volume of blood trapped in this compartment would be significant. The increase in the diameter of the intestinal veins would likely result in increased tension in the wall of the vessels, which may explain the preponderance of gastrointestinal bleeding in patients with DSS.
Using the diameter of the portal vein and Doppler velocity of the flow in the right portal vein, a modified portal vein congestion index was calculated. We used the velocity of the right portal vein instead of the common portal vein because of its ease of measurement by Doppler ultrasound. The common portal vein is difficult to see from a right lateral abdominal scan because of its distance from the ultrasound probe, and measuring flow from subcostal view may be inaccurate because of the acute angle between the vein and the ultrasound beam. The method is the modification of the portal vein congestion index previously reported to correlate with the severity of liver involvement in patients with hepatitis and cirrhosis (9). The index also correlated with the portal venous pressure (9). Although the absolute value may not be comparable between this and the previous study, comparisons within the same patient and between groups in this study should be possible. The results suggested higher elevation of portal venous pressure and/or more severe degree of liver involvement in patients with DHF (especially with shock) compared with DF.
Portal vein congestion index has been shown to be increased in patients with viral hepatitis A, B, or C infection, especially in patients who developed ascites (19). However, the degree of portal vein dilation in these patients appeared to be less than in patients with DHF in our study. We speculate that the liver pathology in DHF may involve the area closer to the hepatic sinusoid, thus causing more vascular obstruction and splanchnic congestion compared with viral hepatitis A, B, or C. This speculation is supported by an immunohistochemistry study of liver tissues of patients with dengue virus infection that found viral antigens mainly in the endothelium and Kupffer cells lining hepatic sinusoids (20). Liver pathology in patients who died of DHF frequently demonstrated swelling of Kupffer cells and, occasionally, sinusoidal infiltration with megakaryocytes, lymphocytoid cells, and other dislodged cells (17), which may indicate the potential role of these cellular responses in causing sinusoidal obstruction. We postulate that this sinusoidal obstruction, coupled with increased vascular permeability, causes vascular pooling and leakage in the splanchnic organs that are generally not seen in viral hepatitis A, B, or C. This theory could explain the frequent gastrointestinal symptoms and splanchnic organ edema (21) seen in patients with DHF as well. These data demonstrate the potential importance of hepatosplanchnic venous system in the pathophysiology of DHF (and possibly other viral hepatic diseases) that deserves further study.
The authors thank Dr. Ananda Nisalak (The Armed Force Research Institute of Medical Sciences) for providing serologic data, Dr. Wanla Kulwichit for providing PCR data, and Prof. Dr. Usa Thisyakorn for her encouragement and advice. The authors thank Dr. Atchara Mahayosnant for advice on the ultrasonographic technique and the Venerable Dr. Mettanando Bhikkhu of Foundation of King Rama IX, The Great, Bangkok, for editorial advice.
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