Dengue haemorrhagic fever: A pathological study

682 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.

Vol. 62.

No. 5.

1968.

D E N G U E H A E M O R R H A G I C FEVER: A P A T H O L O G I C A L S T U D Y T. BURKE

Classical dengue is usually a self-limiting disease, and if uncomplicated its mortality rate is almost nil (MANsoN-BAHR, 1966). Since 1953, however, there have been reports of a severer form of dengue from South and South-East Asia affecting mainly children, and characterized by haemorrhagic manifestations and shock, and a mortality rate of about 10% (I-hLSTEaD, 1966). A ndlder form was recognized in Singapore in 1960 (CHEw et al., 1961), but it affected mainly young adults and there were no fatalities. Since then, however, there have been seasonal outbreaks similar to those seen in Thailand, and affecting mainly children and adolescents. This communication describes the pathological features of 12 patients who died from dengue haemorrhagic fever in Singapore. Materials and m e t h o d s The necropsy protocols of all 12 patients were reviewed, and histological sections were cut at 6-7 t~ from formalin-fixed tissues. Sections were stained with haematoxylin and eosin, and where appropriate by Perls's stain for iron, by yon Kossa's silver technique for calcium, Lendrum's acid picro-Mallory for fibrin, and Gomori's reficnlin stain. Acutephase and post-mortem sera were tested by complement-fixation against the following viral antigens: dengue types 1, 2, 3, and 4, and Japanese encephalitis. Virus isolation was attempted from blood and post-mortem tissues, and dengue virus was isolated in 3 cases. The methods used have been described elsewhere (GOLDSMXTHet al., 1965), and the results from the present series are shown in Table I (CHAN, 1968). Clinical data There were 9 females and 3 males (Table I I ) a g e d 5-26 years, mean 10 years. The total duration of illness from the time of onset of symptoms till death varied from 3 to 7 days, mean 4.5 days. 2 patients were in hospital for 4 days, but all others died within 24 hours of admission to hospital. GOLDSMITHet al. (1965) described the clinical features of this severe type of dengue haemorrhagic fever in Singapore; the somewhat different clinical features in this series are shown in Figure 1. Since many of the patients died within a few hours of admission, full haematological examination was not always possible, but in those who were haematologically investigated severe thrombocytopenia I Wish to express my indebtedness to my colleagues in both the University and Government Departments of Pathology for their generosity in allowing me to use their cases, and for their co-operation throughout this study. I also wish to thank Dr. Chan Yow Cheong of the Department of Bacteriology, University of Singapore, for' carrying out the serological studies and virus isolation on these patients.

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

Department of Pathology, University of Singapore

683

T . BURKE TABLE I.

Virus isolated

Case

Virus isolations and titres of complement-fixlng antibodies

Dengue 1 -:--Acute Post phase m o r t e m

Dengue 2 Acute phase

-I

Post

mortem

Dengue 3,

Dengue 4

Acute-[ Post--[ Acute phase [mortem l phase

Post mortem

Japanese Encephalitis Acute phase

mortem

Post

1 2 3

[ [ [

----

1:128

1:256

i :256

ND

ND

ND

ND

1:32

1':256

1:1024

ND

1:1024

ND

1:512

ND

1:1024

ND

1:512

ND

1:512

ND

1:1024

ND

1:1024

ND

1:2048

ND

1:1024 '

4

[

--

1:512

1:256

1:512

i :256

1:256

1:256

1:512

1:256

1:256

ND

1:512

ND

1:256

ND

1:256

ND

1:64

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

5 [ ---6---l-Dengue 2(a)

1:256 I ND

1:64

ND 1:128

i ,

I

-

1:128

ND

1:512

ND

ND

ND

ND

ND

1:32

ND

8

I Dengue 2(b)

1:256

1:512

1:256

1:512

1:128

ND

1:512

ND

1:32

1:32 1:512

9

ND

1:512

ND

1:512

ND

ND

ND

ND

ND

I0

1:128

ND

1:128

ND

ND

ND

ND

ND

1:16

II

1:128

IqD

1:128

ND

ND

ND

ND

ND

1:8

ND

ND

ND

1:256

ND

ND

ND

ND

12

D e n g u e 2(c)

(a)

Isolated from Isolated from Isolated from ND--Examination (b) (c)

ND

ND

. 1:16 1:32

post-mortem lung. acute-phase blood. post-mortem liver. not done.

TABLE II

kge

T

1

9 19 11 12

Sex

17 18

Race Chinese Chinese Chinese Chinese Chinese Chinese Chinese Chinese Chinese Chinese Chinese Chinese

Duration of illness 6 3 6 7 5 3 3 4 7 3 3 4

days days days days days days days days days days days days

Length of stay in hospital 4 days 2 hours 18 hours 9 hours 19 hours Dead on arrival 1 day 4 days 3 hours 3 hours 1 day Less than 1 hour

was the rule, with platelet counts of 20,000-75,000 per c.mm., mean 51,000. The total leucocyte count was normal but a consistent finding was the presence of "atypical mononuclears" in the peripheral blood smears. In one patient 22~/o of the leucocytes in the differential white cell count were classified as "atypical mononuclears." The morphological appearance of these cells was described by HANAM (1964). Investigation of the coagulation mechanism was carried out in only 4 cases, and showed slight prolongation of the one-stage prothrombin time and bleeding time; the whole blood coagulation time was normal.

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

7

684

DENGUE HAEMORRHAGIC FEVER: A PATHOLOGICAL STUDY

CLINICAL FEATURES FEVER SHOCK

m

l

_ _

m

SEROUS EFFUSION PETECHIAL SKIN HAEMORRHAGES NAUSEA & VOMITING GASTRO-INTESTINAL I-IAEMORRHAGE !

HEADACHE

CONSTIPATION RETINAL HAEMORRHAGES !

DIARRHOEA ABDOMINAL PAIN BLEEDING FROM GUMS

!

FITS GENERALISED MUSCLE PAINS 1

2

3

4 5 6 7 8 9 10 NUMBER OF PATIENTS

11 12

FIG. 1. Clinical features of the 12 patients in this series.

Necropsy findings Gross e x a m i n a t i o n Petechial skin haemorrhages or rashes present during life were not always seen at necropsy, but when present they were more frequent over the neck region, upper limbs, and anterior aspect of the trunk. There was no evidence of jaundice in any case. A striking feature in 9 subjects was large serous effusions into the peritoneal and pleural cavities, on average 250 ml. and 150 ml. regpectively. This fluid had a protein content of 3.5-5.5 grammes per 100 ml.; by electrophoresis it was shown to be very similar to serum (Figure 2). in about one-third of the cases there was a marked retroperitoneal oedema, best seen at the root of the mesentery where it appeared almost like a loculated effusion. Oedema was also seen occasionally in the pancreatic bed, but retroperitoneal oedema was not seen in the absence of ascites. There was one instance of haemoperitoneum, and although the peritoneal cavity contained 1,000 ml. of fluid blood no local source of bleeding could be demonstrated. C a r d i o v a s c u l a r system Petechial haemorrhages were almost invariably present on the epicardial surface of the heart, especially posteriorly and around the roots of the great vessels. Small focal haemorrhages measuring 1 mm. in diameter were present in the myocardium, and they also showed subendocardial haemorrhage situated most frequently in the posterior wall of the left ventricle and adjacent portion of the interventricular septum.

Respiratory, system Though the larynx was usually normal, the trachea and main bronchi were invariably injected and often contained a thin film of lightly blood-stained mucus. The lungs

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

INTRACRANIAL HAEMORRHAGE

T. BURKE

685

were heavier than normal, and subplenral petechial haemorrhages were invariably present. On section they were congested and moderately oedematous and the cut surface had a characteristic glassy appearance. Areas of intrapulmonary haemorrhage were often seen, but were rarely larger than 5 ram. in diameter. Haemorrhages were also seen in the diaphragm, particularly at the junction of the muscular and tendinous portions.

Digestive system The oesophageal mucosa was frequently congested. The stomach usually showed a large number of petechiai haemorrhages, and in 10 patients contained altered blood. The amount of blood was greater than might be expected from the petechial haemorrhages, but careful examination failed to reveal any bleeding point and there were no erosions. In most of these 10 patients the intestines also contained a small quantity of fresh blood, but less than in the stomach. In the ileum the Peyer's patches often had a haemorrhagic appearance. The liver was slightly enlarged in all cases and was usually congested. Small focal areas of haemorrhage were sometimes seen scattered throughout its substance, and in 4 cases pale areas of necrosis could be seen contrasting with the congested unaffected liver tissue. The pancreas was of normal consistency, but its cut surface often showed minute focal areas of haemorrhage. The gelatinous oedema affecting the pancreatic bed, when present, was striking.

Lymphoid and haemopoietic systems The spleen was invariably slightly enlarged, congested and friable and its cut surface often showed unusual prominence of the Malpighian bodies. There was a moderate generalized enlargement of the lymph nodes in 8 patients, best seen in the mesenteric group, and less frequently in the cervical group. The bone-marrow appeared normal.

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

FIG. 2. Electrophoretic strip of pleural effusion fluid, with quantitative protein estimation.

686

DENGUE HAEMORRHAGIC FEVER" A PATHOLOGICAL STUDY

Endocrine system The pituitary, thyroid, and adrenals were normal, and showed no gross evidence of haemorrhage. G e n l t o - u r i n a r y system The kidneys were of normal size and weight. They were congested and often showed subcapsular petechial haemorrhages; less commonly small focal haemorrhages could be seen within their calyces. The ureters and bladder were normal. No abnormalities were seen in the genital organs.

Microscopical examination Heart There was no evidence of myocarditis. Focal haemorrhages were seen around small blood vessels, but the blood vessels themselves appeared to be quite normal. Lung In all 12 cases the alveolar capillaries were engorged, and in nearly all small foci of intra-alveolar haemorrhage could be seen. There was evidence of an interstitial pneumonids, in all cases, characterized by broadening of the alveolar septa and infiltration by mononuclear cells. The severity of this varied, and even in the same patient sections taken from different portions of the lungs showed different degrees of involvement. The inflammatory infiltrate was predominantly lymphocytic, but larger cells resembling those seen in the spleen and lymph nodes were also present. The alveolar spaces usually contained some oedema fluid, but no hyaline membrane. In a few cases there was a tendency for the alveolar epithelium to assume a cuboidal appearance, but this was focal and never extensive. Megakaryocytes were present in all cases in the alveolar capillaries, and were present in the lungs in larger numbers than in any other tissue. In 3 cases the deposition of fibrin in the subintimal layer of subpleural vessels was observed, and a fine layer of fibrin was often present over the pleural surface. In no case was there evidence of bacterial pneumonia. Liver

In general the liver showed sinusoidal congestion, and frequently there was a mild to moderate degree of fatty metamorphosis. In 4 cases, however, there were zonal areas of necrosis. These necrotic areas were paracentral in position and were eccent~caUy placed in relation to the central vein (Figure 3). They were always separated from the central vein by a border of surviving liver cells at least 2 cells thick. By serial sectioning it was shown that although within any particular lobule two adjacent areas of necrosis might coalesce, they invariably maintained their original eccentric position relative to the central vein and did not cross over to the opposite side of the lobule. Occasionally coalescence of adjacent necrotic areas resulted in a "horse-shoe" area of necrosis occupying

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

Central nervous system The brain was heavier than normal in all cases, and the leptomeninges were congested and oedematous. Petechial haemorrhageS were frequently seen in the white matter. In 4 patients there was gross evidence of intracranial haemorrhage. This took the form of localized subarachnoid haemorrhage in one patient, subdural haemorrhage in another, and focal areas of intracerebral haemorrhage with extension into the ventricles in the remaining two. The spinal cord was not examined.

T° BURKE

687

FIG. 3.

a paracentral position around the central vein; but encirclement of the central vein was not observed. In all 4 cases the necrotic areas showed a mild inflammatory reaction, the inflammatory cells being a mixture of lymphocytes and neutrophils. The individual liver cells within these areas showed a variety of changes ranging from marked swelling of cells with cytoplasmic vacuolation to complete cellular necrosis. Another feature was the appearance of hyaline acidophilic masses within the cytoplasm of affected cells, resembling the Councilman bodies seen in yellow fever. They could also be recognized lying free among the necrotic debris, when they tended to assume an ovoid rather than spherical shape, and were about twice the size of an erythrocyte. They were rather laminated in appearance with a paler staining central area, and were unlike the more uniformly staining "hyaline globules" occasionally seen in infectious hepatitis. A narrow crescentic margin of nuclear material was sometimes seen associated with the Councilman-like bodies, as if they had been engulfed by a Kupffer cell. The areas of hepatic necrosis were not associated with haemorrhage. The portal tracts showed a slight increase in periportal lymphocytes. In 2 of the 4 cases showing zonal necrosis, intranudear inclusion bodies could be seen. They were extremely scanty, and in one ease an intranudear inclusion body was seen only after closely scrutinizing 60 serial sections of liver. The inclusion body almost completely filled the nucleus, and was acidophilic with a narrow almost colourless halo separating it from the the nuclear margin (Figure 4). Measuring approximately 7 ~t in diameter, the inclusions closely resembled the Torres inclusion body seen in yellow fever. Even in livers without zonal necrosis, scattered foci of necrosis involving individual cells or groups of 2 or 3 cells were sometimes seen.

Gastro-intestinal tract The submucosal vessels were usually congested and small perivascular haemorrhages were common. No mucosal ulceration was seen. Spleen Microscopically there was little evidence of follicular hyperplasia even in those spleens in which the Malpighian bodies were unusually prominent. Although the

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

EccentricaUy placed area of paracentral necrosis of the liver in relation to a central vein (V). H and E. x 150.

688

DENGUE HAEMORRHAGIC FEVER: A PATHOLOGICAL S T U D y

FIE. 4. Liver, showing an intranuclear inclusion body (A). H and E × 500

FIG. 5. Splenic lymphoid foRicle showing a large pale germinal centre consisting of reticulum ceUs, and surrounded by only a narrow rim of lymphocytes. An area of fibrin deposition is seen in the upper right quadrant of the germinal centre. HandE. × 150.

appeared to be a focal hyalinization of arterioles, but this hyaline material was shown to consist of fibrin by Lendrum's method (Figure 6). This subintimal deposition of fibrin was also seen within the trabecular Vessels. The red pulp usually showed a marked hyperplasia of the reticular tissue, and in most cases large mononuclear cells were present within the sinuses. These cells were approximately 5 times the size of an erythrocyte, "with a hyperchromatic vesicular nucleus, a prominent nucleolus, and a finely vacuolated cytoplasm. The mitotic activity of these cells was rather high, and

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

centres of the follicles were often large, they showed little or no mitotic activity, being composed mainly of reticulum ceils with a smaller number of lymphocytes scattered throughout. In several there were small foci of hyaline material within the germinal centres which gave a positive staining reaction for fibrin with Lendrum's acid picroMallory method (Figure 5). A striking feature present in almost all cases was what

T. BURKE

689

binucleate forms were occasionally seen. These cells were also studied in splenic imprints, which allowed more cytological detail than is seen in histological preparations. In imprints their cytoplasm was blue in colour when stained with Giemsa's stain, and the cytoplasmic vacuoles were larger and more prominent (Figure 7). They probably represent the precursors of the "atypical mononuclears" seen in peripheral blood films.

FIG. 7. Spleen showing a large mononuclear cell with finely vacuolated cytoplasm, at the centre of field. H and E. × 500. Inset shows a similar type of cell in a splenic imprint. Giemsa. × 1,200.

Lymph nodes These showed similar changes to the spleen, the sinus tissue showing greater prominence and the follicles being inconspicuous.

Bone-marrow

J

Histological sections showed a normal marrow, and the megakaryocytes were present in adequate numbers and appeared morphologically normal.

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

FIG. 6. Splenic arteriole with an area of fibrin deposition in its wall. Lendrum's acid picro- Mallory. × 500.

690

DENGUE H A E M O R R H A G I C FEVER : A P A T H O L O G I C A L STUDY

Kidney Proteinaceous material was found within the glomerular capsular space adjacen~ to the capsular epithelium in several cases, together with the extravasation of a few erythrocytes. In half of the cases proteinaceous material was present within tubules, but not very extensive in amount. A common finding was degeneration of the proximal tubules, varying from severe cloudy swelling to hyaline droplet change. Small interstitial haemorrhages, as well as haemorrhage into the tubular lumina were frequently seen. Skin No inflammatory or vascular change, other than extravasation of erythrocytes, was seen even in those cases showing florid petechial haemorrhages.

Discussion An interesting feature in this series is the predominance of the females (3F:I M), unusual in infectious disease during childhood. This is unlikely to be due to any discrepancy in the necropsy rates among males and females, because in Singapore there is a slight excess of males in the necropsy population under the age of 25 years (MuiR, 1964). Though no sex difference was noted in the morbidity rate in Thailand (HALS~LU, 1966) a significantly higher mortality rate was observed among females during the epidemics of both 1958 and 1960 (SIDDTmCHAI, 1962). This predominance of females was also observed by PIYASATN (1961) in his series of 18 fatal cases, of which only 3 were males. Paracentral zonal necrosis of the liver was described in dengue haemorrhagic fever by B ~ R A V A T I (1962) who considered it to be the direct result of viral action. It was present in 43 of 63 cases and showed a similar histological picture to the lesions described in this communication. Councilman-like bodies were present, b u t intranuclear inclusions were not observed. AIr,AT et al. (1964) described paracentral zonal necrosis in one case but attributed this to haemorrhage and terminal hypotension; they considered that the lack of a cellular response in their case precluded a specific viral aetiology. This criterion alone, however, does not appear valid, because one of the characteristic features of the specific zonal necrosis which occurs in yellow fever is lack of an inflammatory reaction (KLOTZ and BELT, 1930a). The zonal necrosis in the liver bears a striking resemblance to that seen in yellow fever, and a similar histological pattern has been described in several other human and animal infections in which arboviruses have been incriminated. The massive zonal necrosis occurring in sheep in Rift Valley fever (DAUB~Y et al., 1931; FIre)LAY, 1932), and the liver lesions in mice infected with Guama virus (DE PAOLA, 1963) resemble the hepatic lesions present in this series. Irsg (1966) has described similar changes in Kyasanur Forest disease; and CHILD et al. (1967) noted focal hepatic necrosis in Bolivian haemorrhagic fever. It is difficult to explain why the liver should be involved only in certain cases if this were due to hypotension, since all the patients in this series died in a severe state of shock. It is tempting to postulate the existence of a hepatotropic strain of dengue virus, and the presence of intranuclear inclnsions certainly supports the possibility of 'a direct viral action.

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

Brain In all cases the capillary vessels were engorged, and extravasated erythrocytes could often be seen in the perivascular spaces. No evidence of vascular damage could be seen in the 4 patients with frank intracranial haemorrhage.

T. Btmm~

691

Summary The necropsy findings in 12 cases of dengue haemorrhagic fever are described. Interstitial pneumonitis was present in all 12, and protein-rich serous effusions were found in 9. In most patients deposits of fibrin could be demonstrated within blood vessel walls in the spleen, and less frequently in pancreas and lung. Although the pathogenesis of this fibrin deposition is unknown, it is believed to be a manifestation of increased vascular permeability. Paracentral areas of zonal necrosis were found in the liver in 4 cases; Councilman-like bodies were present in these 4, and intranuclear inclusion bodies were observed in 2 of them.

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

The interstitial pneumonitis is probably also the resuk of direct viral action, and has been consistently noted in most other reports. This pneumonitis has no specific features. The deposition of fibrin within vessel walls in lung, spleen, and pancreas has not previously been described in this disease although hyalinization of vessels has been reported by PIYAP,aTN (1961), and REXr~S(1966). In sections stained with haematoxylin and eosin the deposited fibrin is hyaline in appearance and without recourse to histochemical or empirical stains for fibrin might easily be disregarded as non-specific "hyalinization." The fibrin has presumably leaked through the endothelium from the lumen of the vessel, and is deposited in the form of fibrin within the vessel wall. Although the exact pathogenesis of the fibrin deposition in this disease cannot yet be explained, it is certainly a manifestation of increased vascular permeability and accounts for the proteinrich effusion and retroperitoneal oedema. It is interesting to note that KLOTZand BELT (1930b) found a similar "hyalinization" in the arterioles of the spleen in yellow fever. They described the arteriolar wall as "transformed into a pale homogeneous hyaline substance which spreads out as if it were in a plastic state." This description might have been applied to the vascular changes seen in the present series, and it would be interesting to know whether fibrin can also be demonstrated in the splenic vessels in yellow fever. The hyperplasia of reticulo-endothelial cells, including the large atypical mononuclear cells, represents a non-specific reaction to the viral infection. The large mononuclear cells in lymph nodes and spleen are probably the precursors of the "atypical mononudears" seen in peripheral blood smears, and the striking cytoplasmic vacuolation seen in both would tend to support that view. The major cause of death in all 12 patients appears to have been a severe degree of peripheral circulatory collapse resulting in irreversible shock. The phenomena leading to the irreversible shock which characteristically occurs in fatal dengue haemorrhagic fever have been studied by COHEN and H~a~ST~.~ (1966), who postulate that capillary vascular damage allows the escape of fluid and protein from the vascular compartment, resulting in hypoproteinaemia and hypovolaemia. The extensive protein-rich effusion which was found in 9 patients would have resulted in an average loss of 20 grammes of plasma protein. The interstitial pneumonitis present in all 12 patients would have further embarrassed the already precarious circulatory state by causing hypoxia and acidosis. Although the haemorrhagic manifestations present a rather florid clinical picture, it is unlikely that they contribute in any important way to the state of shock because the total volume of blood lost was not great. In those 4 patients in whom intracranial haemorrhage occurred, however, this must have been a significant factor in causing death.

692

D E N G U E H A E M O R R H A G I C FEVER : A P A T H O L O G I C A L S T U D Y

REFERENCES

Downloaded from http://trstmh.oxfordjournals.org/ at La Trobe University on October 1, 2016

AIKAT, B. K., KONOR, N. R. & BANERJEE,G. (1964). Indian.7. med. Res., 52, 660. BHAMARAPRAVATI,N. (1962). In: Symposium on Haemorrhagic Fever, 1961. Bangkok. p. 76. (SEATO Medical Research Monograph No. 2). CHAN, Y. C., (1968). Personal Communication. CH~W, A., LENG, G. A., YUEN, H., TErn, K. O., KIAT, L. Y., HoNe, L. C. & WELLS, R. (1961). Lancet, 1, 307. CHILD, P. L., MACKENZIE,R. B., VALVERDE,L. R. &-JOHNSON,K. M. (1967). Arch. Path., 83, 434. COr~N, S. N. & HALSTEAD,S. B. (1966)..7. Pediat., 68, 448. DAy,mY, R. & HUDSON, J. R. (1931)..7. Path. Bact., 34, 545. DE PAOLA, D. (1963). Anais Microbiol., 11, 187. FINDLAY, G. M. (1932). Trans. R. Soc. trop. Med. Hyg., 25, 229. GOLDSMITH, R. S., WONG,H. B., PAUL,F. M., CHAN,K. Y., LOH,T. F. & CHAN,Y. C. (1965). Lancet, 1, 333. HALSTEAD, S. B. (1966). Bull. Wld Hlth Org., :i5, 3. HAN~t, E. (1964). Singapore med..7., 5, 73. IYER, C. G. S. (1966). Int. Path., 7, 12. KLOTZ, O. & BELT, T. H. (1930 a). Am..7. Path., 6, 663. &- (1930 b). 1bid., 6, 655. MANSON-B.th'R, P. H. (1966). Manson's Tropical Diseases, 16th Ed., p. 304. London: Bailli6re, Tindall and Cassell. MUIR, C. S. (1964). Singapore reed..7., 5, 96. PIYaltaTN, P. (1961). Am..7. trop. Med. Hyg., 10, 767. RsYEs, V. M. (1966). Bull. PYld Hlth Org., .'15, 49. SIDDTHICI-IAI,P. (1962). In: Symposium on Haemorrhagic Fever, 1961. Bangkok. p. 20. (SEATO Medical Research Monograph No. 2).