Thrombocytopenia progression in dengue cases during the 2010 outbreak in Indian capital metropolis

To the editor

Platelet therapy is a standard clinical practice for dengue (DEN) patients with severe thrombocytopenia [1]. However, during introductory screening, platelet count is not being done immediately and follows serological screening. During the 2010 spurt in the incidence of DEN in New Delhi [2], simultaneous screening for the dengue virus (DENV) non-structural protein NS1, IgM and IgG antibody and platelet enumeration was launched at a multi-disciplinary, private hospital. During different stages of the illness, evolution of thrombocytopenia was associated with tremendous variation in platelet counts and lower counts in NS1-positive cases.

During the interval between August and December 2010, blood samples from 1886 patients were examined at the Sant Parmanand Hospital, a 140-bed tertiary care center in Delhi, for the population in the national capital and adjoining townships. Individual blood samples were drawn into Ethylenediaminetetraacetic acid (EDTA) tubes and tested for DENV components employing the single-step immunochromatigrahic One step dengue NS1 Ag and IgG/IgM test, Dengue Duo, in accordance with the manufacturer's instructions (Standard Diagnostics, Inc., Ingbert, Germany). Platelet enumeration was in a 5- or 3-differential analyser, Coulter^RA^c T™ 5diff Autoloader (Beckman Coulter, Fullerton, CA, USA) or BC-3000Plus (Mindary, Shenzhen, China).

The patients’ ages ranged from 2 to 92 years (mean 31.2 years; standard deviation: 15.5; SEM: 0.36: 678). Of these patients, 1,208 were NS1-positive and NS 1-negative in different combinations. There were 942 cases that were negative for NS1, IgM, and IgG (triple-negatives). The platelet counts in triple-negatives showed wide variations (Table I) though the platelet counts in NS1-positives were lower than the NS1 negatives (mean 116.77 ± 2.6 × 10⁹/L and 167.24 × 10⁹/L, p < 0.0001). On average, the counts in 394 exclusive NS1-positive cases were highest, but lowest in 29 cases who were also IgG positive (139.8 ± 3.6 × 10⁹/L and 66.8 ± 9.04 × 10⁹/L (Table I), p < 0.0001).

Table I.  Platelet counts (10⁹/L) observed in 1886 suspected dengue cases during the 2010 outbreak at the Sant Parmanand Hospital, Delhi.

Antibody status	NS1 status	Cases	Mean ± standard error; Standard deviation	Minimum count	Maximum count	Cases with counts of <20 × 10^9/L	P value
IgM positive§	Positive*	145	100.01 ± 4.39; 52.9	11	2.74	1	0.004
	Negative	48	136.52 ± 9.71; 63.54	13	302	3
IgG positive	Positive*†	29	66.83 ± 9.04; 48.6	10	210	3	0.002
	Negative‡	96	114.32 ± 8.65; 84.7	9	364	12
IgM and IgG positive	Positive*	110	69.25 ± 4.3; 45.1	10	206	12	0.02
	Negative	122	86.07 ± 7.03; 77.66	7	339	15
IgM and IgG Negative	Positive*	394	139.87 ± 3.66; 72.6	10	481	4	<0.0001
	Negative**	942	184.71 ± 29.4; 90.25	5	693	12

*Earlier phase of illness.

†Secondary infection.

‡Indeterminate status.

§Earlier phase of primary infection.

**May have been negative for dengue virus infection.

In 942 apparent triple-negatives ‘DEN-negative’ cases patient platelets were highest. They declined in 394 exclusive NS1-positive cases with primary infection. There was an added decline among the 145 cases after they had acquired IgM antibodies. In 48 cases who were NS1-negative but IgM-positive, counts were higher than the above 145 cases. Nevertheless, the number of patients with counts <20 × 10⁹/L were higher; 1 and 3, respectively; Fisher's P = 0.0482. In patients with a secondary DEN infection, still lower counts were recorded. There was a marked decline in 29 cases that were positive for NS1 and IgG; the counts in the 110 cases that were triple-positive for NS1, IgM, and IgG were also very low (Table I).

The platelet count 12 triple-negative DENV cases were <20 × 10⁹/l (Table I). It would not be possible to rule out DENV infection in the above cases without testing for viral replication in cell culture and/or molecular investigations, immunofluorescence, or immunohistochemistry [3]. This would help to differentiate between patients with dengue-associated thrombocytopenia and those with severe bleeding episodes associated with trauma, invasive intensive care procedures, or emergency surgery. Moreover, in patients with platelet count <20 × 10⁹/l, decreased megakaryocytic production, splenic sequestration, non-immune or immune destruction of platelets would have to be examined.

The search for circulating dengue virus non-structural glycoprotein NS1 was an asset since it indicates a viremic phase of illness. NS1 is detectable from pre-fever phase to the fifth day of illness. Dengue IgM would be detected only after 5–6 days in primary infection while in secondary infection IgG would be the first antibody to appear.

There has been uncertainty regarding the clinical utility of platelet function assessment though a wide variety of available platelet function tests, including flow cytometry, which use different methodologies, have been offered [4]. Such test formats would be desirable in DENV patients at different stages of the illness. An explanation of the observed wide variation in platelet counts in the present report is required (Table I). Such tests are not being conducted by us at the moment.

Acknowledgements

The technical assistance of Mr. Govind Singh and Ms Geeta Kaushik is acknowledged.

Declaration of interest: The author's report no conflicts of interest.