Journal of Postgraduate Medicine Medknow Publications and Staff Society of Seth GS Medical College and KEM Hospital, Mumbai, India ISSN: 0022-3859 EISSN: 0972-2823 Vol. 49, Num. 3, 2003, pp. 218-221

Journal of Postgraduate Medicine, Vol. 49, No. 3, July-Sept, 2003, pp. 218-221

Prognostic Implications of White Cell Differential Count and White Cell Morphology in Malaria

Jadhav UM, Singhvi R, Shah R

Departments of Critical Care Medicine, MGM New Bombay Hospital, Vashi, New Mumbai
Correspondence Address: MGM New Bombay Hospital, Sector 3, Vashi. New Mumbai - 400703 udayj@bom8.vsnl.net.in

Code Number: jp03062

ABSTRACT

BACKGROUND: Malaria is of immense importance amongst the tropical diseases in India. There is a need to develop newer diagnostic aids and research is necessary to identify new prognostic markers for prediction of the course and complications. AIMS: To evaluate the white cell differential count and morphology in Plasmodium vivax and Plasmodium falciparum malaria and study their prognostic utility. SUBJECTS AND METHODS: Two hundred and sixty-four adult patients in the age range of 20 to 65 years presenting to the hospital over a period of 4 months with clinical features of malaria and a positive peripheral smear examination were studied. RESULTS: No statistically significant difference was noted in the white blood cell (WBC) count and neutrophil count in P.vivax versus P. falciparum malaria. Band cells were more frequently noted in P. falciparum malaria than in P.vivax malaria (p < 0.0001). Toxic granulation of the neutrophils was noted in 9.5% of the patients and exclusively in P. falciparum malaria. Presence of toxic granulation of the polymorphs in subjects with P. falciparum malaria was significantly associated with anaemia (p=0.019), jaundice, cerebral involvement, adult respiratory distress syndromes, renal dysfunction and death (p < 0.0001 for all these parameters). CONCLUSION: Band cells were seen in P. vivax and P. falciparum malaria, although in higher numbers in P. falciparum malaria. Toxic granulation of the neutrophils was noted only in the presence of P. falciparum malaria in this study and correlated with severity.

Keywords: P. falciparum malaria, P. vivax malaria, Band cells, Toxic granules

Malaria is one of most important of the parasitic diseases in humans with transmission in 103 countries affecting more than 1 billion people and causing between 1 and 3 million deaths each year.[1] Malaria afflicts today, as it has been for centuries, a heavy burden on tropical communities, and is a threat to non-endemic countries, and a danger to travellers.

Various prognostic biochemical markers have been studied in malaria and attempts have been made to correlate them with the severity of the infection and mortality. These include raised serum concentration of Tumour Necrosis Factor-alpha (TNF-alpha),[2] elevated plasma cytokines,[3] hyperlactataemia, metabolic acidosis, raised lactate/pyruvate ratio,[4] elevated serum procalcitonin concentration,[5] hypocalcaemia,[6] and changes in plasma amino acid pattern.[7] Leucocytosis has been shown to correlate with severe malaria,[8] however there are limited studies on white cell morphology in malaria. The present study evaluated the white cell morphology in P. vivax and P. falciparum malaria and assessed whether the morphology had any prognostic value.

SUBJECTS AND METHODS

A total of 264 adult patients consecutively presenting to the hospital outpatient services or hospitalised over a period of 4 months were analysed. The diagnosis of malaria was confirmed by a positive peripheral smear examination with conventional microscopy using Wright smear. Rings and the trophozoite phase of the parasite were used for the diagnosis of malaria by microscopy. Parasite densities were calculated in subjects with clinical features suggestive of severe malaria. Band neutrophil (stab form) were diagnosed on the basis of more condensed chromatin in the nucleus and uniform elongated shape with distinct lobes not deciphered. Toxic granules were identified as dark blue or purple cytoplasmic granules in the band/polymorph cells. Blood culture studies for pyogenic organisms were done in subjects with toxic granulation of the polymorph cells. Subjects with associated leptospirosis, dengue and any obvious bacterial infection were excluded from the study. P. falciparum antigen test (Paracheck) and the laboratory profile for multi-system involvement were performed and chest radiographs were taken based on clinical and therapeutic decisions. The laboratory profile included blood sugar values, serum bilirubin, aspartate transaminase and alanine transaminase, prothrombin time, blood urea nitrogen, serum creatinine, and platelet count. Statistical analysis was performed with SPSS Version 10. Continuous variables were analysed using the student's t-test and proportion with the chi-square test as appropriate. P values of less than 0.05 were considered significant. The study was approved by the ethics committee of the institution.

 RESULTS

The age range was 20-65 years with a mean of 38.6 years. Subjects included 140 men and 124 women.

P. falciparum was detected in 42.4% (n=112), P. vivax in 44.7% (n=118) and mixed infection in 12.9% (n=34). P. falciparum antigen test was performed in 68 subjects. P. P. falciparum antigen test was negative in all the 22 subjects suffering from P. vivax malaria in whom the test was performed to rule out an associated P. falciparum infection. The test was also negative in 5 subjects in whom the smear was positive for P. falciparum. [Figure-1] depicts the WBC count in both the types of malaria. Mean WBC count was 6120/cumm (SD 2107) in P.vivax malaria and 6010/cumm (SD 3760) in P. falciparum malaria (p=0.40). WBC count in the normal range of 4000 to 11000/cumm was noted in 81.1% of all cases. WBC count less than 4000/cumm and WBC count exceeding 11000/cumm was noted in 14% and 4.9% of the cases, respectively. WBC count less than 4000/cumm was more common in P.vivax than in P. falciparum malaria (15.2% vs. 10.7%), while a WBC count exceeding 11000/cumm was less common in P.vivax than in P. falciparum malaria (1.7% vs. 6.8%, Pearson chi-square 7.25, p = 0.12). A higher number of patients with mixed infection had a WBC count beyond the normal range. Neutrophil count in the range of 45 to 80% was noted in 79.3% of all patients. Neutrophil count of less than 45% and neutrophil count exceeding 80% was noted in 5.6% and 15.1% of the patients respectively as shown in [Figure-2]. Neutrophil count exceeding 80% was noted in 7.9% in subjects with P.vivax malaria, 19.4% in subjects with P. falciparum malaria and 22.7% in subjects with mixed infestations; but the difference was not statistically significant (Pearson chi-square 8.179, p = 0.08).

A total of 24.6% (n=65) smear-positive subjects with malaria had band cells on microscopic examination as shown in [Table-1]. Band cells were noted in 10.2% (n=12) subjects with P.vivax malaria, 40.2% (n=45) subjects with P. falciparum malaria and 23.5% (n=8) subjects with combined parasitaemia, which was statistically significant (p<0.0001). The band cell count varied from 1-20% in P.vivax malaria, 1-27% in P. falciparum malaria and 2-23% in mixed parasitic infestation.

Toxic granulations of the neutrophils were noted in 9.5% of the subjects (n=25), and exclusively in P. falciparum malaria. Blood culture for pyogenic organism was negative in all these 25 subjects. Parasite densities exceeded 1000 parasites/ml in the 25 subjects with clinical features suggestive of severe malaria and toxic granulations of the leucocytes. The mean random blood sugar levels at presentation were 78 mg/dl in subjects with toxic granulation as against 96 mg/dl in those without toxic granulation of the leucocytes. Mortality was 24% (n=6) in subjects with P. falciparum malaria and toxic granulation of the leucocytes. Presence of toxic granulation of the leucocytes in subjects with falciparum malaria was significantly associated with anaemia (p=0.019), jaundice (p < 0.0001), cerebral involvement (p < 0.0001), adult respiratory distress syndrome (p < 0.0001), renal dysfunction (p < 0.0001) and death (p < 0.0001) in comparision with subjects without toxic granulation, as noted in [Table-2]. No mortality was noted in subjects with P.vivax malaria or those without toxic granulation.

DISCUSSION

Toxic granules are dark blue or purple cytoplasmic granules in the etamyelocyte/band/polymorph cells. Toxic granules are azurophilic that retain basophilic staining reaction by lack of maturation or increased basophilia. The granules in the neutrophil represent packages of enzymes, which are involved in the killing of ingested microbes and the digestion of phagocytosed material. Based on differences in enzyme content, the granules can be classified into two different subtypes. The so-called primary lightly basophilic staining granules contain, for example, perioxidase plus acid hydrolytic enzymes, while secondary granules contain alkaline phosphatase and certain other enzymes. Peroxidase is a major neutrophil protein, which catalyses the reaction between the hydrogen peroxide generated in the phagocytosing neutrophil and chloride and iodide ions to chlorinate, or iodinate, respectively. This process is believed to contribute to the microbiocidal function of the neutrophils. Peroxidase has been shown to lessen resistance to mycotic and bacterial infection.[9] Tertiary granules also referred to as C particles are released at the leading front of neutrophils during chemotaxis and are thought to be the source of enzymes that promote the migration of cells through the basement membranes and tissue. Both toxic granulation and Dohle bodies are characteristic of benign or reactive processes.[10] Toxic granules indicate a generalised process of severe infection.

There is limited number of studies reporting about the presence or importance of band cells or toxic granulation in malaria. The present study should evoke interest amongst researchers spurring them to undertake larger studies on the subject. One of the probable explanations for the presence of toxic granulation of the neutrophils could be the presence of an element of sepsis, which accompanies severe falciparum malaria. Translocation of the bacterial flora from the gut may be related to mesenteric ischaemia produced by parasitised erythrocytes.[11] In fact, gram-negative bacteraemia with secondary sepsis has been observed in the algid form of P. falciparum malaria. In the absence of any prior studies addressing this issue specifically in malaria, we do not have any other plausible explanation.

REFERENCES

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