The association of intraleucocytic malaria pigment and disease severity in Papua New Guinean children with severe P. falciparum malaria

ABSTRACT

Background

Plasmodium falciparum pigment-containing leucocytes (PCLs) are associated with adverse clinical manifestations of severe malaria in African children. However, limited data exist on the association of PCLs in settings outside of Africa.

Methods

Thin films on peripheral blood slides obtained from children ages 6 months–10 y with severe malaria were examined for PCLs. The intraleucocytic pigment data were correlated with clinical phenotypic data such as severe anaemia, metabolic acidosis and coma to determine the association of PCLs with clinical phenotypes of severe malaria and outcome.

Results

Of the 169 children with severe P. falciparum malaria confirmed by microscopy, 76% (129/169) had PCLs. Compared with children without PCLs, the presence (adjusted odds ratio [AOR] 3.2 [95% confidence interval {CI} 1.5 to 6.9], p≤0.01) and quantity (AOR 1.0 [95% CI 1.0 to 1.1], p=0.04) of pigment-containing monocytes (PCMs) was significantly associated with severe anaemia, while the quantity of both PCMs (AOR 1.0 [95% CI 1.0 to 1.1], p≤0.01) and pigment-containing neutrophils (AOR 1.0 [95% CI 1.0 to 1.1], p=0.01) was significantly associated with metabolic acidosis. Plasma P. falciparum histidine-rich protein-2 level negatively correlated with the platelet count (r=−0.5, p≤0.01) in patients with PCLs and no PCLs.

Conclusions

In Papua New Guinean children with severe P. falciparum malaria, the presence and quantity of PCLs are predictors of disease severity, severe anaemia and metabolic acidosis.

Introduction

In malaria-endemic countries, severe malaria remains a significant cause of morbidity and mortality, particularly in young children. In patients with severe Plasmodium falciparum malaria, predominant clinical features include severe anaemia, metabolic acidosis and coma.¹ The prevalence of these clinical features may differ across geo-epidemiological settings.^2–4 Most complications associated with severe malaria are often associated with the sequestration of mature blood-stage forms of P. falciparum in the microvasculature of vital organs.^5,6

Since sequestered parasite burden cannot be diagnosed by microscopy, which may or may not reflect peripheral parasitaemia,^5,6 other low-cost methods of diagnosing total parasite biomass and its association with clinical outcome and prognosis need evaluation. Mature forms of the P. falciparum parasite release free malaria pigments called haemozoin into the host circulation during schizogony. The free haemozoin and/or infected erythrocytes with late-stage parasites containing pigments are ingested through phagocytosis by host neutrophils and monocytes. These white blood cells are referred to as pigment-containing leucocytes (PCLs).^7–12 PCLs can be easily visualised and quantified as part of standard malaria microscopy. African studies have used this technique to investigate the association of PCLs with adverse clinical manifestations of severe P. falciparum malaria in children.^7–9,11,12

However, there have been no such studies from Papua New Guinea (PNG), a country in the Pacific that currently accounts for 86% of the malaria burden in the Western Pacific region.¹³ We investigated the association of PCLs with adverse clinical manifestations and outcomes in young PNG children hospitalised with severe P. falciparum malaria.

Methods

Study setting and design

We re-examined malaria blood slides collected as part of a prospective observational study conducted between October 2006 and December 2009 at Modilon Hospital in Madang Province on the northern coast of mainland PNG.² Children 6 months–10 y of age were screened for eligibility and details of recent treatment with antimalarials and/or antibiotics and past medical history were documented prior to enrolment and administration of parenteral antimalarial treatment. Inpatients on admission were treated with 3.2 mg/kg/day intramuscular artemether and 1.6 mg/kg/day until oral antimalarial could be tolerated.² In the present study, only those with peripheral blood slides that were positive for P. falciparum mono-infections by light microscopy at enrolment were included. The children were followed until discharge or death, as previously described.¹⁴ Written informed consent was obtained from a parent or guardian prior to enrolment.

Study participants

Patients with P. falciparum mono-infections who had matched peripheral blood slides, full blood counts and clinical data available were eligible for inclusion in this study. In a subset of children, plasma P. falciparum histidine-rich protein-2 (PfHRP2) concentrations were measured, as previously reported.⁶ Eligible participants were those who fulfilled any of the following criteria in accordance with the World Health Organization (WHO) criteria for severe malaria:¹ impaired consciousness/coma (Blantyre Coma Score [BCS] <5), prostration (inability to sit/stand unassisted), multiple seizures (two or more episodes), hyperlactataemia (blood lactate >5 mmol/L), severe anaemia (haemoglobin level <70 g/L),¹⁵ dark urine, hypoglycaemia (blood glucose <2.2 mmol/L), jaundice, respiratory distress, persistent vomiting, abnormal bleeding or signs of shock. In addition, a parasitaemia threshold of ≥1000 parasites/µl, a negative blood culture and/or cerebrospinal fluid culture were used to reduce the likelihood of including patients with non-malarial severe illnesses.² As per the recent WHO definitions, anaemia was defined as haemoglobin concentration <110 g/L and categorised as mild (90–109 g/L), moderate (70–89 g/L) and severe (<70 g/L).¹⁵ Malnutrition was defined as mid-upper arm circumference ≤12.5 cm and fever was defined as an axillary temperature >37.5°C. Spleen size was determined using Hackett's grading system for palpable splenomegaly.¹⁶ A BCS ≤2 was considered coma and a BCS <5 as impaired consciousness.

Clinical and laboratory investigations

On admission, demographic and clinical data were recorded on prepared case report forms. Venous blood samples were collected for malaria microscopy and rapid diagnostic testing (ICT Diagnostics, Brookvale, NSW, Australia). Other onsite investigations included whole blood glucose (Hemocue, Angelholm, Sweden), lactate (Lactate Pro, Arkray, Japan), full blood count (Coulter Ac'T diff, Beckman Coulter, Brea, CA, USA),² liver function tests, renal indices¹⁷ and plasma PfHRP2 levels.⁶

Peripheral thick and thin blood films collected before antimalarial administration were Giemsa-stained and examined for malaria parasite speciation and density quantification using established methods.¹⁸

Evaluation of PCLs

The Giemsa-stained blood slides were examined for PCLs using the thin film. Pigments were detected on thin films by counting a total of 100 neutrophils and a total of 30 monocytes, then from these totals the proportion of cells containing pigment was determined.⁸ The following formulas were used for pigment quantification: total pigmented neutrophils/mm³=(number of pigmented neutrophils/100)×(absolute white blood cells×percent of neutrophils). Similarly, total pigmented monocytes/mm³=(number of pigmented monocytes/30)×(absolute white blood cells×percent of monocytes). Microscopists were blinded to patients’ clinical presentations and outcomes. For quality control, 10% of slides were read by an independent microscopist.

Statistical analysis

Comparison of proportions was performed using the Fisher's exact test or χ² test and comparisons of continuous variables using Student's t-test. Logistic regression analysis was performed to assess adverse clinical manifestations and outcomes associated with PCLs. Multivariate analysis was with a choice of independent variables based on biological plausibility and/or p<0.10 on bivariate logistic analysis. Spearman's rank correlation coefficient was used to compare for relationship between log plasma PfHRP2 concentration and platelet counts between those with and without PCLs. Sensitivity, specificity, positive predictive value and negative predictive value were calculated to determine the predictive value of PCLs compared with reference tests. All tests were two-tailed and the confidence level was set at 95% with p<0.05 used throughout. All statistical analyses were performed using Stata 11 (StataCorp, College Station, TX, USA).

Results

Of the 169 children with P. falciparum malaria, 129 (76%) had PCLs and 40 (24%) did not (Table 1). The mean (±standard deviation [SD]) age of children with PCLs was 48±29 months and 52% (88/169) were males. Compared with children without PCLs, a higher proportion of children with PCLs had fever, headache, vomiting, convulsions, severe anaemia, a palpable spleen, hyperlactatemia, prostration and were malnourished, but only hyperlactatemia and severe anaemia were statistically significant clinical manifestations (Table 1). Of the subset of patients with plasma PfHRP2 concentrations, children with PCLs (mean PfHPR2 1491.9±2238.8 ng/ml) had, on average, levels 981 ng/ml higher than those who did not have any PCLs (mean PfHRP2 511.0±1190.4 ng/ml) (Table 1). A quarter of children with P. falciparum malaria had only pigment-containing monocytes (PCMs) [25% (42/169)], 5% (9/169) had only pigment-containing neutrophils (PCNs) and 46% (78/169) had both PCMs and PCNs (Figure 1). In total, there were 120 children with PCMs and 87 with PCNs (Figure 1). Children with intraleucocytic Plasmodium vivax pigment were not included in this analysis due to the small sample size.

Table 1.

Characteristics of children with and without intraleucocytic P. falciparum pigment

		PCLs
Characteristicsa	Values, n/N	Pigmented cell	No pigmented cell	p-Value
Age (months), mean±SD	48.4±28.8	48.4±28.8	48.3±29.3	>0.99
Weight (kg), mean±SD	12.2±5.3	12.1±5.3	12.7±5.5	0.51
Male, n (%)	88/169	62 (48.1)	26 (65.0)	0.07
Fever, n (%)	156/169	121 (93.8)	35 (87.5)	0.20
Headache, n (%)	40/169	32 (24.8)	8 (20.0)	0.67
Vomiting, n (%)	70/169	54 (41.9)	16 (40.0)	0.86
Convulsion, n (%)	90/169	68 (52.7)	22 (55.0)	0.86
Pulse (per min), mean±SD	131.6±23.9	132.3±21.7	129.2±30.2	0.47
Respiratory rate (per min), mean±SD	35.5±11.0	35.6±11.3	35.0±9.9	0.75
Oxygen saturation, n (%)	10/169	9 (7.0)	1 (2.5)	0.46
Malnutrition, n (%)	15/169	13 (10.1)	2 (5.0)	0.53
Prostration, n (%)	146/169	112 (86.6)	34 (85.0)	0.79
Hypoglycaemia, n (%)	1/169	0 (0.0)	1 (2.5)	0.24
Hyperlactataemia, n (%)	36/169	32 (24.8)	4 (10.0)	0.05
Palpable spleen, n (%)	112/169	85 (65.9)	27 (67.5)	>0.99
Mild anaemia, n (%)	17/169	8 (6.2)	9 (22.5)	0.01
Moderate anaemia, n (%)	72/169	53 (41.1)	19 (47.5)	0.58
Severe anaemia, n (%)	76/169	64 (49.6)	12 (30.0)	0.03
Impaired consciousness, n (%)	44/169	30 (23.3)	14 (35.0)	0.33
Coma, n (%)	26/169	22 (17.1)	4 (10.0)	0.33
PfHRP2,b mean±SD	1292.9±2238.8	1491.8±2381.1	511.0±1190.4	0.03
Length of hospital stay (days), mean±SD	3.0±4.2	2.9±3.8	3.3±5.3	0.56
Death, n (%)	4/169	2 (1.6)	2 (5.0)	0.24

^aFever: axillary temperature >37.5°C; malnutrition: mid-upper arm circumference ≤12.5 cm; hypoglycaemia: blood glucose <2.2 mmol/L; hyperlactataemia: blood lactate >5 mmol/L; palpable spleen: Hackett's grading; anaemia: haemoglobin level <110 g/L (mild, 90–109 g/L; moderate, 70–89 g/L; severe, <70 g/L); impaired consciousness: BCS <5; coma: BCS ≤2; PfHRP2 (ng/ml).

^bSubset of children that have plasma PfHRP2 results.

Figure 1.

Flowchart showing the proportion of PCMs and/or PCNs in children with severe malaria according to Plasmodium species.

In patients with PCLs, the prevalence of severe anaemia, hyperlactatemia and coma differs, including the overlaps between these clinical syndromes. In children with PCLs, 50% (64/129), 25% (32/129) and 17% (22/129) presented with severe anaemia, hyperlactatemia and coma, respectively. There was no difference observed in the prevalence of severe anaemia, hyperlactatemia and coma when PCLs were further stratified as PCMs and PCNs. Of those with PCMs, 53% (63/120), 25% (30/120) and 18% (21/120) presented with severe anaemia, hyperlactatemia and coma, respectively, while in those with PCNs, 44% (38/87), 26% (23/87) and 18% (16/87) presented with severe anaemia, hyperlactatemia and coma, respectively. Three of the four children who died had severe anaemia and two of them had both PCMs and PCNs. In 143 children with P. falciparum malaria, the plasma PfHRP2 concentration was significantly negatively correlated with the platelet count in those with PCLs and those without PCLs (r=−0.5, p<0.01; Figure 2).

Figure 2.

Scatter plot of plasma PfHRP2 concentrations vs platelet counts in 143 children with severe malaria who have PCLs or no PCLs. The blue circles represent children with PCLs and the orange circles those without PCLs.

A significantly higher proportion of children with severe anaemia had PCMs compared with those without (83% [63/76] vs 17% [13/76], p≤0.01). When quantifying PCLs, children with severe anaemia were more likely to have a high quantity of PCMs compared with those who were not anaemic (9.6/mm³ vs 3.5/mm³; p≤0.01). In univariate analysis, the presence (unadjusted odds ratio [UOR] 3.1 [95% confidence interval {CI} 1.5 to 6.3], p≤0.01) and quantity (UOR 1.0 [95% CI 1.0 to 1.1], p=0.03) of PCMs was associated with severe anaemia compared with those without. In multivariate analysis, the presence of PCMs (adjusted odds ratio [AOR] 3.2 [95% CI 1.5 to 6.9], p≤0.01) and the quantity of PCMs (AOR 1.0 [95% CI 1.0 to 1.1], p=0.04) remained a significant predictor of severe anaemia (Table 2). When leucocytes were further stratified according to the respective white cells with ingested malaria pigments, patients with only PCMs had fourfold increased odds of having severe anaemia (AOR 4.1 [95% CI 1.6 to 11.0], p≤0.01) (Table 2).

Table 2.

Association of intraleucocytic P. falciparum pigment between adverse clinical manifestations and outcome in children with severe malaria, adjusted for confounding variables

		Adverse clinical manifestations						Clinical outcome
Pigmented leucocytes		Hyperlactataemia		Severe anaemia		Coma		Deatha
Pigment present	n	AOR (95% CI)b	p-Value	AOR (95% CI)b	p-Value	AOR (95% CI)b	p-Value	AOR (95% CI)b	p-Value
PCMsc	120	2.2 (0.8 to 5.9)	0.11	3.2 (1.5 to 6.9)	≤0.01	2.3 (0.8 to 6.9)	0.14	0.4 (0.0 to 3.0)	0.35
PCNsd	87	1.9 (0.9 to 4.2)	0.10	0.9 (0.5 to 1.7)	0.72	1.8 (0.7 to 4.4)	0.22	1.0 (0.1 to 7.6)	>0.99
PCMs only	42	2.1 (0.6 to 7.7)	0.28	4.1 (1.6 to 11.0)	≤0.01	1.7 (0.4 to 7.3)	0.45	–	–
PCNs only	9	2.2 (0.3 to 15.3)	0.42	0.3 (0.0 to 2.6)	0.27	1.0 (0.1 to 11.4)	>0.99	–	–
PCMs plus PCNs	78	3.0 (0.9 to 9.8)	0.06	2.2 (0.9 to 5.1)	0.07	2.6 (0.7 to 9.0)	0.13	0.5 (0.1 to 4.0)	0.52
No pigments	40	Ref		Ref		Ref		Ref
Pigment quantity
PCMsc	120	1.0 (1.0 to 1.1)	≤0.01	1.0 (1.0 to 1.1)	0.04	1.0 (0.9 to 1.0)	0.92	1.0 (0.9 to 1.0)	0.48
PCNsd	87	1.0 (1.0 to 1.1)	0.01	1.0 (0.9 to 1.0)	0.92	1.0 (0.9 to 1.0)	0.82	1.0 (0.9 to 1.0)	0.57

Data are AOR (95% CI) for logistic regression model.

Ref: reference group.

^aTwo of the patients who died had both PCMs and PCNs, while the other two have no pigmented leucocytes.

^bMultivariate analysis variables include pigmented leucocytes, gender, fever, headache, vomiting, convulsion, malnutrition, prostration and splenomegaly.

^cExcludes nine patients with only PCNs.

^dExcludes 42 patients with only PCMs.

Similarly, a high proportion of children with hyperlactatemia had PCMs compared with those without (83% [30/36] vs 17% [6/36], p≤0.01). When examining the quantity of PCLs, children with hyperlactatemia were more likely to have a high quantity of PCMs compared with those without (19.9/mm³ versus 4.9/mm³; p≤0.01). In addition, children with hyperlactatemia were more likely to have a high number of PCNs compared with those without (20.2/mm³ vs 0.0/mm³; p≤0.01). In univariate analysis, the number of PCMs (UOR 1.0 [95% CI 1.0 to 1.1], p=0.01) and PCNs (UOR 1.0 [95% CI 1.0 to 1.1], p=0.01) was significantly associated with hyperlactatemia. In multivariate analysis, the number of PCMs (AOR 1.0 [95% CI 1.0 to 1.1], p≤0.01) and PCNs (AOR 1.0 [95% CI 1.0 to 1.1], p=0.01) remained significantly associated with hyperlactatemia (Table 2).

A significantly higher proportion of children who presented with coma on admission had PCMs compared to those without (81% [21/26] vs 19% [5/26]; p=0.01). Although these children had increased odds of having coma compared with those without PCMs (AOR 2.3 [95% CI 0.8–6.9], p=0.14), this association was not statistically significant (Table 2).

Intraleucocytic malaria pigment had a relatively good sensitivity of 89%, 84%, 85% and 50% but a poor specificity of 27%, 30%, 25% and 23% in predicting hyperlactatemia, severe anaemia, coma and death, respectively (Table 3).

Table 3.

Diagnostic accuracy of intraleucocytic P. falciparum pigment in detecting adverse clinical manifestations and outcome of severe malaria in children

	Intraleucocytic P. falciparum pigment		% (95% CI)
Clinical features	Positive (n=129), n	Negative (n=40), n	Sensitivity	Specificity	PPV	NPV
Hyperlactataemia
Positive (n=36)	32	4	88.9 (73.9 to 96.9)	27.1 (19.7 to 35.9)	24.8 (17.6 to 33.2)	90.0 (76.3 to 97.2)
Negative (n=133)	97	36
Severe anaemia
Positive (n=76)	64	12	84.2 (74.0 to 91.6)	30.1 (21.0 to 40.5)	49.6 (40.7 to 58.5)	70.0 (53.5 to 83.4)
Negative (n=93)	65	28
Coma
Positive (n=26)	22	4	84.6 (65.1 to 95.6)	25.2 (18.3 to 33.1)	17.1 (11.0 to 24.7)	90.0 (76.3 to 97.2)
Negative (n=143)	107	36
Death
Positive (n=4)	2	2	50.0 (6.8 to 93.2)	23.0 (16.8 to 30.2)	1.6 (0.2 to 5.5)	95.0 (83.1 to 99.4)
Negative (n=165)	127	38

NPV: negative predictive value; PPV: positive predictive value.

Discussion

This study shows that in PNG children with severe P. falciparum malaria who had PCLs identified on microscopy, the clinical utility of PCLs varied according to the types of pigmented leucocytes, with the presence and quantity of PCMs being significantly associated with severe anaemia while only the quantity of both PCMs and PCNs was significantly associated with hyperlactatemia. The plasma PfHRP2 concentration was significantly negatively correlated with platelet counts in those with PCLs and no PCLs. However, the specificity and positive predictive value of PCLs in predicting adverse clinical manifestations of severe malaria and outcome were low.

More than 80% of children with severe malarial anaemia in this study had PCMs, and this is consistent with findings reported in African studies.^7–9,12 However, in our study, the presence of both PCMs and PCNs was higher in children with severe anaemia combined with hyperlactatemia, whereas in African children, only the presence of PCMs was associated with anaemia.^7–9,12 Since the half-life of monocytes is 8–9 d, with a longer clearance rate,^8,19 in some children this may reflect the chronology of the infection. In addition to the fact that severe anaemia is a common clinical manifestation of P. falciparum malaria,^2,20 in our setting, non-malarial causes of severe anaemia in children are also common and the causes can be multifactorial, including malnutrition and dietary insufficiencies such as vitamin A deficiency and iron deficiency, helminthic infestations as well as viral infections such as parvovirus B19.²¹

Furthermore, the quantity of both PCMs and PCNs was significantly associated with hyperlactatemia in our children with severe malaria. More than 80% and 60% of children with hyperlactatemia had PCMs and PCNs, respectively. The high numbers of both PCMs and PCNs is likely a reflection of the infected erythrocytes rupturing during schizogony and phagocytosis of parasitised red cells. This process contributes to the total parasite biomass often documented in P. falciparum sequestration studies.^5–7 Among other underlying pathophysiology of metabolic acidosis or hyperlactatemia, the main causes of this clinical phenotype in severe malaria are parasite sequestration and microvascular obstruction,²² which increases plasma bicarbonate and/or lactate normally through parasite metabolism and skeletal muscle during convulsions,²⁰ which was common in our study (Table 1). More than half of the children with severe malaria in this study had convulsions and this may have contributed to the increased plasma bicarbonate and/or lactate in those with metabolic acidosis or hyperlactatemia, respectively.

In support of these findings, patients with PCLs have high plasma PfHRP2 concentrations, which was negatively associated with platelets. Both the P. falciparum malarial pigment and HRP2 are produced in parallel normally by mature blood-stage parasites, and these two parameters reflect the sequestered parasite biomass. The presence of PCLs can be a prognostic biomarker for total sequestered parasite biomass often associated with adverse clinical manifestations in children with severe malaria.^5,6,11 In addition, the combined measurement of platelet count and plasma PfHRP2 showing an inverse relationship substantially improved the specificity of the diagnosis of severe P. falciparum malaria in African children with severe illness.²³ The negative relationship between PfHRP2 concentrations and platelet counts observed in children with PCLs in our study supports these findings and validates PCLs as a simple diagnostic marker for severe P. falciparum malaria. Our study shows that PCLs can be considered an ideal predictor for sequestered parasite biomass, including disease severity, and can be used as a simple laboratory test that can provide diagnostic information.

Patients in our study had a very low rates of bacterial co-infections² compared with African children with severe malaria where bacterial co-infections were associated with leucocytosis,²⁴ which may have influenced the recruitment of phagocytic cells. Despite the differences in factors influencing PCLs and peripheral parasitaemia, the results in this study confirm the significant association of P. falciparum pigment with adverse clinical features of severe malaria in PNG children.

This study had several limitations. First, even though P. vivax is common in our setting,² we were unable to include children with severe P. vivax malaria in this analysis because of low numbers. Second, in anaemic patients, the blood smear on the slide is often too thin, and this may have influenced the presence or quantity of PCLs. Third, the mortality rate due to severe malaria is low in this setting and it was not possible to determine whether the presence of PCLs could predict mortality, although pooled data from similar studies in Africa and Asia have shown PCNs to be predictive of in-hospital mortality.¹¹ In the future, longitudinal studies with greater numbers will be required to determine the association of PCLs with mortality in this setting.

In conclusion, the present study shows that microscopic diagnosis of a high proportion and quantity of PCLs on thin peripheral blood smears in patients with severe P. falciparum malaria can be used as a predictor of disease severity, severe anaemia and hyperlactatemia in PNG children.

Authors’ contributions

ML, LJR and EL conceived the study. ML, LM, JW and SA enrolled and managed patients. EL and LL performed microscopy. EL and ML analysed the data. EL wrote the first draft of the manuscript. All authors read, edited and approved the final version of the manuscript.

Funding

This work was supported by the Asia-Pacific International Centers of Excellence for Malaria Research (ICEMR) program, funded by the National Institutes of Health, Bethesda, MD, USA (grant U19 AI129392-01) and the Southwest Pacific ICEMR program funded by the National Institutes of Health (grant U19 AI089686). The authors also acknowledge funding support from the Malaria Genomic Epidemiology Network Consortium. EL was supported by a PNG Institute of Medical Research Training Office Masters Scholarship. LJR was supported by an Australian National Health and Medical Research Council Research Fellowship (GNT1161627).

Competing interests

None declared.

Data availability statement

Data can be made available as per the requirements of the journal.

Ethical approval

The research was approved by the PNG Institute of Medical Research Institutional Review Board (IRB 1607) and the Medical Research Advisory Committee of the PNG Health Department (MRAC 16.23).