Plasma parasitemia as assessed by quantitative PCR in relation to clinical disease severity in African adults with falciparum malaria with and without HIV co-infection.

PURPOSE: When considering malaria disease severity, estimation of parasitemia in erythrocytes is important, but sometimes misleading, since the infected erythrocytes may be sequestered in peripheral capillaries. In African children and Asian adults with falciparum malaria, parasitemia as assessed by quantitative PCR (qPCR) in plasma seems to be a valuable indicator of disease severity, but data on African adults as well as the impact of co-infection with HIV is lacking. METHODS: In 131 patients with falciparum malaria in a public tertiary teaching hospital in Mozambique, plasma malaria parasitemia as assessed by qPCR, compared to qualitative malaria PCR in blood cell fraction, was related to malaria disease severity and HIV co-infection. RESULTS: Of the 131 patients with falciparum malaria, based on positive qualitative PCR in the blood cell fraction, 93 patients (72%) had positive malaria qPCR in plasma. Patients with severe malaria as defined by the WHO criteria had higher malaria quantitative plasma parasitemia (median 143 genomes/µL) compared to those with uncomplicated malaria (median 55 genomes/µL, p = 0.037) in univariate analysis, but this difference was attenuated after adjusting for age, sex and HIV co-infection (p = 0.055). A quarter of the patients with severe malaria had negative qPCR in plasma. CONCLUSIONS: This study of adult African in-patients with falciparum malaria with and without HIV co-infection, neither confirms nor rejects previous studies of malaria qPCR in plasma as an indicator of disease severity in patients with falciparum malaria. There is a need for further and larger studies to clarify if parasitemia as assessed malaria qPCR in plasma could be a surrogate marker of disease severity in falciparum malaria.

Soluble markers of neutrophil, T-cell and monocyte activation are associated with disease severity and parasitemia in falciparum malaria.

BACKGROUND: The immune response during P. falciparum infection is a two-edged sword, involving dysregulation of the inflammatory responses with several types of immune cells participating. Here we examined T-cell, monocyte/macrophage and neutrophil activation during P. falciparum infection by using soluble activation markers for these leukocyte subsets. METHODS: In a prospective cross-sectional study clinical data and blood samples were collected from adults in Mozambique with P. falciparum infection, with (n = 70) and without (n = 61) co-infection with HIV-1, as well as HIV-infected patients with similar symptoms but without malaria (n = 58) and healthy controls (n = 52). Soluble (s)CD25, sCD14, sCD163 and myeloperoxidase (MPO) as markers for T-cell, monocyte/macrophage and neutrophil activation, respectively as well as CX3CL1, granzyme B and TIM-3 as markers of T-cell subsets and T-cell exhaustion, were analyzed. RESULTS: All patient groups had raised levels of activation markers compared with healthy controls. Levels of sCD25 and MPO increased gradually from patient with HIV only to patient with malaria only, with the highest levels in the HIV/malaria group. In the malaria group as a whole, MPO, sCD14 and in particular sCD25 were correlated with disease severity. sCD163, sCD25 and in particular MPO correlated with the degree of parasitemia as assessed by qPCR. Patients with falciparum malaria also had signs of T-cell subset activation (i.e. increased granzyme B and CX3CL1) and T-cell exhaustion as assessed by high levels of TIM-3 particularly in patients co-infected with HIV. CONCLUSION: Our data support a marked immune activation in falciparum malaria involving all major leukocyte subsets with particular enhanced activation of neutrophils and T-cells in patients co-infected with HIV. Our findings also support a link between immune activation and immune exhaustion during falciparum malaria, particularly in relation to T-cell responses in patients co-infected with HIV.

Challenges in diagnosing paediatric malaria in Dar es Salaam, Tanzania.

BACKGROUND: Malaria is a major cause of paediatric morbidity and mortality. As no clinical features clearly differentiate malaria from other febrile illnesses, and malaria diagnosis is challenged by often lacking laboratory equipment and expertise, overdiagnosis and overtreatment is common. METHODS: Children admitted with fever at the general paediatric wards at Muhimbili National Hospital (MNH), Dar es Salaam, Tanzania from January to June 2009 were recruited consecutively and prospectively. Demographic and clinical features were registered. Routine thick blood smear microscopy at MNH was compared to results of subsequent thin blood smear microscopy, and rapid diagnostics tests (RDTs). Genus-specific PCR of Plasmodium mitochondrial DNA was performed on DNA extracted from whole blood and species-specific PCR was done on positive samples. RESULTS: Among 304 included children, 62.6% had received anti-malarials during the last four weeks prior to admission and 65.1% during the hospital stay. Routine thick blood smears, research blood smears, PCR and RDT detected malaria in 13.2%, 6.6%, 25.0% and 13.5%, respectively. Positive routine microscopy was confirmed in only 43% (17/40), 45% (18/40) and 53% (21/40), by research microscopy, RDTs and PCR, respectively. Eighteen percent (56/304) had positive PCR but negative research microscopy. Reported low parasitaemia on routine microscopy was associated with negative research blood slide and PCR. RDT-positive cases were associated with signs of severe malaria. Palmar pallor, low haemoglobin and low platelet count were significantly associated with positive PCR, research microscopy and RDT. CONCLUSIONS: The true morbidity attributable to malaria in the study population remains uncertain due to the discrepancies in results among the diagnostic methods. The current routine microscopy appears to result in overdiagnosis of malaria and, consequently, overuse of anti-malarials. Conversely, children with a false positive malaria diagnosis may die because they do not receive treatment for the true cause of their illness. RDTs appear to have the potential to improve routine diagnostics, but the clinical implication of the many RDT-negative, PCR-positive samples needs to be elucidated.

A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen, Norway.

BACKGROUND: Nested PCR is a commonly used technique in diagnosis of malaria owing to its high sensitivity and specificity. However, it is time-consuming, open to considerable risk of contamination and has low cost-efficiency. Using amplification targets presented in multiple copies, such as rRNA 18S, or mitochondrial targets with an even higher copy number, might increase sensitivity. METHODS: The sensitivity and specificity of two newly designed Plasmodium genus-specific single-round amplification PCR programmes, based on previously published primers targeting 18S and mitochondrial genome, were compared with a widely used nested 18S PCR. Analyses of dilution series from Plasmodium falciparum reference material were performed, as well as retrospective analyses of 135 blood samples, evaluated by routine microscopy, from 132 fever patients with potential imported malaria. Sequencing of the 220 bp mitochondrial PCR products was performed. RESULTS: At the threshold dilution 0.5 parasites/µl, the sensitivity of the mitochondrial PCR was 97% (29/30 parallels), that of the single-round 18S PCR 93% and the reference nested 18S PCR 87%. All three assays detected as low as 0.05 p/µl, though not consistently. In the patient cohort, malaria was diagnosed in 21% (28/135) samples, defined as positive by at least two methods. Both single-round amplification assays identified all malaria positives diagnosed by nested PCR that had sensitivity of 96% (27/28). The mitochondrial PCR detected one additional sample, also positive by microscopy, and was the only method with 100% sensitivity (28/28). The sensitivity and specificity of the mitochondrial PCR were statistically non-inferior to that of the reference nested PCR. Microscopy missed two infections detected by all PCR assays. Sequencing of the genus-specific mitochondrial PCR products revealed different single nucleotide polymorphisms which allowed species identification of the 28 sequences with following distribution; 20 P. falciparum, six Plasmodium vivax, one Plasmodium ovale and one Plasmodium malariae. CONCLUSIONS: In this study, design of PCR programmes with suitable parameters and optimization resulted in simpler and faster single-round amplification assays. Both sensitivity and specificity of the novel mitochondrial PCR was 100% and proved non-inferior to that of the reference nested PCR. Sequencing of genus-specific mitochondrial PCR products could be used for species determination.