High prevalence of impaired glucose metabolism among children and adolescents living with HIV in Ghana.

BACKGROUND: Antiretroviral therapy (ART)-associated metabolic abnormalities, including impairment of glucose metabolism, are prevalent in adults living with HIV. However, the prevalence and pathogenesis of impaired glucose metabolism in children and adolescents living with HIV, particularly in sub-Saharan Africa, are not well characterized. We investigated the prevalence of impaired glucose metabolism among children and adolescents living with perinatally infected HIV in Ghana. METHODS: In this multicentre, cross-sectional study, we recruited participants from 10 paediatric antiretroviral treatment clinics from January to June 2022 in 10 facilities in Greater Accra and Eastern regions of Ghana. We determined impaired glucose metabolism in the study sample by assessing fasting blood sugar (FBS), insulin resistance as defined by the homeostatic model assessment for insulin resistance (HOMA-IR) index and glycated haemoglobin (HbA1c) levels. The prevalence of impaired glucose metabolism using each criterion was stratified by age and sex. The phenotypic correlates of glucose metabolism markers were also assessed among age, sex, body mass index (BMI) and waist-to-hip ratio (WHR). RESULTS: We analysed data from 393 children and adolescents living with HIV aged 6-18 years. A little over half (205/393 or 52.25%) of the children were female. The mean age of the participants was 11.60 years (SD = 3.50), with 122/393 (31.00%) aged 6-9 years, 207/393 (52.67%) aged 10-15 years, and 62/393 (15.78%) aged 16-18 years. The prevalence rates of glucose impairment in the study population were 15.52% [95% confidence interval (CI): 12.26-19.45], 22.39% (95% CI: 18.54-26.78), and 26.21% (95% CI: 22.10-30.78) using HbA1c, HOMA-IR, and FBS criteria, respectively. Impaired glucose metabolism detected by FBS and HOMA-IR was higher in the older age group, whereas the prevalence of abnormal HbA1c levels was highest among the youngest age group. Age and BMI were positively associated with FBS and HOMA-IR (p < 0.001). However, there was negative correlation of WHR with HOMA-IR (p < 0.01) and HbA1c (p = 0.01). CONCLUSION: The high prevalence of impaired glucose metabolism observed among the children and adolescents living with HIV in sub-Saharan Africa is of concern as this could contribute to the development of metabolic syndrome in adulthood.

Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity.

Background: The epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains. Methods: The study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14+ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection. Results: We observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4 = 1.2, L4 = 1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs. Conclusion: Our results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC.

Comparison of the impact of allelic polymorphisms in PfAMA1 on the induction of T Cell responses in high and low malaria endemic communities in Ghana.

BACKGROUND: Malaria eradication requires a combined effort involving all available control tools, and these efforts would be complemented by an effective vaccine. The antigen targets of immune responses may show polymorphisms that can undermine their recognition by immune effectors and hence render vaccines based on antigens from a single parasite variant ineffective against other variants. This study compared the influence of allelic polymorphisms in Plasmodium falciparum apical membrane antigen 1 (PfAMA1) peptide sequences from three strains of P. falciparum (3D7, 7G8 and FVO) on their function as immunodominant targets of T cell responses in high and low malaria transmission communities in Ghana. METHODS: Peripheral blood mononuclear cells (PBMCs) from 10 subjects from a high transmission area (Obom) and 10 subjects from a low transmission area (Legon) were tested against 15 predicted CD8 + T cell minimal epitopes within the PfAMA1 antigen of multiple parasite strains using IFN-γ ELISpot assay. The peptides were also tested in similar assays against CD8 + enriched PBMC fractions from the same subjects in an effort to characterize the responding T cell subsets. RESULTS: In assays using unfractionated PBMCs, two subjects from the high transmission area, Obom, responded positively to four (26.7%) of the 15 tested peptides. None of the Legon subject PBMCs yielded positive peptide responses using unfractionated PBMCs. In assays with CD8 + enriched PBMCs, three subjects from Obom made positive recall responses to six (40%) of the 15 tested peptides, while only one subject from Legon made a positive recall response to a single peptide. Overall, 5 of the 20 study subjects who had positive peptide-specific IFN-γ recall responses were from the high transmission area, Obom. Furthermore, while subjects from Obom responded to peptides in PfAMA1 from multiple parasite strains, one subject from Legon responded to a peptide from 3D7 strain only. CONCLUSIONS: The current data demonstrate the possibility of a real effect of PfAMA1 polymorphisms on the induction of T cell responses in malaria exposed subjects, and this effect may be more pronounced in communities with higher parasite exposure.

Measurement of ex vivo ELISpot interferon-gamma recall responses to Plasmodium falciparum AMA1 and CSP in Ghanaian adults with natural exposure to malaria.

BACKGROUND: Malaria eradication requires a concerted approach involving all available control tools, and an effective vaccine would complement these efforts. An effective malaria vaccine should be able to induce protective immune responses in a genetically diverse population. Identification of immunodominant T cell epitopes will assist in determining if candidate vaccines will be immunogenic in malaria-endemic areas. This study therefore investigated whether class I-restricted T cell epitopes of two leading malaria vaccine antigens, Plasmodium falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), could recall T cell interferon-γ responses from naturally exposed subjects using ex vivo ELISpot assays. METHODS: Thirty-five subjects aged between 24 and 43 years were recruited from a malaria-endemic urban community of Ghana in 2011, and their peripheral blood mononuclear cells (PBMCs) were tested in ELISpot IFN-γ assays against overlapping 15mer peptide pools spanning the entire CSP and AMA1 antigens, and 9-10mer peptide epitope mixtures that included previously identified and/or predicted human leukocyte antigen (HLA) class 1-restricted epitopes from same two antigens. RESULTS: For CSP, 26 % of subjects responded to at least one of the nine 15mer peptide pools whilst 17 % responded to at least one of the five 9-10mer HLA-restricted epitope mixtures. For AMA1, 63 % of subjects responded to at least one of the 12 AMA1 15mer peptide pools and 51 % responded to at least one of the six 9-10mer HLA-restricted epitope mixtures. Following analysis of data from the two sets of peptide pools, along with bioinformatics predictions of class I-restricted epitopes and the HLA supertypes expressed by a subset of study subjects, peptide pools that may contain epitopes recognized by multiple HLA supertypes were identified. Collectively, these results suggest that natural transmission elicits ELISpot IFN-γ activities to class 1-restricted epitopes that are largely HLA-promiscuous. CONCLUSIONS: These results generally demonstrate that CSP and AMA1 peptides recalled ELISpot IFN-γ responses from naturally exposed individuals and that both CSP and AMA1 contain diverse class 1-restricted epitopes that are HLA-promiscuous and are widely recognized in this population.

High Levels of IL-10 and CD4+CD25hi+ Treg Cells in Endemic Burkitt's Lymphoma Patients.

BACKGROUND: The interplay between Epstein-Barr virus infection, malaria, and endemic Burkitt's Lymphoma is not well understood. Reports show diminished EBV-specific Th1 responses in children living in malaria endemic areas and deficiency of EBNA1-specific IFN-γ T cell responses in children with endemic Burkitt's Lymphoma (eBL). This study, therefore, examined some factors involved in the loss of EBNA-1-specific T cell responses in eBL. METHODS: T-cell subset frequencies, activation, and IFN-γ- or IL-4-specific responses were analyzed by flow-cytometry. Plasma cytokine levels were measured by ELISA. RESULTS: CD4+ and CD8+ cells in age- and sex-matched healthy controls (n = 3) expressed more IFN-γ in response to all immunostimulants than in pediatric endemic BL (eBL) patients (n = 4). In healthy controls, IFN-γ expression was higher than IL-4 expression, whereas in eBL patients the expression of IL-4 by CD4+ cells to EBNA-1 was slightly higher than IFN-γ. Moreover, the blood levels of TNF-α was significantly lower (p = 0.004) while IL-10 was significantly higher (p = 0.038), in eBL patients (n = 21) compared to controls (n = 16). Additionally, the frequency of CD4+CD25hi+ T cells was higher in both age-matched acute uncomplicated malaria (n = 26) and eBL (n = 14) patients compared to healthy controls (n = 19; p = 0.000 and p = 0.027, respectively). CONCLUSION: The data suggest that reduced Th1 response in eBL might be due to increased levels of IL-10 and T reg cells.

Recombinant ESAT-6-CFP10 Fusion Protein Induction of Th1/Th2 Cytokines and FoxP3 Expressing Treg Cells in Pulmonary TB.

BACKGROUND: Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are Mycobacterium tuberculosis (Mtb)-specific antigens that are secreted by actively metabolising bacteria and contribute to the virulence of the bacteria. Their ability to induce Treg and Th2 responses, particularly during the first two weeks of treatment, has not been comprehensively examined to date. The purpose of this work was to characterise Th1, Th2 and Treg responses to rESAT-6-CFP10 fusion protein in TB patients before and during the intensive phase of treatment and in healthy M.bovis BCG vaccinated donors. METHODS: Forty-six newly diagnosed, HIV-negative, smear-positive pulmonary TB patients and 20 healthy donors were recruited in the UK and Ghana. Their peripheral blood mononuclear cells (PBMC) were used in ex vivo ELISPOT and in vitro cultures to identify immunological parameters of interest. RESULTS: The study confirmed that protective immune responses to rESAT-6-CFP10 are impaired in active TB but improved during treatment: circulating antigen-specific IL-4-producing T-cells were increased in untreated TB but declined by two weeks of treatment while the circulating antigen-specific IFN-γ producing T cells which showed a transient rise at one week of treatment, persisted at baseline levels at two months of treatment. In vitro T cell proliferation and IFN-γ production were reduced, while IL-4 and CD4(+)FoxP3(+)CD25(hi) cell expression were increased in response to rESAT-6-CFP10 fusion protein in untreated TB. These responses were reversed during early treatment of TB. CONCLUSIONS: These observations support further investigations into the possible utility of these parameters as markers of active disease and favourable treatment outcomes.

High plasma levels of soluble intercellular adhesion molecule (ICAM)-1 are associated with cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is responsible for most of the malaria-related deaths in children in sub-Saharan Africa. Although, not well understood, the pathogenesis of CM involves parasite and host factors which contribute to parasite sequestration through cytoadherence to the vascular endothelium. Cytoadherence to brain microvasculature is believed to involve host endothelial receptor, CD54 or intercellular adhesion molecule (ICAM)-1, while other receptors such as CD36 are generally involved in cytoadherence of parasites in other organs. We therefore investigated the contributions of host ICAM-1 expression and levels of antibodies against ICAM-1 binding variant surface antigen (VSA) on parasites to the development of CM. METHODOLOGY/PRINCIPAL FINDINGS: Paediatric malaria patients, 0.5 to 13 years were recruited and grouped into CM and uncomplicated malaria (UM) patients, based on well defined criteria. Standardized ELISA protocol was used to measure soluble ICAM-1 (sICAM-1) levels from acute plasma samples. Levels of IgG to CD36- or ICAM-1-binding VSA were measured by flow cytometry during acute and convalescent states. Wilcoxon sign rank-test analysis to compare groups revealed association between sICAM-1 levels and CM (p<0.0037). Median levels of antibodies to CD36-binding VSA were comparable in the two groups at the time of admission and 7 days after treatment was initiated (p>0.05). Median levels of antibodies to CD36-binding VSAs were also comparable between acute and convalescent samples within any patient group. Median levels of antibodies to ICAM-1-binding VSAs were however significantly lower at admission time than during recovery in both groups. CONCLUSIONS/SIGNIFICANCE: High levels of sICAM-1 were associated with CM, and the sICAM-1 levels may reflect expression levels of the membrane bound form. Anti-VSA antibody levels to ICAM-binding parasites was more strongly associated with both UM and CM than antibodies to CD36 binding parasites. Thus, increasing host sICAM-1 levels were associated with CM whilst antibodies to parasite expressing non-ICAM-1-binding VSAs were not.

Insights into deregulated TNF and IL-10 production in malaria: implications for understanding severe malarial anaemia.

BACKGROUND: Severe malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients. METHODS: The IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLA-DR+ and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined. RESULTS: Monocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR+ T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL-10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .001). CONCLUSIONS: These data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.

Generation of HIV-1 primary isolates representative of plasma variants using the U87.CD4 cell line.

In order to obtain HIV-1 primary isolates in settings with limited access to donor PBMCs, a culture method was developed where patient PBMCs infected with HIV-1 were cultured together with U87.CD4 cells. Using this non-laborious method, it is possible to harvest virus solely on the basis of syncytia formation and circumventing monitoring of viral replication by CA-p24 ELISA. Primary isolates from 23 out of 33 patients (70%) were isolated successfully. From PCR amplification and sequencing of the V1V5 region of the viral gp120 envelope gene, primary isolates were compared with variants obtained from plasma and PBMCs of 13 patients. The primary isolates of seven patients (54%) resembled closely the plasma viral quasispecies, whereas different variants were isolated from the other patients (46%). Three patients harboured a dual infection, while this remained unnoticed from sequencing the plasma or PBMC compartment. The primary isolates were highly infectious for TZM-bl cells and could infect CD4-enriched lymphocytes. This study demonstrates that it is possible to grow viral isolates using a non-laborious and simple method. These isolates may be used in the field for studies on antiretroviral therapy or for vaccine trials.

Cerebral malaria is associated with low levels of circulating endothelial progenitor cells in African children.

Damage to the cerebral microvasculature is a feature of cerebral malaria. Circulating endothelial progenitor cells are needed for microvascular repair. Based on this knowledge, we hypothesized that the failure to mobilize sufficient circulating endothelial progenitor cells to the cerebral microvasculature is a pathophysiologic feature of cerebral malaria. To test this hypothesis, we compared peripheral blood levels of CD34 (+)/VEGFR2(+) and CD34 (+)/CD133(+) cells and plasma levels of the chemokine stromal cell-derived growth factor 1 (SDF-1) in 214 children in Accra, Ghana. Children with cerebral malaria had lower levels of CD34 (+)/VEGFR2(+) and CD34 (+)/CD133(+) cells compared with those with uncomplicated malaria, asymptomatic parasitemia, or healthy controls. SDF-1 levels were higher in children with acute malaria compared with healthy controls. Together, these results uncover a potentially novel role for endothelial progenitor cell mobilization in the pathophysiology of cerebral malaria.

Complement activation in Ghanaian children with severe Plasmodium falciparum malaria.

BACKGROUND: Severe anaemia (SA), intravascular haemolysis (IVH) and respiratory distress (RD) are severe forms of Plasmodium falciparum malaria, with RD reported to be of prognostic importance in African children with malarial anaemia. Complement factors have been implicated in the mechanism leading to excess anaemia in acute P. falciparum infection. METHODS: The direct Coombs test (DCT) and flow cytometry were used to investigate the mean levels of RBC-bound complement fragments (C3d and C3balphabeta) and the regulatory proteins [complement receptor 1 (CD35) and decay accelerating factor (CD55)] in children with discrete clinical forms of P. falciparum malaria. The relationship between the findings and clinical parameters including coma, haemoglobin (Hb) levels and RD were investigated. RESULTS: Of the 484 samples tested, 131(27%) were positive in DCT, out of which 115/131 (87.8%) were positive for C3d alone while 16/131 (12.2%) were positive for either IgG alone or both. 67.4% of the study population were below 5 years of age and DCT positivity was more common in this age group relative to children who were 5 years or older (Odds ratio, OR = 3.8; 95%CI, 2.2-6.7, p < 0.001). DCT correlated significantly with RD (beta = -304, p = 0.006), but multiple regression analysis revealed that, Hb (beta = -0.341, p = 0.012) and coma (beta = -0.256, p = 0.034) were stronger predictors of RD than DCT (beta = 0.228, p = 0.061). DCT was also not associated with IVH, p = 0.19, while spleen size was inversely correlated with Hb (r = -402, p = 0.001). Flow cytometry showed similar mean fluorescent intensity (MFI) values of CD35, CD55 and C3balphabeta levels on the surfaces of RBC in patients and asymptomatic controls (AC). However, binding of C3balphabeta correlated significantly with CD35 or CD55 (p < 0.001). CONCLUSION: These results suggest that complement activation contributed to anaemia in acute childhood P. falciparum malaria, possibly through induction of erythrophagocytosis and haemolysis. In contrast to other studies, this study did not find association between levels of the complement regulatory proteins, CD35 and CD55 and malarial anaemia. These findings suggest that complement activation could also be involved in the pathogenesis of RD but larger studies are needed to confirm this finding.

V beta profiles in African children with acute cerebral or uncomplicated malaria: very focused changes among a remarkable global stability.

T cells are thought to play a critical role in cerebral malaria pathogenesis. However, available evidences are restricted to rodent models in which V beta specific T cell expansion has been associated with neurological syndrome suggesting involvement of superantigens or dominant antigens. Using flow cytometry, we studied the peripheral V beta T cell repertoire of Ghanaian children with cerebral malaria, uncomplicated malaria and asymptomatic control children, to look for either expansion or deletion of specific V beta associated with cerebral malaria. At admission, the general pattern of the repertoire of the patients was very similar, with no major distortion compared to the control group a part a significant increase of the frequency of the V beta 21.3 subset correlating with disease severity and attributed to the CD4 subset. During convalescence very limited fluctuations were observed including a significant decrease of the V beta 21.3 subset and increase of the V beta 20 subset, a subset not detected at admission. The remarkable stability of the V beta repertoire observed in acute malaria either cerebral or uncomplicated argues against the idea that cerebral malaria would result from a T cell-mediated inflammatory shock syndrome driven by some dominant super-antigenic activity(ies). The significance of the reproducible increase of the CD4+V beta 21.3T cell subset deserves further investigations.

Increased levels of inflammatory mediators in children with severe Plasmodium falciparum malaria with respiratory distress.

BACKGROUND: Respiratory distress (RD), a symptom of underlying metabolic acidosis, has been identified as a major risk factor for mortality in children with severe malaria in Africa, yet the molecular mediators involved in the pathogenesis of RD have not been identified. METHODS: We studied circulating levels of mediators of inflammation--including the cytokines tumor necrosis factor (TNF)- alpha and interleukin (IL)-10; the chemokines macrophage inflammatory protein (MIP)-1 alpha , MIP-1 beta , and IL-8; and the immune activation marker neopterin--in children with RD, severe malarial anemia (SMA), cerebral malaria (CM), and uncomplicated malaria (UM). RESULTS: Children with RD had significantly higher plasma levels of TNF- alpha , IL-10, and neopterin and a significantly higher TNF- alpha : IL-10 ratio than those without RD. In addition, the results demonstrated that, relative to UM, CM was associated with increased levels of TNF- alpha and decreased levels of MIP-1 alpha , whereas SMA was associated with decreased levels of IL-10. Circulating levels of neopterin were inversely correlated with hemoglobin, whereas levels of MIP-1 beta were positively correlated with parasitemia. CONCLUSIONS: We conclude that distinct clinical presentations of severe malaria are associated with specific patterns of inflammatory mediators. In particular, we show, to our knowledge for the first time, that patients with malaria and RD have a strong and unbalanced proinflammatory response that may be involved in the pathogenesis of the underlying metabolic acidosis.

High CD4/CD45RO+ and CD8/CD45RO+ frequencies in children with vaccine-modified measles.

BACKGROUND: Despite availability and wide vaccine coverage, measles infections still occur especially in developing countries. An outbreak of measles occurred among previously immunized older Ghanaian children who had milder clinical symptoms with measles-specific IgG antibodies that could have been attributed to secondary vaccine failure, suggesting that the infection was vaccine-modified measles (VMM). METHODS: Two-color immunophenotyping of the peripheral blood mononuclear cells was performed at acute, recovery and convalescence phases for 19 VMM patients (mean age 6.2 +/- 3.5 years) using flow cytometry, and compared with that of 20 healthy, sex- and age-matched controls. RESULTS: The results showed a significantly higher memory helper (CD4(+)/CD45RO(+)) cell frequency and increased suppressor cell (CD8(+)/CD45R0(+)) frequency in VMM patients compared to healthy controls. There were no complications and all the patients recovered completely. CONCLUSIONS: These findings show that the mild symptoms in patients with VMM may have correlated with the increase of memory T cells, which is in sharp contrast with previous reports on acute measles infection. This may suggest that the intact immunologic memory cells could have been crucial for the resolution of VMM.

Plasma levels of Th1 and Th2 cytokines in Ghanaian children with vaccine-modified measles.

To understand the pathogenesis of vaccine-modified measles (VMM), we measured plasma levels of IFN-gamma and IL-2 (Th1 cytokines), IL-4 and IL-10 (Th2 cytokines), IL-12, TNF-alpha and TGF-beta1 in children with uncomplicated measles, who had anti-measles IgG antibodies and with a history of immunization on admission (day 0), day 14 and day 60. We compared these to levels in healthy, age-matched, immunized children. Plasma levels of IFN-gamma, IL-2 and IL-12 were significantly higher in VMM patients on day 0 compared to healthy controls (p = 0.023; p = 0.018; p = 0.001) respectively. In contrast, plasma IL-4 was lower in VMM patients on day 0 when compared to the controls (p = 0.009). Plasma levels of IL-12 remained consistently high on days 14 and 60 (p = 0.001; p = 0.04), whilst IL-10 levels fell significantly on the same days (p = 0.002; p = 0.001) respectively. Kinetically, IFN-gamma and IL-10 levels decreased consistently from day 0 to days 14 and 60 in VMM patients. In contrast, IL-4 levels increased from day 0 to day 14 and day 60. Our results therefore suggest that VMM is associated with an early up-regulation of Th1 cytokine production and a down-regulation of Th2 cytokine production. The strong Th1 response may be associated with the induction of IL-12 and memory cells, thus contributing to the early resolution of the infection and lack of complications.