Severe malaria may be complicated by the acute respiratory distress syndrome (ARDS), which is associated with a high mortality. In the present report, a series of three cases of imported malaria complicated by refractory severe ARDS supported with extracorporeal membrane oxygenation (ECMO) is presented.Plasmodium falciparum infection and one patient had Plasmodium vivax and Plasmodium ovale co-infection. Anti-malarial therapy consisted in intravenous quinine (in two patients) and intravenous quinidine (in one patient), plus clindamycin or doxycycline.One female and two male adult patients (ages 39 to 53) were included. Two patients had 2 to FiO2 ratio 68) and venovenous ECMO was then initiated. In one patient, a bicaval dual-lumen cannula was inserted; in the two other patients, a two-site configuration was used. Two patients survived to hospital-discharge (duration of ECMO support: 8.5 days) and one patient died from nosocomial sepsis and multi-organ failure after 40 days of ECMO support.Despite lung protective ventilation, a conservative strategy of fluid management, corticosteroids (two patients), prone position (two patients) and inhaled nitric oxide (one patient), refractory severe ARDS supervened (PaOECMO support allowed adequate oxygenation and correction of hypercapnia under lung protective ventilation, therefore reducing ventilator-induced lung injury. ECMO referral should be considered early in malaria complicated by severe ARDS refractory to conventional treatment. Plasmodium falciparum as the predominant organism [P. falciparum and in 1 to 10% of patients with severe Plasmodium vivax[Malaria is responsible for more than one million deaths annually, the majority of which occur in sub-Saharan Africa, with organism . In non-organism ,3. Such organism ,4-6. Acuium vivax.Extracorporeal membrane oxygenation (ECMO), referring to an extracorporeal circuit that directly oxygenates and removes carbon dioxide from the blood, may be considered in patients with ARDS whose pulmonary injury is so severe that positive-pressure ventilation alone is insufficient to maintain adequate gas exchange, or when adherence to lung-protective ventilation strategies results in unacceptable levels of hypercapnia and acidaemia . In mostP. falciparum (2% parasitaemia) and severe thrombocytopaenia . The patient was initially admitted in a regular ward and oral quinine sulphate plus doxycycline were administered. However, shortly after admission, he developed vomiting with intolerance to oral drugs. In that context, intravenous quinine and doxycycline were started. No arrhythmia occurred during quinine treatment. The patient developed progressive hypoxemic respiratory failure requiring invasive mechanical ventilation (IMV) on hospital day 3 despite antimicrobials, diuresis, and clearance of his parasitaemia  was also performed before ECMO treatment, the results being consistent with ARDS  extravascular lung water index (ELWI) and increased  pulmonary vascular permeability index (PVPI)), without intravascular volume overload  intrathoracic blood volume index (ITBI) and decreased  global enddiastolic volume index (GEDI)). Cardiac index  and systemic vascular resistance index  were also recorded. On day 7 of IMV, his PaO2 was 69 mmHg with a FiO2 of 1.0 and PEEP of 13 cm of water, without improvement with a conservative strategy of fluid management, prone positioning or corticosteroids. ECMO was initiated with placement of a 21Fr right femoral venous drainage cannula and a 15Fr right internal jugular venous re-infusion cannula. The ventilator was then set to a lung-protective strategy  and thrombocytopaenia . Taking into account the presence of vomiting with intolerance to oral drugs, the patient was treated with intravenous quinine and clindamycin. Despite negative cultures and clearance of his parasitaemia, hypoxemic respiratory failure developed on hospital day 6, requiring IMV   and low-fractional oxygen concentrations, limiting ventilator-induced lung injury [In this report, three cases of imported malaria complicated by severe ARDS supported with ECMO are presented. Severe ARDS may be a complication of  malaria . In all g injury . Howeverg injury  and thatg injury . The useFluid management is crucial in patients with severe malaria. Taking into account that in these patients pulmonary edema secondary to increased pulmonary vascular permeability has been shown to be frequent, unpredictable and exacerbated by fluid loading, a conservative strategy of fluid management was followed in all three patients before and after ECMO initiation . Moreove2O of peak inspiratory pressure) or high FiO2 (>0.8) ventilation for more than seven days; intracranial bleeding; any other contraindication to limited heparinization) criteria for ECMO consideration in malaria and severe ARDS were used in the present case series.ECMO is not without risks and there are no definitive criteria for ECMO treatment in adult ARDS. Therefore, ECMO should only be considered in adult patients with malaria and severe ARDS refractory to conventional treatment. The recently conducted CESAR trial showed favourable results of ECMO referral for adult patients with severe ARDS . In thatP. falciparum infection, given that in P. vivax malaria there is no substantial lung sequestration. Severe ARDS in patients with malaria has a poor prognosis, with mortality as high as 69 to 95% in observational ICU case series [P. falciparum infection and is rarely a complication of P. vivax or P. ovale infection. This could be related to differences in cyto-adhesion of infected erythrocytes [Severe imported malaria is an important problem in the western world due to the increasing number of travellers returning from endemic countries who are non-adherent to malaria chemoprophylaxis . Most ime series ,25. Sevehrocytes -29. HoweRisk factors associated with the development of ARDS in malaria include advanced age, lack of specific immunity, immunocompromised status, and pregnancy. A high parasitic burden is not necessary . In thisP. falciparum was identified as the causal organism and ECMO was instituted within seven days for refractory hypoxaemia, but only one patient survived [P. falciparum was also identified as the causal organism and extracorporeal carbon elimination was instituted on the 17th day, being discontinued after twelve days. The patient was weaned from mechanical ventilation and successfully discharged home [There are only two prior cases reported separately on the use of ECMO for severe ARDS in patients with malaria, as well as one case on the use of extracorporeal carbon dioxide elimination ,12,13. Isurvived ,12. In tged home -35. Earlged home ,36-38.Patients with severe forms of malaria are highly susceptible to bacterial infections and concomitant antibacterial therapy is recommended ,39. All ECMO-related complications in the present case series included cannula migration into a hepatic vein, which was identified by transesophageal echocardiogram and corrected at the bedside. Thrombus formation in the oxygenator was ultimately the cause for oxygenator exchange in one patient. However, the rate of thrombosis-associated oxygenator failure requiring device exchange is low, even at low levels of anticoagulation . Major bP. falciparum, P. vivax, and P. ovale infection. In regions where the technology is available, ECMO referral should be considered for patients with malaria complicated by severe ARDS refractory to conventional treatment. Taking into account the limited experience with ECMO in severe malaria yet, its use should be performed in experienced ECMO centers. In fact, severe malaria can cause haemolysis, disseminated intravascular coagulation and low platelets, which might be exaggerated by the use of extracorporeal devices.In conclusion, severe ARDS can complicate The authors declare that they have no competing interests.CA, JTC, NP, DA, RRA and DB wrote the paper. CA, PF, LS, AS, JAP and RRA were the physicians responsible for Patient 1 and Patient 2. JTC, NP, DA, MB, MLW and DB were the physicians responsible for Patient 3. RRA and DB conceived the study, its design and coordination. All authors have read and approved the final manuscript.


Plasmodium falciparum infection, and anaemia is an inevitable outcome. This study examines the influence of malaria status and altitude on haematologic parameters in school-aged pupils.Haematologic abnormalities are features in A cross-sectional study was conducted among 728 school pupils aged between four and 15 years at three different altitudinal ranges along the slope of the Mount Cameroon region. The investigative methods included the use of questionnaire, clinical evaluation and laboratory investigations. Blood sample collected from each child was used for the preparation of blood films for detection of malaria parasites and assessment of malaria parasite density as well as full blood count determination using an automated haematology analyzer.9/L) than those with CM (5.9 ± 2.3 × 109/L). Age, altitude and malaria parasitaemia was of significant influence on several haematological parameters.The prevalence of malaria in the study population was 33.8% and 64.2% (158/246) of these were asymptomatic (AM). Pupils in lowlands had a significantly (P <0.05) prevalence  of malaria  than those in middle belt  and highlands , while those in middle belt had significantly higher geometric mean parasite density (475) than those in lowlands (233) and highlands (388). The prevalence of malaria was significantly higher in children that presented with fever  when compared with afebrile subjects . Pupils with AM had a higher prevalence of leucopaenia , microcytosis , hypochromasia  and thrombocytopaenia  when compared with those with clinical malaria (CM). All mean haematological parameters were comparable in pupils with CM and AM, except for the mean white blood cell (WBC) counts. Pupils with AM had significantly lower (P = 0.02) mean WBC counts (5.1 ± 2.5 × 10Altitude influenced the distribution and density of malaria parasites and was of confounding influence on the haematologic profiles. These results highlight the insidious effects of AM on the haematologic components. Malaria still remains a major public health problem in sub-Saharan Africa in spite of an increase in control measures. In Cameroon it is estimated that 41% of the population has at least one episode of malaria each year with children under five years and pregnant women mostly affected .Although fever usually has a high sensitivity but poor specificity for the diagnosis of malaria, it still serves as the entry point for presumptive treatment of malaria in African children who, if left untreated, run the risk of severe complications and even death. The presumptive treatment of all fevers is the most risk-adverse approach to managing "malaria" across Africa .Plasmodium falciparum infection. The severity of haematologic disease caused by Plasmodium is related to the ability of the parasites to invade and grow in different red cell populations as well as the intrinsic growth rate of the parasite  and highlands  as shown in Table Out of the 728 pupils, 218 (29.9%) were febrile. The prevalence of malaria was significantly higher  in children that presented with fever  when compared with the afebrile subjects  than in the afebrile (269 /μL blood).The prevalence of leucopaenia and microcytosis was significantly higher P <0.001) in malaria parasitaemia-positive pupils  than those negative , respectively. In contrast, hypochromasia was significantly higher (P <0.001) in pupils who were malarial parasite negative  than those positive  as shown in Figure  in malar9/L) when compared with those with CM (5.9 ± 2.3 × 109/L). The mean difference (0.78 × 109/L) was statistically significant  at 95% CI (0.14-1.4).Overall, all mean haematological parameters were comparable in pupils with CM and AM but for the mean WBC counts. Pupils with AM had lower mean WBC counts (5.1 ± 2.5 × 10Children with AM had significantly lower (P <0.05) mean Hb, Hct, RBC, WBC, MCV and platelet counts than their negative counterparts Table . Whilst The parasite density category did not have any significant effect on the mean Hct, WBC, RBC, platelet counts and MCH. On the other hand, while the mean Hb and MCHC significantly (P <0.05) decreased with an increase in parasite density category, a significant (P <0.01) increase was observed in MCV with parasite density category Table .A statistically significant decrease (P <0.001) in prevalence of leucopaenia with altitude was observed irrespective of the status of malaria parasitaemia. On average the prevalence of leucopaenia in pupils in lowlands was 47.3% (CI = 42.1–53.6%), 8% (CI = 4.5–14.5%) in middle belt and 6.3% (CI = 2.7–8.2%) in highlands.Overall, the prevalence of anaemia was significantly highest (χ2 = 16.4 P <0.001) in pupils in lowlands when compared with those in middle belt and highlands  in children with CM in the lowland than their asymptomatic counterparts . In the 246 malaria parasitaemia positive children; altitude significantly (P <0.05) influenced the Hct, WBC, MCV and MCHC; age had an effect on the Hb, Hct, WBC and MCV; while parasitaemia influenced the Hb, Hct RBC and WBC counts  models were run with each haematological variable as the dependent variable to examine the influence of altitude, age, sex, parasitaemia, temperature, febrile status and SES on each haematologic variable. Overall, the sex, temperature and febrile status had no significant (P >0.05) influence on the haematological parameters under study. On the other hand, the age (P <0.001), altitude (P <0.001), SES (P = 0.04) and clinical malaria status (P = 0.001) significantly influenced the haematological parameters  were malaria parasite negative. The high temperatures in these pupils could be attributed to other infections, such as co-infections with other parasites, viruses or bacteria, which were beyond the scope of this study. Nonetheless, this is a cause for concern because presumptive treatment for malaria with fever as a marker is the order of the day amongst most parents in the Mount Cameroon region. These parents administer treatment themselves at home and only seek medical attention when the fever persists . It is tet al[In line with Achidi et al malaria et al,25 reporet al.It is worth noting that the pupils in this study were seemingly healthy, with regular school attendance, even though a pupil with temperature as high as 39.1°C was recorded. Also, the majority (64.2%) of the pupils positive for malaria parasites were asymptomatic and remarkably the length of period they were parasitaemic unclear. This may have contributed to the higher prevalence of leucopaenia, microcytosis and thrombocytopaenia observed in children with AM when compared with those with CM and those negatives. This highlights the insidious effects AM may have on the haematological components of children living in an area of intense malaria transmission.The presence of malaria parasites in these pupils was found to be associated with lower Hb, Hct, RBC and WBC counts as confirmed by some of the negative beta values in the models. The higher prevalence of AM than CM most likely would have exacerbated the reduction in the red cell indices as asymptomatic parasitaemia and protracted malaria infections have been associated with a marked reduction in Hb concentration  and withPlasmodium infection is thought to lower erythrocyte counts and Hb levels by inducing haemolysis, decreased erythropoiesis and increased clearance of both parasitized and non-infected erythrocytes, findings from the study revealed no significant effect of the parasite density on the mean RBC count. The significant decrease in mean Hb levels with an increase in parasite density without a corresponding significant decrease in RBC count may be related to the extraction of P. falciparum parasites from RBCs, leaving the RBCs intact. Kumaratilake et al[in vitro are capable of extracting P. falciparum parasites from RBCs, leaving the RBCs intact and the removal of intra-erythrocytic parasites occurs naturally in vivo when the host immune system can act. Also, parasitized RBCs treated with anti-malarial drugs have been recorded extruding dead trophozoites [Even though ake et al reportedhozoites .Further observations from the study revealed the altitude not to be of any significance on the Hb levels and RBC counts, rather these haematological measures were influenced mostly by the age and parasite density. Majority (184/246) of the pupils with malaria parasitaemia were above seven years of age with 87.5% (161/184) of them having parasite density <1000/μl of blood. The age related decrease in malaria parasite density observed is probably related to the acquisition of protective immunity due to repeated infections as children grow older in high transmission areas. This protective immunity may have permitted the host immune system to act consequently the non-significant decrease in mean RBC counts.Plasmodium infection on WBC counts are conflicting. Consistent with previous investigations [et al[Reports on the impact of igations ,11,31,32ns [et al reportedet al[Findings from the study revealed that thrombocytopaenia is common among pupils with malaria parasitaemia and a significant  relationship exists between the platelet counts and the WBC counts. The prevalence of thrombocytopaenia (14.9%) in pupils with AM in the study population is comparable to those obtained by Ladhani et al in a simet al, which iet al.An evaluation of the effect of parasite density category on MCV showed an increase in MCV with an increase in parasite density category. Parasitized red cells are known to develop knobs, which facilitate their adhesion to endothelial cells of capillaries , hence nThe decrease in prevalence of anaemia with increase in altitude observed in the study is not unexpected as most red cell indices have a positive relationship with altitude as indicated by their β value. The level of Hb (<11 g/dL) used as an indicator of anaemia was not significantly influenced by the altitude. However, there were similarities in trend in the prevalence of anaemia and malaria parasite prevalence with altitude. Furthermore, pupils in the middle belt had the highest GMPD with significant variations in the mean haematological values when compared with the other altitudes. Higher parasite intensity may lead to lower mean Hb levels and consequently anaemia although the density of malaria infection is not a significant factor in determining disease severity .The significant differences in haematological parameters with clinical status observed in children in the middle belt Table  may be aWith a decline in the burden of malaria in school-aged pupils, there is a need for differential diagnosis of all fevers to corroborate the role of malaria in the febrile presentation. Altitude influenced the distribution and density of malaria parasites and was of confounding influence on the haematologic profiles. The results highlight the insidious effects of AM on the haematologic components. However, further studies to assert the protective role of leucopaenia in asymptomatic malaria is necessary.AM: Asymptomatic malaria; CM: Clinical malaria; CI: 95% confidence interval; GMPD: Geometric mean parasite density; Hb: Haemoglobin; Hct: Haematocrit; ITNs: Insecticide-treated bed nets; LLINs: Long-lasting insecticidal nets; MCH: Mean corpuscular haemoglobin; MCHC: Mean corpuscular haemoglobin concentration; MCV: Mean corpuscular volume; MLR: Multiple linear regression; RBC: Red blood cell; SES: Socio economic status; WBC: White blood cell.The authors declare that they have no competing interests.HKK conceived the study, participated in the design and co-ordination, data collection and revision of the manuscript; IUNS conceived and participated in the design, data analysis, interpretation, and write-up of the manuscript; MN participated in the collection of data and laboratory analysis; JKAK participated in supervision and revision of the manuscript; EL participated in the collection of data and laboratory analysis; YN participated in data collection, LMN participated in coordination and revision of manuscript; HN carried out laboratory analysis; LGL participated in co-ordination and revision of manuscript. All authors read and approved the final manuscript.


Plasmodium malariae and P. falciparum parasites in 45% and 55% of microscopy-positive samples, respectively. Although 36% of microscopy-positive samples were negative by rapid diagnostic test, 75% showed P. malariae or P. ovale parasites by PCR, indicating that routine diagnostic testing misses many non–P. falciparum malarial infections.A survey of asymptomatic children in Uganda showed  P. falciparum  and remain undetected for longer periods because of milder clinical symptoms  than for P. falciparum infections, enabling persistent transmission of non–P. falciparum infections /pan-Plasmodium lactate dehydrogenase (pLDH) RDT . Nested PCR was performed on all RDT- or microscopy-positive samples. Predictors of malaria were selected a priori .Stratified, 2-stage cluster sampling was used to select study participants. We administered questionnaires to gather information about standard knowledge, attitudes, and practices related to malaria and collected blood for testing with microscopy, RDT, and PCR . RDT wasWe surveyed 631 children with a mean age of 2.4 years . Bed netP. falciparum infections than P. falciparum monoinfections. Of 22 microscopy-positive samples, 9 (40.9%) were P. falciparum monoinfections, 7 (31.8%) were P. malariae monoinfections, 3 (13.6%) were P. ovale monoinfections, and 3 (13.6%) were P. falciparum/P. malariae mixed infections. Most P. malariae monoinfections occurred in Isingiro district. Sixteen (72.7%) of 22 blood-smear readings correlated directly with PCR results . Speciation by microscopy revealed a higher proportion of non– results  Table 2.P. falciparum monoinfections and mixed infections. A comparison of the diagnostic performance of RDT and microscopy (uncorrected by PCR) indicates that agreement of results from these methods was high (>94%); however, agreement was poor in detecting non–P. falciparum infections (κ = 0.15) compared with detecting overall infection (κ = 0.41) and P. falciparum infection (κ = 0.33)  . PCR det = 0.33)  Table 2.P. malariae or P. ovale monoinfections; all had parasite densities <1,060/μL (Approximately one third (8/22) of children with microscopy-positive cultures had negative RDT results  Table 3.1,060/μL  Table 2.P. falciparum malaria have continued in Uganda. Compared with data from 2010, P. falciparum prevalence by microscopy had a 4- and 5-fold decrease in urban and rural villages, respectively  have also reported a rise in non–P. falciparum infections, particularly P. malariae .One possible reason for the nearly equivalent prevalence of asymptomatic P. malariae and P. ovale infections is critical because P. malariae has been associated with chronic infections that can persist for years, including a chronic nephrotic syndrome that, once established, is unresponsive to treatment , which affords higher sensitivity in detecting low-level parasitemia, especially P. malariae infections that tend to exhibit lower parasite densities than P. falciparum and P. vivax (From a malaria control perspective, the performance of the pLDH/HRP-2–based RDT was suboptimal in our data, indicating a need for accurate diagnostic methods aimed at detecting Detailed methods and tables describing risk factors for asymptomatic malaria infections and results of PCR-corrected samples found to be positive by microscopy and rapid diagnostic test.


The nature of protective immune responses elicited by immunization with the candidate malaria vaccine RTS,S is still incompletely understood. Antibody levels correlate with protection against malaria infection, but considerable variation in outcome is unexplained .E between March and August 2007. We evaluated the association between the AI and the subsequent risk of clinical malaria. We selected 19 cases  and 42 controls , matching for anti-CS antibody levels and malaria exposure. We assessed their sera collected 1 month after the third dose of the vaccine, in March 2008 (range 4–10 months after the third vaccine), and at 12 months after the third vaccine dose. The mean AI was 45.2 (95% CI: 42.4 to 48.1), 45.3 (95% CI: 41.4 to 49.1) and 46.2  at 1 month, in March 2008 (4–10 months), and at 12 months after the third vaccination, respectively (p = 0.9 by ANOVA test for variation over time). The AI was not associated with protection from clinical malaria . The AI was higher in children with high malaria exposure, as measured using the weighted local prevalence of malaria, compared to those with low malaria exposure at 1 month post dose 3 (p = 0.035).We measured the avidity index (AI) of the anti-CS antibodies raised in subgroup of 5–17 month old children in Kenya who were vaccinated with three doses of RTS,S/AS01E-vaccinated children residing in malaria endemic countries, the avidity of anti-circumsporozoite antibodies, as measured using an elution ELISA method, was not associated with protection from clinical malaria. Prior natural malaria exposure might have primed the response to RTS,S/AS01E vaccination.Our data suggest that in RTS,S/AS01 P. falciparum circumsporozoite protein (CS) fused to hepatitis B surface antigen (HBsAg), and co-expressed with unfused HBsAg in Saccharomyces cerevisiae cellsRTS,S consists of 19 copies of the central tandem repeats and C-terminal region of the RTS,S is highly immunogenic Even in trials where correlations between anti-CS titers and outcome were observed, considerable variation in outcome remained unexplained . Some studies suggest an association between cell-mediated immune responses  and protection against clinical malaria in children, albeit of lesser importance than antibody responses Haemophilus influenza type b vaccine, Hepatitis B vaccine and Pneumococcal conjugate vaccine In addition to the quantity of antibodies, quality of antibodies may determine protection following vaccination against diverse pathogens E.To date, no study has investigated the role of avidity of RTS,S-induced anti-CS antibodies in protection against malaria infection among RTS,S vaccinees in the field. Here we report the results of such study in children 5–17 month residing in Kilifi, Kenya who were immunized with RTS,S/AS01E against Plasmodium falciparum clinical malaria in 5–17 month old children were used  E between March and August 2007. The candidate vaccine was given intramuscularly in the right deltoid area in a 0, 1, 2 month schedule. Blood samples were collected at screening, at 1 month after the third dose of vaccine, in March 2008 (range 4–10 months (mean 8 months) post dose 3) and at 12 months after the third dose of vaccine for the assessment of antibodies to P. falciparum CS repeat region (anti-CS antibodies).Serum samples from a phase IIb randomized controlled trial originally designed to determine the efficacy of RTS,S/AS01Informed written consent was obtained from parents of the study participant using approved Swahili or Giriama consent forms. All the parents signed the informed consent and were provided with the copy of informed consent and participant information sheet. Illiterate parents thumb printed the forms with independent literate witness countersigning. The original study was approved by the Kenya Medical Research Institute National Ethics Committee, Western Institution Review Board and Oxford Tropical Research Ethics Committee.rd dose of vaccine while controls were children who did not experience any clinical malaria episodes.A nested case-control study was conducted to investigate the association between vaccine-induced anti-CS antibody avidity and protection from clinical malaria. Cases were defined as children who had at least one episode of clinical malaria  during the 15 months of follow-up beginning 2 weeks after the 3The study was conducted in villages of Junju and Pingilikani in Kilifi district. The two areas have moderate malaria transmission based on parasite prevalence rates Due to cost and allowable time to accomplish the study, only a fraction of the available samples could be analyzed. We randomly selected 19 cases and 42 controls from 295 RTS,S/AS01E vaccinees with immunogenicity data matching for the level of peak anti-CS levels and malaria exposure. The selected sample size was enough to provide 80% power to detect an odds ratio of 3 between cases and controls given 50% probability of exposure (antibody avidity with protective effect) among controls and an alpha of 0.05. In an exploratory analysis we compared the avidity indices of controls (protected children) who had low antibody level (in the lower tertile) and high malaria exposure with cases to determine if the avidity index could explain the protection. All selected cases and controls had their samples assessed for the anti-CS avidity at three time points i.e. 1 month, March 2008 (range 4–10 months) and at 12 months post dose three. Avidity was not measured in screening samples because anti-CS titers were undetectable before vaccination.15]2LR and antibody titers were calculated using a reference standard curve and expressed in ELISA units (EU) per mL. A cut-off point for positive titers was 0.5 EU/mL.Anti-CS antibody titers were determined by standard enzyme-linked immunosorbent assay (ELISA) developed by GSK Biologicals 4SCN) elution ELISA to determine the avidity of the polyclonal anti-CS antibodies. Polystyrene microtiter plates were coated overnight at 4°C with 2.5 µg/ml R32LR protein in coating buffer. Following washing, the plates were blocked with 200 µl PBS-5% skim milk per well for 1 hour at 25°C in a horizontal orbital shaker (Skatron 300).We used a single concentration ammonium thiocyanate , negative control  and positive control  were added in each plate and serially diluted like samples. The plates were then incubated for 2 hours at 37°C.4SCN, and one untreated plate. Both plates were washed twice with 0.05% Tween-20 in PBS (wash buffer). A 1 M solution of NH4SCN in laboratory grade water was added in the treatment plate while 0.05% Tween-20 in PBS was added in the control plate and both were incubated for 30 min at 25°C. The plates were then washed three times with wash buffer. After a third wash, anti-human IgG conjugated to horseradish peroxidase (HRP) was added and incubated for 30 min at 25°C before washing. After 30 min incubation with chromogen substrate  at 25°C (yielding a blue color), the reaction was stopped with 50 µl of 1 N sulphuric acid changing the color to yellow. The intensity of the color was proportional to the titer of the anti-CS IgG antibodies contained in the sample. Absorbance at 450 nm was read by use of an automatic microtiter plate reader. Samples Optical densities (OD’s) were converted into EU/ml using a standard curve at the linear segment of the curve and mean values used to estimate avidity index. Linearity of the assay was demonstrated by the observation that for a high concentration sample, serially diluted sample was able to reproduce the same Avidity Index (AI) over the entire anti-CS analytical range. The AI is defined as the ratio of the quantity of anti-CS antibodies (in EU/ml) that remained bound to the coated antigen after treatment with NH4SCN divided by the quantity of antibodies (in EU/ml) that remained bound to the coated antigen in the control plate. The AI for each serum represents the composite value across all the dilutions.Each serum sample was processed in two different plates; one treated with NHWhole-blood intracellular staining assay was used to determine the frequency of CD4 T cells producing TNF-α as previous described Anti-CS avidity measures were not transformed in the analysis and were presented as percentage (by multiplying the ratio by 100). The anti-CS titers and avidity indices were presented for each group as arithmetic mean±95% confidence interval per group. Students T-test was used to compare the avidity indices and antibody titers between groups. A one-way analysis of variance (ANOVA) was used to test for the difference in antibody titers and antibody avidity during follow up . Unconditional logistic regression analysis was used to estimate odds ratios (ORs) and 95% Confidence intervals adjusting for TNFα-producing CD4 T cells, bed net use and malaria exposure (high or low). Cluster sandwich covariance matrix estimator was used to account for the multiple entries per child. No adjustment was made for the variable (i.e. anti-CS titers) used to select cases and controls to avoid bias. All analyses were done in STATA .E between March and August 2007.All children received all three doses of RTS,S/AS01A total of 162 sera  and 61 sera obtained 12 month post dose 3) from 61 study participants were available for the analysis.The geometric mean titer of anti-CS titer in a subset of children in this study 1 month after the third vaccination was 732.1 EU/mL  and this fell to 105.3 EU/mL  and 64.7 EU/mL  at 6.5 month and 12 month post vaccination 3 respectively (F test = 125 p<0.001 by ANOVA test). The dynamics of anti-CS titers in cases and controls is shown in The arithmetic mean AI of polyclonal anti-CS antibodies at 1 month, March 2008 (∼8 months) and 12 months post dose 3 was 45.2 (95% CI: 42.4 to 48.1), 45.3 (95% CI: 41.4 to 49.1) and 46.2 , respectively . The AI Correlations were observed between the AI at 1 month post-dose 3 and at ∼8 months post-dose 3 , as well as at 1 month and 12 months post-dose 3  , Panel BThere was no correlation between the AI and anti-CS titers at all three time points  or between the AI and age at vaccination   for each 10% increase in avidity index, indicating that we had power to exclude an OR of <0.89. Multivariable logistic regression showed no association between the avidity index and protection from clinical malaria . TNF-alpThe AI at 1 month post dose 3 was higher in children under high malaria exposure conditions compared to those under low malaria exposure with borderline statistical significance  . This diE vaccinees with similar levels of anti-CS antibodies aged 5–17 months and residing in a malaria endemic country.The nature of protective immune responses elicited by immunization with RTS,S is still incompletely understood. Although RTS,S-induced humoral and cell mediated immune responses correlate with protection against both asymptomatic and symptomatic parasitaemia There was no evidence of anti-CS avidity maturation beyond one month post dose 3. Avidity maturation follows B cells activation in a CD4 T cell dependent manner and is the hallmark of immunologic memory P. berghei) suggests that induction of sterile immune responses by CSP-based subunit vaccines depends on inducing antibody of the appropriate isotype, specificity and avidity P. yoelii) found protection against sporozoite challenge to be independent of antibody avidity and/or isotype E had exceeded a minimum avidity threshold required for protection and hence we could not detect any variation in outcome. For instance, Bachmann et al. observed lack of correlation between antibody avidity and protection against vesicular stomatitis virus (VSV) once the antibody avidity had reached a minimum threshold We found no association between avidity and protection from clinical malaria. Data in one animal model . However CD4 T cells are known to play an important role in antibody affinity maturation through their interactions with cognate B-cells There are other qualitative aspects of the antibody responses that we did not examine; such as the IgG isotypes in the children and ability of antibodies to inhibit hepatocyte infection in functional assays. IgG3 and IgG1 are the most effective isotypes at mediating antibody-dependent cellular mechanisms like phagocytosis and complement fixation against blood stage malaria antigens, and have been associated with protection from clinical malaria in the field Our assays measured average avidity of specific polyclonal serum antibodies which comprise of a mixture of antibody subpopulations with different avidities. Avidity indices can vary considerably with each sample and this can only be well depicted by avidity distribution. We cannot comment on this possibility given that we did not store the data in a manner to enable the performance of this analysis.E vaccinees with similar levels of anti-CS titers Antibody avidity was higher in children with high malaria exposure suggesting a possible priming effect of natural infection on the RTS,S-induced response.In conclusion, antibody avidity did not predict protection in RTS,S/AS01


Plasmodium falciparum infections during a dramatic malarial decline.Malaria is receding in many endemic countries with intervention scale -up against the disease. However, this resilient scourge may persist in low-grade submicroscopic infections among semi-immune members of the population, and be poised for possible resurgence, creating challenges for detection and assessment of intervention impact. Parasite genotyping methods, such as the molecular barcode, can identify specific malaria parasite types being transmitted and allow tracking and evaluation of parasite population structure changes as interventions are applied. This current study demonstrates application of pre-amplification methods for successful detection and genotyping of residual Plasmodium falciparum DNA was performed. Plasmodium falciparum infections were genotyped using a 24 SNP-based molecular barcode assay using real-time PCR. Submicroscopic parasitaemia samples were subjected to pre-amplification using TaqMan PreAmp Master Mix following the manufacturer’s instructions before SNP barcode analysis.The study was a prospective cross-sectional design and based on a 2,000 sq km vicinity of Macha Mission Hospital in southern Zambia. Willing and predominantly asymptomatic residents of all ages were screened for malaria by microscopy during the 2005 and 2008 transmission seasons, with simultaneous collection of dried blood spots (DBS) on filter paper, and extraction of p <0.0005, n = 84). Furthermore, nine samples negative for parasites by microscopy and standard quantitative PCR amplification were positive after pre-amplification.There was a dramatic decline of malaria between 2005 and 2008, and the geometric mean parasite density (95% CI) fell from 704/μL  in 2005 to 39/μL (23–68) in 2008, culminating in a large proportion of submicroscopic infections of which 90% failed to yield ample DNA for standard molecular characterization among 2008 samples. Pre-amplification enabled successful detection and genotyping of 74% of these low-grade reservoir infections, overall, compared to 54% that were detectable before pre-amplification (Pre-amplification allows analysis of an otherwise undetectable parasite population and may be instrumental for parasites identification, tracking and assessing the impact of interventions on parasite populations during malaria control and elimination programmes when parasitaemia is expected to decline to submicroscopic levels. Through support from the WHO Global Fund Rollback Malaria (RBM) programme, the President’s Malaria Initiative (PMI), Malaria Control and Evaluation Partnership (MACEPA) and other public-private partnerships, endemic countries are scaling up effective interventions against malaria -4. DecliPlasmodium falciparum populations during scaled up interventions.However, the malaria decline has not been universal, both between and within countries , posing The study was based in a 2,000 sq km vicinity of the Malaria Institute at Macha (MIAM). This area is intrinsically hyperendemic for malaria and is transitioning to hypoendemicity under scaled up interventions against the disease. The resident population comprises essentially stable subsistence farming communities of the Batonga tribe. Ethical approval was obtained for the use of these samples for parasite genotypic analysis.The study consisted of prospective cross-sectional field surveys for malaria from 2005 and 2008. Surveys were conducted in villages that were selected by simple random procedure each year, with individuals of all ages assembling at pre-arranged meeting points for malaria screening.Malaria screening was conducted by microscopy (2005 and 2008) or rapid diagnostic test  following the manufacturer’s instructions. Real-time DNA quantification was performed on resultant sample extracts to determine DNA content, as described by Daniels et al.[P. falciparum strains were used as standards in the quantitative real-time reaction with a total reaction volume of 5 μL.P. falciparum infection samples from 2005 and 2008 were subjected to polymorphic analysis using a 24 SNP-based parasite molecular barcode assay on an ABI 7900 real-time PCR system, also as described by Daniels et al.[P. falciparum genome to be selectively neutral; concatenating the genotypes from these independent assays generates what is known as the molecular barcode. Samples from low-grade infections, which gave DNA concentrations below the detectable threshold for the barcode assay (0.005 ng/μL), were subjected to pre-amplification  to enhance the template concentration specifically in the regions interrogated by the molecular barcode, following the manufacturer’s instructions. Pre-amplification reactions comprised 5 μL template DNA extract, 1X TaqMan assay mixture containing 0.2x TaqMan barcoding and quantification primers, and 1X Preamp master mix in 20 μL total volumes. Following pre-amplification, samples were re-quantified as described above.The ls et al.. The molSeveral other master mixes were also evaluated to determine which offered the best performance, testing a master mix from another vendor  and a standard genotyping master mix  under the same pre-amplification thermal cycler conditions described above.t-test for quantitative data. Temporal changes in parasitaemia and demographic profiles were examined using Student’s t-test and Mantel-Haenzel’s Chi-square, respectively.Differences in DNA yields from field DBS extracts for protocols with and without pre-amplification were tested using McNemar’s Chi-square and the paired P. falciparum infections exhibited substantially lower parasite densities in 2008 compared to 2005  . The standard genotyping master mix did not improve the product yield compared to both of these pre-amplification products  amplicon assays in a single reaction, thus allowing genotyping of multiple loci. The amplification conditions, primarily the low primer concentration, limited number of amplification cycles, and increased extension time, together reduce chimera formation and maintain sample integrity. This strategy is not specific to TaqMan-based assays, and pre-amplification has been successfully incorporated for used in gene-specific assays as well as high-resolution melting assays for drug resistance genotyping [Here the application of a TaqMan pre-amplification assay demonstrated that this approach significantly increased the proportion of low-parasitaemia field samples that could undergo molecular genotyping, leading to successful analysis of subpatent notyping .By overcoming the detection challenge posed by these low-grade silent infections, pre-amplification may aid in monitoring intervention efficacy, tracking of the last bastions of residual parasites and thwarting potential resurgences during malaria control and elimination programs. As malaria-endemic regions reduce malaria transmission and work toward elimination, it will become critical to have tools and strategies to identify and track asymptomatic carriers, with highly reduced parasitaemia, that contribute to incident infections.P. falciparum infections during malaria control and elimination programmes.The TaqMan pre-amplification assay provides a means for sensitive detection and tracking of residual DBS: Dried blood spot; DNA: Deoxyribo nucleic acid; LEV: Low elution volume; PCR: Polymerase chain reaction; SNP: Single nucleotide polymorphism; ABI: Applied Bioystems Inc; CI: Confidence interval; RBM: Roll Back Malaria; PMI: US President’s Malaria Initiative; MACEPA: Malaria Control and Evaluation Partnership; MIAM: Malaria Institute at Macha; RDT: Rapid diagnostic test; qPCR: quantitative PCR; bp: base pair; GMPD: Geometric mean parasite density.The authors declare that they have no competing interests.SM participated in the study planning, design, field data collection, laboratory assays, and performed data analysis and writing of the manuscript. RD introduced the pre-amplification assay and developed the protocol as well as ran laboratory assays and contributed to the manuscript. AS was a co-investigator and contributed genomics technical guidance as well as manuscript write up. DW provided guidance for the project. PT was the local field site project director and contributed to the manuscript. SV planned the science, supervised the molecular assays and provided overall guidance of the research and manuscript write up. All authors read and approved the final manuscript.Real-time PCR cycle threshold (Ct) values for barcode SNPs, at decreasing concentrations of input Plasmodium falciparum 3D7 DNA, either with or without pre-amplification. Eight samples (numbered 1 through 8) of 3D7 DNA at variable input concentrations ranging from 15 to 0.0000015 nanograms (ng) are shown with or without pre-amplification (PA). Resulting Ct values for each molecular barcode assay (numbered 1 through 24) are shown with UD (undetermined) indicating that a Ct value could not be determined due to low amplicon signal.Click here for fileGenotyping results for barcode SNPs, at decreasing concentrations of input Plasmodium falciparum 3D7 DNA, either with or without pre-amplification. Eight samples (numbered 1 through 8) of 3D7 DNA at variable input concentrations ranging from 15 to 0.0000015 nanograms (ng) are shown with or without pre-amplification (preamp). Resulting calls for each molecular barcode assay (identified by SNP assay chromosome and coordinate) are shown with a “dash” (-) indicating that the genotyping call could not be determined for that assay. “Blank” indicates a no template control.Click here for fileGenotyping results from alternative pre-amplification kits. Amplification of the 24 molecular barcode assays (identified by chromosome and coordinate number) from control samples  or Roche samples  at various concentrations of input 3D7 P. falciparum DNA ranging from 1 to 0.0001 nanograms (ng). A “dash” (-) indicates that the genotyping call could not be determined for that assay.Click here for file


Leishmania donovani complex and Plasmodium falciparum in naturally co-infected patients altered the immunological response elicited by the two pathogens individually.The immune system plays a critical role in the development of co-infections, promoting or preventing establishment of multiple infections and shaping the outcome of pathogen-host interactions. Its ability to mediate the interplay between visceral leishmaniasis (VL) and malaria has been suggested, but poorly documented. The present study investigated whether concomitant infection with L. donovani and P. falciparum to mutually interact at the immunological level. Progressive polarization towards type-1 and pro-inflammatory cytokine patterns characterized the co-infected patients, whose response partly reflected the effect elicited by VL  and malaria , and partly resulted from a synergistic interaction of the two diseases upon each other (IL-17A). Significantly reduced levels of P. falciparum parasitaemia (P <0.01) were detected in the co-infected group as opposed to the malaria-only patients, suggesting either a protective or a non-detrimental effect of the co-infection against P. falciparum infection.Circulating levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, IL-17A and tumor necrosis factor (TNF) were assessed in sera of patients infected with active VL and/or malaria and healthy individuals from Gedarif State, Sudan. Comparative analysis of cytokine profiles from co- and mono-infected patients highlighted significant differences in the immune response mounted upon co-infection, confirming the ability of L. donovani and P. falciparum co-infect the same patient, with potential implications on the course and resolution of these diseases.These findings suggest that a new immunological scenario may occur when  Co-infection of a host by multiple parasite species is a commonly observed condition, where individual susceptibility and infectiousness are affected at various levels -3. InterLeishmania donovani complex successfully colonize macrophages and other reticulo-endothelial cells of the lymphoid system, by altering signaling pathways associated with parasite killing and adaptive immunity engagement [Leishmania parasites are incapacitated to function as cytolytic and T-cell priming effectors, causing immune dysfunction and tissue injury. Resistance to infection is conferred by development of effective T helper cell 1-type (Th1) responses, mounted upon release of a pleiotropic interleukin (IL)-12 and interferon (IFN)-γ cytokine network, and boosted by pro-inflammatory (tumor necrosis factor (TNF), IL-23, IL-17A) and Th2-promoting (IL-4) mediators [L. donovani appears to be blunted by induction of the regulatory T cell subset Tr1, rather than Th2 or Th3 clusters, through an IL-10 mediated mechanism [Plasmodium falciparum infections [Plasmodium spp., with clinical implications depending on the type and timing of cytokine release. Early type-1 responses, dominated by IFN-γ, IL-2 and TNF, have been reportedly associated with inhibition of liver stage development [Plasmodium-specific CD8+ and CD4+ cells [Visceral leishmaniasis (VL) and malaria are two major parasitic diseases which overlap geographically and may co-exist in the same patients ,10. Partgagement ,12. As aediators -17. Thusechanism -20. Anerfections -25, whicelopment -31, resoelopment -34 and delopment , as confelopment -38. Releelopment -41 and s4+ cells ,37,39, r4+ cells ,43. Simi4+ cells ,44,45.P. chabaudi chabaudi and L. infantum[P. yoelii and L. mexicana amazonensis[Plasmodium-triggered release of splenic IL-4, as assessed by gene expression [L. infantum strains resulted in a reduced proliferation of P. berghei, with no aggravation of the Leishmania infection [Leishmania and Plasmodium spp. interactions upon co-infection.Despite VL and malaria co-infection cases being encountered across co-endemic areas, little work has been done so far to examine the dynamics of this co-infection and its effect on host immunity. Studies performed in co-infection murine models of  infantum, and of azonensis,48 have pression . Conversnfection . Whilst P. falciparum parasitaemia, emphasizing the importance of immune-mediated interactions in poly-parasitism.In the present study, the cytokine profiles of naturally co-infected patients were examined. Blood samples from patients actively infected with VL and/or malaria and from healthy individuals were collected during an exploratory survey conducted in Gedarif State, Sudan, and the level of nine different cytokines selected from across the four major response arms of the immune system were assessed simultaneously. The comparative analysis between co- and mono-infected groups highlighted substantial differences in the cytokine profile of these patients and their levels of L. donovani, located in Gedarif State, Sudan. Patients were recruited at Tabarak Allah Hospital, which hosts a VL treatment center managed by Médecins sans Frontières since January 2010. Seasonal and unstable malaria prevails in the area, where co-infection rates of 18% to 45% were recorded amongst Tabarak Allah VL in-patients (2005-2010) [The sample collection was performed in February 2011 in the village of Tabarak Allah, an endemic area of All individuals included in the study originated from Gedarif State and aged six years or above. Eligibility for the study was precluded to children up to age six, due to immature status of their immune system ,51. IndiP. falciparum parasitaemia was performed by microscopy, counting the total number of parasites per 200 WBCs, as previously described [Leishmania were measured in sera or, when unavailable, filter paper-spotted blood using the DAT and two commercially available rk39 tests, the DiaMed IT-Leish®  and the Kalazar Detect™ . The following conditions were considered indicative of VL infection: a) DAT titers ≥3,200, with or without positive rk39 test outcomes; b) DAT titers =1,600 with at least one confirmatory rk39 test; c) DAT titers <1,600 with positive result in the field  and a positive rk39 test. Samples that did not fulfill these criteria were excluded from the study (n = 8) or re-categorized (n = 3), if tested positive for malaria only. Thin and thick blood smears of all study participants were microscopically re-assessed to confirm or exclude presence of P. falciparum parasites. When slide re-examination resulted in discordant outcomes, a rapid test  for detection of P. falciparum and Plasmodium spp. was performed on the corresponding serum sample. Positive results obtained with the serological test were considered confirmatory of malaria cases (n = 4), while specimens which tested negative were excluded from the analysis (n = 2). In addition, five other cases  were excluded from the study, due to poorly reliable test outcomes, missing samples or diagnosis of non-P. falciparum malaria. From the 102 participants included at study entry, 15 were excluded because they did not match the diagnostic criteria, narrowing the sample size to 87 cases.For categorization of study subjects, the following diagnostic algorithm was implemented. All patients presenting at the study hospital with symptoms of VL and/or malaria, including fever, weight loss, hepato-splenomegaly and anemia, were given physical examination. Finger-prick blood was assessed by microscopy for diagnosis of malaria and by direct agglutination test (DAT) for diagnosis of VL. Assessment of escribed . Artemisescribed . Accordiescribed , and havn = 29), defined as VL-seropositive individuals who fulfilled the clinical case definition of VL and tested negative for malaria.Primary VL cases (n = 21). This group included parasitologically-confirmed cases of P. falciparum malaria who presented at hospital with clinical symptoms, such as fever, hepato-splenomegaly and anemia, and lacked Leishmania-specific antibodies.Clinical malaria patients (n = 15), defined as VL-seropositive individuals diagnosed with a P. falciparum malaria infection.VL and malaria co-infected patients (n = 22) with a VL-seronegative profile and no microscopically detectable malaria in peripheral blood.Healthy endemic controls (n = 83) and, when unavailable, plasma samples (n = 5), since exclusion of plasma assessments had no effect on the outcome of the analysis.Cytokine levels in patients’ samples were determined using a 9-milliplex magnetic bead-based immunoassay , performed according to manufacturer’s instructions. Briefly, 25 μL of magnetic beads internally labeled with multiple fluorophores and coated with specific capture antibodies against one of the nine cytokines  was added to 25 μL of patient sample and an equal amount of assay buffer. Standards and quality controls for each cytokine were mixed likewise. After an overnight incubation followed by extensive wash to remove unbound proteins, 25 μL of biotinylated detection antibodies was added and the fluorescence of the streptavidin-phycoerythrin complex measured by a MAGPIX® . A minimum of 50 beads per cytokine was measured. Interpolation of sample concentrations using a five-parameter logistic standard curve was performed with the MILLIPLEX® Analyst 5.1 software . The lower detection limits of the assay were: 0.53 pg/mL for TNF, 0.54 pg/mL for IL-2, 0.34 pg/mL for IL-4, 0.97 pg/mL for IL-6, 0.59 pg/mL for IL-10, 0.74 pg/mL for IL-12p70, 0.57 pg/mL for IL-13, 0.22 pg/mL for IL-17A and 0.17 pg/mL for IFN-γ. Comparative analysis of cytokine profiles included both serum  rank correlations were computed to assess statistical dependence between cytokine levels and the corresponding patient demographic/clinical characteristics and between each cytokine pair. Data analysis was conducted with STATA software .Group-wise comparison of cytokine values was performed using nonparametric statistics. Mann-Whitney U test and Kruskal-Wallis test were used to examine whether continuous variables from two or multiple groups, respectively, originated from the same distribution, whilst comparison of categorical variables was performed using the Chi-square test. P. falciparum infection intensity of patients diagnosed with VL and/or malaria are summarized in Table P <0.01), no major differences in the baseline distribution of these variables were observed, with patients sharing most of their demographic and clinical characteristics. Mild anemia with normal leukocyte counts characterized most of the actively infected population, who largely consisted of young boys. Co-infected patients displayed some intermediate features between the VL and the malaria groups, including lower male to female ratio, younger age and milder anemia as compared with the VL patients.The sex, age, hemoglobin level, WBC count and P <0.0001) : undetectable in all, but one control, this interleukin reached or exceeded the detection limit (0.74 pg/mL) in nearly half of the VL patients     and, to a less extent, IFN-γ and IL-17A (P <0.01), IL-4 and IL-12p70 (P <0.05)    between the two groups . None of the three variables displayed a systematic confounding effect on the cytokines examined here, whose variations amongst the three study groups remained overall stable .To exclude that the cytokine profiles observed for VL and/or malaria infected patients may have been biased by differences in their demographic and clinical features, the study population was stratified by sex, age or ia Table . For agevs. IL-6) and type-1 cytokines (IFN-γ vs. IL-12p70), as well as for type-1 vs. pro-inflammatory , and type-2 vs. pro-inflammatory  and type-1 cytokines (IL-13 vs. IL-12p70 & IFN-γ). Conversely, no correlation could be identified between the examined cytokines and the DAT titers as well as the P. falciparum parasitaemia of co-infected patients (data not shown).Specific mechanisms cross-regulate the production of cytokines, with positive and negative feed-back loops to control secretion of signaling molecules. To understand the link interconnecting each of the cytokines examined here, their correlation was investigated using the Spearman’s correlation rank test. As shown in Table Leishmania and Plasmodium parasites to manipulate host immunity and co-inhabit part of the same lymphoid tissues suggests the possibility that the two diseases may interact with each other, when co-occurring in the same host. This is demonstrated for the first time in naturally co-infected patients by the pilot study presented here. Comparative analysis of cytokine profiles from co- and mono-infected patients highlighted substantial variations in the immune response mounted upon co-infection, confirming the ability of L. donovani and P. falciparum to mutually interact at the immunological level. Patients harboring both leishmanial and malarial parasites responded with an overall increase in type-1 and pro-inflammatory cytokine release, which partly reflected the effect elicited by VL  and malaria (IL-2), and partly resulted from a synergistic interaction of the two diseases upon each other (IL-17A). Secretion of IL-13 in co-infected patients significantly exceeded the amounts found in VL patients and displayed positive correlations with most of the examined cytokines. Although this trend can be seen as an attempt of the immune system to contain the effects elicited by type-1 and pro-inflammatory cytokines , the finding remains poorly representative, reflecting the response of 4 patients only, with the remaining co-infected patients (n = 11) displaying negligible levels of IL-13 just as most of VL and malaria patients.The ability of + Th17 cells has been associated with resistance to human VL [L. donovani infections [vs. the VL mono-infected cohort, besides the already elevated IFN-γ and IL-12p70, may be indicative of a favorable, possibly improved, prognosis for VL, though the present data do not allow to draw conclusions in this respect. In support of this speculation is the finding of a recent study conducted in Barbar el Fugarra, a Sudanese village situated only a few tens of kilometers away from Tabarak Allah Hospital (where patients in this study were recruited), in which peripheral blood mononuclear cells (PBMCs) isolated from VL-seropositive individuals who did not develop disease at any time during the 6-year survey, secreted significantly higher IL-17 levels when challenged with L. donovani in comparison with VL-seropositive individuals who became symptomatic within 6 months from the evaluation [+ T cells, CD8+ T cells or macrophages) and related cytokines  has been observed in P. vivax natural infections [P. berghei, P. chabaudi and P. fragile animal models [P. falciparum infections is yet to be demonstrated. Transcriptional profiling of PBMCs isolated from P. falciparum-infected patients has recently highlighted a Th17/Thαβ driven bias in the immune response mounted against malaria, with up-regulation of several Th17- and neutrophil-related genes [via interferon α and β [Plasmodium infection, for the development of malaria adaptive immunity  and the nitric oxygen-dependent suppression of intra-hepatocytic forms. The above-shown data confirm the leading presence of IFN-γ (P <0.0001), followed by TNF (P <0.05) and IL-4 (P <0.05), in the sera of VL patients as compared with the malaria ones, and clearly identify a shift towards type-1/pro-inflammatory polarization when malaria co-occurred with VL. In addition, a significantly reduced P. falciparum infection intensity was observed among co-infected patients, suggesting improved tolerance of these individuals to the malaria disease. Whether this reduced susceptibility resulted from the VL-driven pre-immune response remains to be demonstrated. The pioneer work of Adler et al. on co-infected hamsters [P. berghei for effect of the Leishmania infection, supporting the idea of a VL-triggered cross-immunity against malaria, whereas the more recent mouse model data [Leishmania parasites visceralize in the liver, too). Moreover, mice and hamsters are not equally representative models of the VL disease, whose clinico-pathological features in humans are better reproduced by the golden hamster model [Measurement of IL-17A levels allowed to distinguish the co-infected patients from both VL and malaria mono-infected counterparts, indicating that both diseases synergistically concurred to its up-regulation. Better known for its pro-inflammatory effects in allergic and autoimmune conditions, IL-17 has been recently implicated in the protective immunity towards bacterial, fungal and protozoan infections , where ihuman VL  and posifections , suggestaluation . Interesfections  as well l models -62, wherl models -61. Conved genes ,64 and i α and β . Trigger α and β , necessa α and β ,65-67, phamsters  highlighdel data -48 seem er model .P. falciparum blood parasitaemias. Whether these differences are linked to their particular diagnosis, however, is unknown, as patients were recruited sequentially and discernment between clinical and sub-clinical co-infection cases is not possible if one of the two diseases manifests with symptoms. Therefore, recruitment of asymptomatic, but parasitaemic individuals for each of the two infections may be useful to control for non-homogeneous group-wise comparisons. Absence of pre-existent disorders was based on patient reporting only, with no diagnostic procedure performed, other than those ones aimed to confirm VL or malaria. Finally, longitudinal rather than cross-sectional assessments should be endorsed, as they could help identifying those fundamental associations amongst parasite load, cytokine response and clinical picture which are keys to the interpretation of present data. Similar studies may not only clarify the exact role of the VL-malaria co-infection on P. falciparum proliferation, but they would be pivotal for understanding the clinical implications that arise from the different cytokine profiles.The exploratory nature of this survey implies its design and findings are limited by the small sample sizes and the lack of subject matching between groups, although no significant difference in the distribution of patients’ demographic and clinical features was observed. Diagnosis of VL in clinical suspects was confirmed by serology, according to the national policy, precluding any analysis on parasite loads and their link with cytokine profiles. Assessment of malaria parasitaemia, on the contrary, was performed on peripheral blood films, but the low sensitivity of microscopy observation inevitably limits its reliability as a quantitative assay. Moreover, in the absence of a molecular screening of the recruited individuals, the risk of sub-microscopy malaria infections being carried by the VL patients and/or apparently healthy controls cannot be excluded. Malaria mono- and co-infected patients exhibited different L. donovani complex and P. falciparum appear to shape the immunological response taking place in the co-infected host and possibly the intensity of infections that follow. Similar scenarios have been depicted with other malaria co-infections, indicating that the potential implications arising from multiple pathogen-host relations should be addressed when designing malaria vaccine trials. Careful consideration of parasite interplays should be taken when defining the best strategy for clinical management of VL-malaria co-infections, to ensure that immune homeostasis may be restored without harming patient’s clinical course.Immune-mediated interactions between Plasmodium falciparum; PBMC: Peripheral blood mononuclear cell.VL: Visceral leishmaniasis; IFN-γ: Interferon-gamma; IL: Interleukin; TNF: Tumor necrosis factor; Th: T helper; NK: Natural killer; TGF-β: Transforming growth factor-beta; WBC: White blood cell; WHO: World Health Organization; DAT: Direct agglutination test; MΦ: Macrophage; Hb: Hemoglobin; P.f: The authors declare that they have no competing interests.EvdB designed the study, carried out the cytokine measurement and drafted the manuscript. AT recruited the study participants, performed collection of data and samples and participated to the sample analysis. MS performed the statistical analysis of the data. PM and EM conceived of the study, participated in its design, contributed to the sample analysis and helped to draft the manuscript. MG revised the critical content of the manuscript. BN participated to the study design and coordinated the work in the field. HS conceived of the study, participated in its design and coordination, contributed to the sample analysis and helped to draft the manuscript. All authors read and approved the final manuscript.Cytokine levels (pg/mL) as measured in all patient samples.Click here for file


Assessments of the epidemiology of malaria over time are needed to understand changes in transmission and guide control and elimination strategies.A longitudinal population study was established in 1985 in Nyamisati village in the Rufiji River Delta, Tanzania. A physician and research team lived in the village 1984–2000. Parasite prevalence by microscopy and two PCR methods, spleen rates and haemoglobin levels were measured in repeated cross-sectional surveys between 1985 and 2010. Passive surveillance of malaria cases was maintained until end 1999. Bed nets were distributed after the surveys 1993, 1999 and 2010.msp2-PCR , whereas real-time PCR prevalence remained higher throughout the 1990s . In 2010, parasite prevalence was 17.8% by real-time PCR and 16.3% by msp2-PCR, and estimated to 4.8% by microscopy. Spleen rates in children ages two to nine years decreased earlier than parasite prevalence, from >75 to 42% in the 1980s, to nil during the 1990s. The prevalence of severe and moderate anaemia decreased from 41.1 to 13.1%. No deaths at the time of acute malaria were recorded when the research team lived in the village.In 1985, overall parasite prevalence by microscopy was 70% (90% in children ages two to nine years). The prevalence decreased gradually by microscopy  and A marked decline in malaria transmission was observed over 25 years. The decrease was detected after the arrival of the research team and continued gradually both before and after distribution of bed nets. Spleen rates and microscopy identified early changes when transmission was still intense, whereas real-time PCR was a more sensitive metric when transmission was reduced. The study provides historical data on malaria within a closely monitored rural village and contributes to the understanding of changing epidemiology in sub-Saharan Africa.The online version of this article (doi:10.1186/1475-2875-13-459) contains supplementary material, which is available to authorized users. Malaria remains a major global health problem. The infection is spread in tropical and subtropical regions with the most intense transmission in sub-Saharan Africa. Several countries have reported reduced transmission over the last decade . UnderstPlasmodium falciparum populations [The Nyamisati Malaria Research Project was set up in 1985 in a fishing village in Rufiji District, Tanzania. A medical team established a health clinic in this community, which previously lacked primary health care, and became interested in understanding the impact of malaria. A research project, in which all villagers were invited, aimed to describe the epidemiology and clinical aspects of malaria longitudinally. The first cross-sectional surveys in 1985–1988 revealed a holo-endemic setting with 90% parasite prevalence and >75% spleen rates in two to nine years old children . Repeateulations ; parasitulations , 12; as ulations –16, and ulations , 18. Repulations  inspiredP. falciparum prevalence were evaluated using microscopy and two PCR methods: species-specific real-time PCR and merozoite surface protein-2 (msp2) genotyping PCR. Moreover, spleen rates, haemoglobin levels and incidence of febrile malaria were assessed over time. The study shows a marked yet gradual decline in malaria transmission in Nyamisati, and demonstrates that different methods to monitor malaria endemicity are informative at different stages of changing transmission intensity.This paper summarizes the epidemiology of malaria in Nyamisati village over 25 years. Long-term changes in Nyamisati village 7°47′S; 39°16′E) is situated in the Rufiji River Delta, the largest area of mangrove forest on the East African coast, in the Pwani region, Tanzania . Emergencies and more complicated medical conditions were treated by the physician or referred to Mchukwi Hospital, 72 km away.Malaria was diagnosed by microscopy and treated with chloroquine during the first years and later on with sulphadoxine-pyrimethamine (SP), alone or in combination with oral quinine. The different anti-malarial drugs administered at the health unit are summarized in Additional file In 1985, 3% of households owned a bed net. Between October 1993 and April 1994, 300 insecticide-treated bed nets (ITNs) were distributed to pregnant women and families with small children. The research team also distributed bed nets after the cross-sectional surveys in 1999 (900 ITNs not re-impregnated) and 2010 . The bed net coverage, assuming 1.8 individuals per net  was 4% iA passive surveillance system for malaria was maintained between 1986–1989 and 1993–1999. The villagers were instructed to come to the clinic in the event of fever. Individuals who presented with fever (axillary temperature >37.5°C), or history of fever (“hot body”) within 24 hours, and had parasites detected by microscopy were administered anti-malarial treatment.Cross-sectional surveys were performed in the population at the start of the long rainy season in 1986–1988, 1993–1999 and 2010. A subset of children aged ten to 19 years was sampled in January 2003 . The croThick and thin films were stained with Giemsa and analysed by conventional light microscopy. Parasite densities were estimated per microlitre (μl) of blood and enumerated against the number of leukocytes assuming 8,000 leukocytes/μl. Blood films were considered negative if no parasites were detected in 100 microscopic fields of the thick smear.Plasmodium species was performed by a multiplex probe-based method [P. falciparum msp2 gene was performed by nested PCR using fluorescently labelled primers targeting the two allelic types of msp2 in separate nested reactions, fragment sizing by capillary electrophoresis in a DNA sequencer (Applied Biosystems), and data analysis by GeneMapper software [msp1 block 2 genotyping PCR from 2003 was available from a previous study [P. falciparum.Parasite prevalence was assessed by PCR at the cross-sectional surveys 1994, 1995, 1999, and 2010 when most complete surveys across ages were performed. DNA was purified from frozen packed cells by Qiagen blood mini kit (Qiagen) , phenol-chloroform extraction (1999), and using an automated Qiagen BioRobot® M48 (2010). Real-time PCR for detection of d method  using thsoftware . Data onus study . The genmsp2-PCR and real-time PCR. Missing prevalence data for msp2-PCR in 1986 and 1993, as well as for microscopy in 2010 (due to staining artefacts after storage), were estimated using the prevalence estimation tool developed by Okell et al., validated for nested PCR data from all age cross-sectional surveys [Data analysis was performed using GraphPad Prism 5.0  and Stata 13.0 . The data from 1985–1988 were retrieved from summarized data . Parasit surveys . Agreeme surveys . To evalAll data and blood samples were collected with informed consent from each participant and/or their guardians. The project was approved by the Nyamisati Village Board, the Ethical Review Board of the National Institute for Medical Research in Tanzania and the Central Ethical Review Board in Stockholm, Sweden.The population of Nyamisati increased from 451 individuals in 1985 to 1,553 individuals in 1999, as recorded by the research team. By 2012, 2,350 individuals lived in the village according to the National Bureau of Statistics in Tanzania . The demP. falciparum in all surveys. In 2010, the microscopy prevalence was estimated at 4.8%  from the corresponding msp2-PCR data  in 1986–1988, 46.3% (95% CI 42.0-50.5%) in 1993, 38.9% (95% CI 35.5-42.4%) in 1994, and 26.7% (95% CI 23.8-29.7%) in 1999 , whereas real-time PCR positivity remained at similar levels . In 2010, real-time PCR prevalence was 17.8% (95% CI 15.1-20.7%) and msp2-PCR 16.3% (95% CI 13.7-19.1%)  and highest in 1999 (17.0%) Table B. Among In two to nine years old children, the age group conventionally used to estimate malaria endemicity , the parmsp1-PCR [The peak parasite prevalence by microscopy gradually shifted to older children. In 1994–1999, the peak prevalence was seen in children aged five to eight years by microscopy and nine to 12 years by PCR. In 2010, the peak PCR prevalence had shifted to the 13–16 years group  and further reduced in 2010 . Multinomial logistic regression analysis showed that the prevalence of severe and moderate anaemia decreased significantly from 41.4% (95% CI 36.4-46.5%) and 29.6% (95% CI 24.9-34.3%) in 1994 to 33.3% (95% CI 30.2-36.4%) and 24.8% (95% CI 22.0-27.7%) in 1999 and further to 27.6 (95% CI 24.5-30.7%) and 13.1% (95% CI 10.7-15.4%) in 2010, respectively. Prevalence of mild anaemia remained stable throughout the period at 10.2% (95% CI 7.1-13.3%), 11.9% (95% CI 9.7-14.0%) and 11.9% (95% CI 9.7-14.2%) in 1994, 1999 and 2010, respectively .The Nyamisati Malaria Research Project provides longitudinal data on the epidemiology of malaria in a rural African village. A substantial, yet gradual, decline from holo- to hypo-endemicity was observed over 25 years, as reflected by changes in parasite prevalence from 90 to <10% in two to nine years old children. Spleen rates decreased before changes in parasite prevalence were observed. Parasite prevalence by PCR, especially real-time PCR, remained high during the 1990s, and then revealed a substantial decrease by 2010.This study adds to a number of reports of changing malaria epidemiology, giving hope for elimination of malaria in some settings , 19–21. The changes in transmission in Nyamisati were detected by different methods over time which demonstrates the advantage of using several metrics, i.e., spleen rates, microscopy and PCR in longitudinal surveillance. Molecular methods, especially PCR, have become increasingly used in epidemiological studies, and often reveal a large proportion of sub-microscopic infections . The advThe first malariometric index to capture changes in the burden of malaria was spleen rate, which decreased down to nil while parasite prevalence by microscopy in the same two to nine age group remained above 75%. Spleen rate (the proportion of children with enlarged spleens) was once the standard method to define malaria endemicity , but is Microscopy showed a gradual decrease in parasite prevalence over time. Among asymptomatic individuals, microscopy detected an even more rapid decrease in parasite prevalence, indicating the value of defining the clinical status of individuals in cross-sectional epidemiological surveys.msp2-PCR allowed estimates of missing microscopy data according to the method by Okell et al., which is based on nested PCR data [msp2-PCR, likely due to differences in targeted genes . Real-tsp2 gene , demonstThe age patterns of parasite prevalence changed over the study period. The age group with the highest prevalence gradually shifted to older children, suggesting reduced immunity as a result of lower exposure as previously described , 39. DurCases of severe and fatal malaria were remarkably rare in the village. The continuous presence of a physician could have contributed to the paucity of severe malaria. However, malaria mortality was also low at the referral hospital in Mchukwi  and abseP. falciparum infections [The proportion of individuals with severe and moderate anaemia declined over time. Increasing haemoglobin levels were also reported in the Pwani region in the Tanzania Demographic and Health Survey in 2010 . There wfections .Declining transmission has been associated with introduction of one or several interventions, such as ITN/LLIN and artemisinin-based combination therapy (ACT) , 31. HowAnopheles vector populations decreased markedly [Anopheles gambiae complex were Anopheles gambiae s.s, Anopheles arabiensis and Anopheles merus in 2003–2004 [Interestingly, the declines in parasite prevalence over two decades in north-eastern Tanzania  and Senemarkedly , along wmarkedly  and entomarkedly . Entomol003–2004 . Changes003–2004 , nonetheThe presence of a research team closely monitoring malaria with prompt and efficacious treatment is likely to have contributed to reducing parasite prevalence in Nyamisati and other closely monitored communities , 31. AlrThe study is limited by data not being equally available for all variables at all time points. Cross-sectional surveys only provide a snapshot of the malaria transmission and incidence data available for only part of the study period. Nonetheless, the major strength of the study is that the continuous malaria surveillance and cross-surveys were performed at the same time of the year (at the beginning of the rainy season) by the same physician and research team , using the same sample collection tubes and processing, thus providing a unique consistency and comparability over this extended period of time. Moreover, only the survey years with complete data and samples available throughout the different ages were included in the analyses. Another limitation might be that DNA extractions were performed by different methods for the different years, which potentially could affect the sensitivity of detection. However, the PCR analyses were recently performed in a highly standardized fashion by the same individual. Moreover, the samples collected 1994 and 2010 were extracted by similar commercial kits  and can thus be considered highly comparable.The study demonstrates declining malaria transmission in this East African village over 25 years. Moreover it demonstrates the value of using different methods to monitor malaria transmission. Spleen rates and microscopy detect early changes when transmission is still intense. Conversely, PCR, especially real-time PCR, does not detect initial changes in transmission, but robustly and sensitively reveals a large proportion of sub-microscopic infections as transmission declines in a population.The gradually declining transmission appears to have started already in the 1980s, with the arrival of the research team in 1985, and continued gradually after that. From the present data it is not possible to determine how much the interventions with ITNs contributed to the decline, partly because of the lack of data between 2000 and 2010. Further studies, using sero-epidemiological tools might contribute to the understanding of patterns and timing of transmission changes in periods when parasite prevalence data is missing. The historical data on malaria within this longitudinally, closely monitored rural village contributes to the understanding of changing epidemiology in sub-Saharan Africa over the last decades.Monthly rainfall (solid line) and temperature patterns, including high (dashed line) and low (dotted line) temperatures, in southeast coastal Tanzania from 1980 to 2010. Data from meteorological stations of Utete, Kingupira, Kilwa Masoko, and Dar es Salaam International Airport, kindly provided by Tanzania Meteorological Agency. (PDF 90 KB)Additional file 1: Type of anti-malarial drugs administered through the health and research unit during the 1990s.(PDF 867 KB)Additional file 2:Agreement between real-time PCR results with microscopy andmsp2-PCR.(DOCX 11 KB)Additional file 3:Malaria incidence in Nyamisati 1993–1999 evaluated using univariate and multivariate generalized estimating equation negative binomial regression models.(DOCX 13 KB)Additional file 4:


Plasmodium falciparum parasites lacking the PfHRP2 protein, the most common target antigen for detection of P. falciparum, have been reported in some regions. Therefore, accurately mapping the presence and prevalence of P. falciparum parasites lacking pfhrp2 would be an important step so that RDTs targeting alternative antigens, or microscopy, can be preferentially selected for use in such regions. Herein the available evidence and molecular basis for identifying malaria parasites lacking PfHRP2 is reviewed, and a set of recommended procedures to apply for future investigations for parasites lacking PfHRP2, is proposed.Malaria rapid diagnostic tests (RDTs) play a critical role in malaria case management, surveillance and case investigations. Test performance is largely determined by design and quality characteristics, such as detection sensitivity, specificity, and thermal stability. However, parasite characteristics such as variable or absent expression of antigens targeted by RDTs can also affect RDT performance.  Malaria remains endemic in 104 countries, representing a major public health problem in many. To imprThe availability of quality-assured malaria RDTs in recent years has dramatically increased access to malaria diagnostics. Malaria RDTs are not only playing an increasing critical role in malaria case management, but also in malaria surveillance and case investigations in malaria elimination countries. They are also widely used in non-endemic settings to diagnose malaria in travellers returning from the tropics.Plasmodium spp.[Plasmodium falciparum, target an antigen that is unique to P. falciparum, the histidine-rich protein 2 (PfHRP2). Many antibodies used to detect PfHRP2 also detect histidine-rich protein 3 (PfHRP3), a protein that shares a high degree of similarity in amino acid sequence with PfHRP2[P. falciparum infections.Malaria RDTs are lateral flow devices that use antibodies to capture and detect parasite proteins by immunochromatography. Currently, over 200 brands of RDTs are commercially available for detecting different dium spp.. Over thdium spp.. One hunh PfHRP2. Indeed,h PfHRP2. Generalh PfHRP2 and tendh PfHRP2. These ch PfHRP2 make PfHP. falciparum alone are suitable for use in much of Western and sub-Saharan Africa where malaria cases are predominantly caused by P. falciparum. However, outside of Africa where P. falciparum co-exists with Plasmodium vivax and other Plasmodium species, RDTs that detect both P. falciparum and non-falciparum spp. are typically selected.RDTs detecting P. falciparum, only tests)[Forty-one of 44 African countries and 49 of 55 countries of other regions have adopted the WHO policy of parasitological diagnosis for all age groups in 2011[The detection sensitivity of quality RDTs is generally similar to that of quality field microscopy. However, variable performance has been reported in field use. False nThe major causes of false negative results can be grouped into five broad categories: poor product design or quality, unsuitable transport or storage conditions, parasite or operator factors and host parasite density/antigen concentration,12 , no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified[Many of the potential causes can be eliminated or minimized by procuring good quality RDTs, by improving quality control of procured RDTs, and by good training of end users. However, host and parasite characteristics could be specific to the region where the RDTs are deployed and are beyond the realms of quality control and training. Therefore, false negative results should be exhaustively investigated, particularly in relation to parasites not expressing target antigens or expressing, as of yet unrecognized, antigenic variants. In regards to the potential contribution of the antigenic variants, Baker entified. Thus, dP. falciparum parasites not expressing PfHRP2 and/or PfHRP3 have been reported both in laboratory and field isolates. In contrast, there are no reports of parasites failing to express pLDH or aldolase, as these targets are essential enzymes for parasite metabolism and survival.When a parasite expresses little or no target antigen, a false negative result will occur. Plasmodium falciparum parasites not expressing PfHRP2 were first reported in culture adapted, cloned laboratory lines: one originating from Papua New Guinea (FCQ27-D10)[pfhrp2 gene were also observed in a parasite line  when cultured in vitro. These subpopulations showed different breaking points, which appear to be random within the exon 2 of pfhrp2[Q27-D10) and the Q27-D10). In bothQ27-D10) or entirQ27-D10) due to bof pfhrp2.pfhrp3[pfhrp3 gene were favoured strongly in a genetic cross between 3D7 and HB3 compared to those without pfhrp3, due to rapid growth of progeny expressing PfHRP3[PfHRP3 was reported missing in a parasite line originating from Honduras (HB3), again due to chromosomal breakage at the subtelomeric region, resulting in the deletion of pfhrp3. Parasitg PfHRP3.pfhrp2 and pfhrp3 genes were observed in a progeny of a genetic cross between Dd2 and HB3[pfhrp2 locus on chromosome 8 and the HB3 parental type at PfHRP3 locus on chromosome 13, thus causing a deletion of both genes. This parasite clone was able to grow and multiply in vitro with these deletions. Interestingly, presence of pfhrp2 was not favoured in inheritance in the progeny[Parasites lacking both  and HB3. This pr progeny.P. falciparum parasites lacking PfHRP2 and/or PfHRP3 was published by Gamboa et al.[P. falciparum isolates collected in Iquitos, Peru, in 2007, eight were found to lack the pfhrp2 and six to lack both the pfhrp2 and pfhrp3 genes when tested by PCR. When tested by ELISA, none of the eight parasites lacking pfhrp2 had detectable PfHRP, but all had detectable pLDH[pfhrp2 and pfhrp3 returned negative results on three different brands of good quality RDTs.The first definitive report of oa et al.. This dible pLDH. Furtherpfhrp2 and pfhrp3 revealed that the lack of PfHRP2 was due to deletions on chromosome 8 which removed pfhrp2 and upstream genes, while the lack of PfHRP3 was mostly due to deletions on chromosome 13 that removed pfhrp3 and its flanking genes[P. falciparum isolates also collected from the Iquitos region[P. falciparum isolates revealed deletions of subtelomeric regions in lengths of 20–25 kb containing several genes including pfhrp2 and pfhrp3[pfhrp3 exon 1 was also seen in two isolates causing non expression of PfHRP3[pfhrp2 occur because of chromosomal breakage and re-join in the subtelomeric regions of chromosome 8. The breaking point could occur in a broad region, often upstream of pfhrp2 or within pfhrp2 [P. falciparum parasites lacking pfhrp2 gene will cause a high rate of false negative results in PfHRP2-detecting RDTs. This is especially true in areas with low malaria transmission where multi-clone infections are rare, i.e. parasite lacking PfHRP2 are less likely co-infected with another strain having PfHRP2 in the same individual, and parasite densities are usually low. These false negative results will cause a major problem for malaria case management.ites/μL). TherefoP. falciparum infections in health centres around Iquitos[pfhrp2 gene. The sensitivity of 10 products based on PfHRP2 detection was approximately 70 – 72%, with none of the products detecting any of the 19 isolates containing parasites without pfhrp2. In contrast, four pLDH-detecting RDTs detected all 19 of these isolates, giving an average sensitivity of 97-99%[P. falciparum infections both there and in returning travellers from regions affected by HRP2 deletions. Instead, quality pLDH-detecting RDTs and microscopy should be used in this region of Peru, and in contiguous regions in bordering countries.This effect is demonstrated in a recent study comparing sensitivities of several PfHRP2- and pLDH-based RDTs for detecting  Iquitos. Samplesf 97-99%. ClearlyP. falciparum parasites lacking pfhrp2 on malaria case management and on procurement decisions regarding the type of RDTs is significant, and as such it is imperative that the presence and prevalence of these parasites are accurately documented and mapped. However, incorrect or unsubstantiated reporting of parasites lacking pfhrp2 may unnecessarily trigger a costly change of RDT product , a process which requires significant logistic and financial support, as well as user retraining. It may also damage overall user confidence in RDTs. So far, the rarity of reports summarized in Table pfhrp2 deletion is not a major problem across broad areas; however, no systematic mapping has occurred. Not to be underestimated is the difficulty of determining the cause of false negative RDT results in the field, or when parasite antigen deletion may be suspected but there is poor capacity to investigate, and lack of guidance on how to conduct the investigation. The latter is addressed in the following sections.The potential impact of pfhrp2 deletion on malaria diagnosis at the clinical, public health and economic levels it is imperative that procedures and standards are developed in order to confirm new geographic foci of pfhrp2-deleted parasites. As a minimum, the following should be collected when reporting or documenting the presence of PfHRP2 deletions.Due to the potential consequences of 1. Initial evidenceP. falciparum, the parasitaemia is counted and all findings are verified by two qualified microscopists and slide archived. The same sample does not produce a positive Pf band on two quality-assured PfHRP2-detecting RDTs taken from the same box of RDTs that produced positive results using a positive control, such as a known RDT positive blood sample. This sample should also be positive for the pan band if the RDT detects Pf and pan Plasmodium spp, and positive as Pf when a pLDH-detecting RDT meeting WHO procurement criteria is used. The requirement for a positive pan-band is because pan-bands are generally less sensitive than HRP2-test bands and therefore a positive pan-band, and negative HRP2 test band makes the possibility of a false negative due to low parasite density less probable.• The sample is confirmed as microscopy positive for P. falciparum by PCR and ideally quantified against the WHO International P. falciparum DNA standard[Plasmodium species by PCR.• The sample is confirmed as standard and the If all of the above initial evidence is observed, a confirmatory investigation should be undertaken. A blood sample (>50 μL) should be stored frozen or dried on filter papers (e.g. Whatman 3MM) or other collection cards as sources of DNA for confirmatory analyses outlined below. Blood slides and used RDTs could also be stored as sources of DNA.2. Confirmatory evidence• Gene deletion analysisa. PCR to amplify the full length of exon 2 and the region across exon 1 and exon 2 of the gene [After reviewing published data we believe that unequivocal evidence exists for g areas),23. TherIn areas outside of South America, further studies should be undertaken to verify the existence, and if present, the prevalence and geographical extent of populations of parasites with gene deletions. Overwhelmingly, operational experience on RDT use indicates that there is no reason why PfHRP2-detecting RDTs should not be used as part of routine clinical case management in most malaria-endemic areas, they are safe and effective for case management-35. InveThe authors declare that they have no competing interests.QC conducted the literature review and drafted the manuscript. MG carried out the data analysis and contributed to data interpretation. JB, JM, DB and PC contributed substantially to data interpretation and revising the draft manuscript. JC conceived of the study, participated in its design, data interpretation and in revising the draft manuscript. All authors read and approved the final manuscript.