Plasmodium vivax malaria. Here we report the evaluation of four rapid diagnostic tests (RDTs) for diagnosis of this disease. A total of 253 subjects were enrolled in the study. The sensitivities, specificities and agreement frequencies were estimated by comparing the four RDTs against the standard of nested-PCR and microscopic examination. The CareStartTM and SD Bioline had higher test sensitivities  compared with the NanoSign and Asan Easy tests . The CareStartTM and SD Bioline tests could detect P. vivax in samples with parasite densities <150/μl, which was a slightly better performance than the other two RDTs. The quantitative accuracy of the four RDTs was also estimated by comparing results with P. vivax counts from blood samples. Lower test price would result in increased use of these RDTs in the field. The results of this study contribute valuable information that will aid in the selection of a diagnostic method for the detection of malaria.South Korea is one of many countries with endemic  Plasmodium vivax accounts for an estimated 80 million cases of malaria each year P. vivax is the only indigenous Plasmodium species in South Korea. Vivax malaria has been endemic in Korea and seasonal transmission has occurred since the disease re-emerged in 1993. From 1994 to 2010, 29,692 cases of endemic vivax malaria were reported. South Korea has embarked on a malaria elimination program based on early and accurate diagnosis followed by radical and rapid treatment. Standardization and use of sensitive diagnostic tools are critically important for a successful program outcome.Worldwide, Plasmodium antigens. RDT performance is affected by the target antigen During the last 100 years, malaria has been diagnosed by microscopic examination of Giemsa-stained thick and thin blood films http://www.finddiagnostics.org/resource-centre/reports_brochures/malaria-diagnostic-test-report.html). Interestingly, some field test results have differed from the laboratory results reported by WHO/FIND TM) is produced in the United States. The SD Bioline and CareStartTM tests performed well in the WHO/FIND comparison RDT performance varies with the environment of employment, geographical location, disease prevalence, and parasite species. RDT sensitivity declines with low parasite densities (<300–500/μl) The study was approved by the ethics committee of the Korean National Institute of Health. An approval form was used to obtain written informed consent from each participant. Each participant also gave permission to provide a 5 ml blood sample.P. vivax negative by microscopic examination and nested-PCR. Mean age of the patients was 47 years, and 75% of them were male. Blood specimens were kept cool and arrived at the KCDC within 24 hours after collection.The study was conducted at the Division of Malaria and Parasitic Diseases, the Korea Centers for Disease Control and Prevention (KCDC), from June 2009–October 2011. Venous blood samples with EDTA were received from 171 patients diagnosed with vivax malaria at local health centers  detected parasite antigen PfHRP-2 specific for P. falciparum in one capture site and pan-pLDH, which detected all four Plasmodium species, in a separate capture line. Individually packed test cassettes and fixed quantified sample buffer in a disposable tube were provided in each package of tests.All blood samples were assayed with each of four commercial RDTs. CareStartPfHRP-2 using a method similar to CareStartTM. However, sample buffer was supplied in a stock bottle and was not included in each package of test cassettes.SD Bioline Ag P.f/pan Rapid , also in individually packed test cassettes, detected parasite antigen P. falciparum  and a second line that detected all Plasmodium species (pan-pLDH antigen).NanoSign Malaria P.f/P.v , a device-type test with one capture line specific for ® malaria P.f/P.v strip , 48 packed strip kits used PfLDH antigen  and Pvaldolase (for P. vivax).Asan Easy TestTM Malaria HRP/pLDH, the SD Bioline Ag P.f/pan Rapid, and the NanoSign Malaria P.f/P.v tests has been evaluated by the WHO/FIND product testing program and results are available on the website (see above) The laboratory performance of the CareStartTest results were recorded as negative if only a control band appeared. The presence of a control and test line indicated a parasite positive result. The test was considered invalid when a control band did not appear. RDT results were recorded by different technicians, but each sample was blinded against the results from the other diagnostic techniques and RDTs.Plasmodium spp. were found during examination of 100 fields. Parasite densities were assessed by counting against 200 leucocytes, converting to parasites per microliter by assuming a standard leucocyte count of 8000/μl. Two technicians counted the parasites under study-blinded conditions.Microscopic examination was performed on 253 blood samples collected from vivax malaria patients (n = 171) and from healthy volunteers (n = 82). The 171 blood samples from patients with malaria symptoms were first examined microscopically at local health centers. In addition, blood smear slides for 62 of these samples were also obtained from local health centers or prepared at the KCDC for further quantitative study. Eighty-two samples from healthy volunteers were confirmed as malaria negative at the KCDC. Thick blood smears were prepared, dried, stained with 10% Giemsa, and examined microscopically. A slide was considered negative if no asexual stage of Nested PCR amplification based on the 18S rRNA gene was performed for all blood samples as described by Snounou et al. http://vassarstats.net/) TM, SD malaria, NanoSign, and Asan Easy tests. The following kappa values were used to categorize the strength of agreement between the each RDT and nested-PCR and microscopic examination: fair (0.40 to 0.61), moderate (0.61 to 0.80), and substantial (>0.8). Whenever possible, Standards for the Reporting of Diagnostic Accuracy (STARD) guidelines were considered during preparation of this report Estimates of sensitivity, specificity, and kappa were calculated with DAG_Stat and VassarStats (Blood samples from 62 patients whose parasite densities were determined by quantification of thick smears were used to assess the quantitative performance of the four RDTs. The positive (sample) line was divided into three groups depending on the ratio of the test line intensity compared to control line intensity . Three technicians read line intensity for all RDTs under conditions in which each sample was blinded against the results from the other diagnostic techniques. These results were compared with parasite counts obtained by thick smear. To facilitate ease of comparison, samples were divided into five groups based on the parasite densities: .P. vivax  and SD Bioline  tests were greater than the sensitivities of the NanoSign  and Asan Easy  tests. Specificity was 100% (CI: 94.4–100) for all of the RDTs. Test agreement was calculated by comparing RDT results with microscopic examination. All RDTs showed high agreements  with microscopic examination , the NanoSign test had a low sensitivity (60%) at a parasite density of 1–150/μl. We wondered whether we could use RDTs to quantify the parasite load in the patient by comparing the ratio of test to control band intensity. Therefore, the ratio of test to control band intensity for the four RDTs was compared to corresponding parasitemia. There was a slight positive correlation between the ratio of the test to control band density for each RDT and the parasitemia counts , a definite pan-LDH line in samples from patients without a travel history to foreign countries could, at least initially, be considered to be vivax malaria positive.In the current study, CareStartTM and SD Bioline tests  estimated by the present study were higher than previously reported by a hospital-based study in South Korea  TM and SD Bioline tests appears to be similar to microscopic examination and nested-PCR, which suggests that these RDTs could be used as alternative diagnostic tools for P. vivax detection. However, except for P. falciparum, these RDTs cannot differentiate Plasmodium species, which is disadvantageous for the diagnosis of imported malaria cases. Thus, we recommend that nested PCR should be performed after a positive CareStartTM or SD Bioline test result to confirm and differentiate the Plasmodium species. These RDTs could be used for malaria screening and diagnosis at local health centers. When appropriate, samples could be sent to a laboratory with PCR capability for further diagnosis.As shown in TM and SD Bioline tests at <150/μl parasite density (both 100%) were higher than previously reported Because transmission rates and parasite densities in South Korea vivax malaria patients are usually low, a high RDT sensitivity is very important. As shown in The malaria endemic regions of South Korea are restricted to near DMZ. Thus, 50% of patients are soldiers and discharged soldiers Continuous product testing is necessary to guide personnel at local health centers and to improve RDT quality. The KCDC intended to summarize published data, including this study, to guide procurement decisions made by personnel at local health centers and hospitals in South Korea. Further, accurate and rapid malaria diagnosis may help to prevent secondary transmission in other countries. This study is expected to help improve the accuracy of malaria diagnosis in the field, especially under conditions of low parasitemia, and to assist with diagnostic method procurement decisions.


Malaria is often considered a cause of adult sepsis in malaria endemic areas. However, diagnostic limitations can make distinction between malaria and other infections challenging. Therefore, the objective of this study was to determine the relative contribution of malaria to adult sepsis in south-western Uganda.Adult patients with sepsis were enrolled at the Mbarara Regional Referral Hospital between February and May 2012. Sepsis was defined as infection plus ≥2 of the following: axillary temperature >37.5°C or <35.5°C, heart rate >90 or respiratory rate >20. Severe sepsis was defined as sepsis plus organ dysfunction . Sociodemographic, clinical and laboratory data, including malaria PCR and rapid diagnostic tests, as well as acid fast bacteria sputum smears and blood cultures were collected. Patients were followed until in-patient death or discharge. The primary outcome of interest was the cause of sepsis. Multivariable logistic regression was performed to assess predictors of mortality.Plasmodium falciparum, two with Plasmodium vivax). Bacteraemia was identified in 41 (19%) patients. In-hospital mortality was 19% (n = 42). In multivariable regression analysis, Glasgow Coma Score <9  and severe sepsis , but no specific diagnoses were statistically associated with in-hospital mortality.Enrollment included 216 participants who were 51% female with a median age of 32 years (IQR 27–43 years). Of these, 122 (56%) subjects were HIV-seropositive of whom 75 (66%) had a CD4+ T cell count <100 cells/μL. The prevalence of malaria was 4% (six with Malaria was an uncommon cause of adult sepsis in a regional referral hospital in south-western Uganda. In this setting, a thorough evaluation for alternate causes of disease in patients presenting with sepsis is recommended. Severe sepsis is an important cause of death in sub-Saharan Africa (SSA) where the global burden of lethal infectious diseases is highest . Due to The time to the initiation of appropriate antimicrobial therapy is an important determinant of the outcome from severe sepsis . On the The study was conducted on the medical ward of Mbarara Regional Referral Hospital (MRRH), which is the teaching hospital for the Faculty of Medicine at the Mbarara University of Science and Technology (MUST) and serves a largely rural population of 1.2-2.5 million people from surrounding districts in south-western Uganda. Mbarara District has an average malaria prevalence of 4% in urban areas and 23% in rural areas. The climate is tropical with a bimodal rainfall pattern in September-January and March-May, and an average precipitation of 1,200 mm per annum .Adult patients (age ≥18 years) admitted to the medical ward during the study period from February-May 2012 were screened consecutively during the hours of 08.00-0.00. Patients were included if they met criteria for sepsis which was defined by clinical suspicion of infection and ≥2 of the following: axillary temperature >37.5°C or <35.5°C, heart rate >90 beats/min, and respiratory rate >20 breaths/min. White blood cell concentration (WBC) was not included in the enrolment criteria due to lack of immediate availability of results. Severe sepsis was diagnosed in patients with evidence of end-organ dysfunction  [A structured questionnaire was administered to patients or their attendants to collect the following data for each participant: demographics, history of presenting symptoms, and HIV serology status. Examination findings, including vital signs and Glasgow Coma Scale (GCS), and in-hospital mortality were also recorded. The study team followed each patient until discharge or death, but medical management was provided by the admitting medical team.Laboratory investigations were obtained for each patient at the time of enrolment including complete blood cell concentration , random blood glucose , and whole blood lactate . Prior to antibiotic administration, a blood sample was obtained aseptically and 10 mL was inoculated into brain heart infusion broth in duplicates and incubated at 37°C at the MUST Microbiology Laboratory. This laboratory is enrolled in the International Organisation for Standardisation and maintained satisfactory performance throughout the study period. Positive blood cultures were observed for turbidity, a clot or haemolysis; Gram’s stain was done after subculturing on blood agar, MacConkey, and Chocolate agar plates for maximum recovery and morphological identification of the organisms. The plates were incubated at 37°C for 18-24 h aerobically and anaerobically for Chocolate agar. Blood cultures that did not show turbidity were further incubated up to 10 days. Biochemical identification of culture isolates was done according to standard methods. Bacteraemia was defined as bacterial isolation from one or more blood culture bottles. Cultures growing coagulase-negative staphylococci were considered contaminated and not included in the final analyses.Patients with suspected tuberculosis had sputum obtained for light-emitting diode fluorescent microscopy (LED-FM). For patients whose HIV status was not known or documented at the time of admission, HIV counselling and testing was provided according to national guidelines. Accordingly, a rapid HIV test was done  followed by a confirmatory test for initially positive results . When the results were conflicting, a tie-breaker  was used.Plasmodium falciparum and Plasmodium lactate dehydrogenase (pLDH) for identification of Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.Malaria blood slides were evaluated via Field’s staining as previously described . For thePlasmodium as described. Species of Plasmodium in positive samples were determined in separate species-specific nest 2 reactions. The resulting PCR products were visualized on 2% agarose gels, with the presence or absence of a band with each species primer pair indicative of the presence or absence of that species in the initial sample. The laboratory technicians performing PCR were blinded to the results of blood smears and RDTs.Blood was also obtained by phlebotomy for PCR testing. Venous blood (100–200 μL) was applied to Whatman FTA Classic Filter paper cards  and left to air dry. These cards deactivate viral DNA/RNA and preserve human and parasite DNA for downstream analyses. DNA extractions were carried out according to the manufacturer's instructions. Briefly, a disc of 1.2 mm in diameter was punched from the centre of each dried blood spotted card and washed three times with Whatman FTA Purification Reagent, and twice with TE buffer. This treated disc was then used directly in subsequent PCR analyses. Nested PCR was performed using the primers and amplification conditions as previously described . Nested χ2 testing was used to compare baseline characteristics between those with and without a definitive diagnosis of malaria. Poisson regression models were fitted to determine factors associated with incident hospital mortality, including demographic characteristics, presenting signs, laboratory results, and discharge diagnoses. Finally, multivariable Poisson regression models which included all the characteristics that were significant in univariable models as determined by a p-value <0.25 were performed.Demographic, clinical and laboratory characteristics of the cohort were summarized. For the primary outcome of interest, the relative contribution of etiologies of sepsis was measured. The causes of sepsis were defined as either definitive or presumptive. Definitive causes were tuberculosis identified by sputum LED-FM, culture-proven bacteraemia, and malaria, as determined by a positive PCR. Presumptive causes of sepsis included malaria , tuberculosis, community acquired pneumonia, enteric fever, fever of unknown origin and other or unspecified diagnosis. Approval was obtained from the institutional review boards of MUST and the University of Virginia prior to the beginning of enrolment in this study.A total of 646 patients were screened and 216 were enrolled  followed by Salmonella species  and streptococcal species . Laboratory evaluation revealed leukocytosis  in 11% (24 of 215), anaemia (haemoglobin <9 mg/dL) in 43% (93 of 216) and thrombocytopenia (platelets <150 cells/μL) in 55% (118 of 215) of participants. Lactic acidosis (lactate >4 mmol/L) was identified in 42% (89 of 211) of patients.The majority of patients were febrile with a temperature >38°C , tachycardic with a heart rate of ≥100 beats/min , and tachypneic with a respiratory rate >20 breaths/min . Half the patients  had severe sepsis with evidence of end-organ dysfunction. Only 4% (8 of 216) of patients had PCR proven malaria whereas bacteraemia was detected in 19% (41 of 216) of patients and 11% (24 of 216) had tuberculosis as defined by positive sputum LED-FM. The most common presumptive diagnosis at discharge was tuberculosis  followed by pneumonia  sensitive and 99.5% (95% CI 97.3-100) specific for PCR proven malaria with an 83.3% positive predictive value and 98.6% negative predictive value. The sensitivity and specificity of a positive blood slide for a PCR proven malaria diagnosis was 25% (95% CI 3.2-65.1) and 100% (95% CI 98.2-100) respectively with a positive predictive value of 100% and negative predictive value of 97.2%. A clinical discharge diagnosis of malaria was 62% (95% CI 24.5-91.5) sensitive and 99.5% (95% CI 97.3-100) specific for PCR proven malaria.Malaria was identified by PCR in eight patients. There were six cases of vs 56%, 116 of 208 in patients without malaria, p = 0.02). A final clinical discharge diagnosis of malaria was given to 50% (4 of 8) of patients with malaria compared to 7% (14 of 208) in those without malaria (p <0.01). Anti-malarial treatment was provided to 63% (5 of 8) of patients with malaria and 23% (48 of 208) of those without malaria (p = 0.01). Antibacterial therapy was provided to 63% (five of eight) and 88% (182 of 208) of patients with and without malaria respectively (p = 0.05).Due to the low prevalence of malaria in the cohort, clinical correlates associated with malaria were difficult to determine; however, cough was less common in those with malaria  and in-hospital death occurred in 19% (42 of 216) of the cohort. Among those with a definitive diagnosis of sepsis, mortality was 26.8% in those with bacteraemia, 25% for those with LED-FM sputum positive tuberculosis, and 12.5% in those with malaria. Mortality rates for those with presumptive diagnoses were 24.6% for tuberculosis, 23.5% for fever of unknown origin, 21.5% for an unknown diagnosis, 12.5% for community acquired pneumonia, 11.1% for malaria, and 10.5% for enteric fever  which may be increasing . AdditioThe reason for the low malaria prevalence in this cohort compared to the general population may be due to several factors. First, epidemiological data are generally derived from children as adults have improved immunological control of malaria and therefore lower prevalence of infection even in the setting of HIV infection . Severe Plasmodium falciparum and pLDH to identify non-falciparum species. HRP-II antigen tests are very sensitive and positive results can persist 14 days or more after treatment whereas pLDH provides a negative RDT result within 3 days of treatment [The RDT used in this study employed HRP-II to identify reatment ,18. TherA diagnosis of malaria was not associated with increased mortality in this study or in similar studies. Moreover, in contrast to receipt of early antibacterial therapy, receipt of anti-malarial drugs did not improve mortality outcomes in this study or a study of fluid resuscitation in severe sepsis performed in Uganda . In a stSalmonella, Streptococcus pneumoniae, and Staphylococcus aureus as common pathogens [Mycobacterium tuberculosis (MTb) as a common cause of bloodstream infection in hospitalized patients and these patients have high mortality with 50% of patients dying within 36 days in one study [This and prior studies of severe sepsis in Uganda have revealed non-Typhi athogens ,19,21. Aathogens . There wne study . Despitene study . In thisThere are several limitations of this study. Patients were not evaluated through an entire calendar year and times of high malaria transmission may have been missed. The enrolment period for this study did include one of the rainiest times of year in Mbarara (March-May) which accordingly is a time of high community malaria prevalence . TherefoSalmonella species. Unfortunately, the vast majority of patients did not have a confirmed microbiological diagnosis for their sepsis. Further studies are needed to discern the role for empirical anti-MTb therapy in patients presenting with sepsis in high HIV prevalent MTb endemic areas. Improved diagnostic capacity, including development of point-of-care testing, for use in regional referral hospitals in resource-limited settings may improve targeted and empirical therapy for sepsis, reduce development of resistance to anti-malarial agents, and improve outcomes. Finally, given the high prevalence of HIV in this region all patients admitted with sepsis should be tested for HIV according to national guidelines.Despite these limitations, data from this study lend strong support to the notion that malaria is an uncommon cause of sepsis in adult inpatients in south-western Uganda. Based on the findings of this study, the following recommendations are made to practitioners providing care to patients with sepsis in similar settings. First, thorough diagnostic evaluations should be pursued where possible to evaluate for non-malarial causes of sepsis. Second, as recommended by the World Health Organization, anti-malarial therapy should be reserved for patients with a positive RDT or blood slide result to minimize over-prescribing and development of resistance. A bacterial cause of sepsis was identified in ~20% of these patients and it is suggested that empirical treatment of sepsis in this region should include coverage of staphylococci, streptococci, and In Mbarara which is located south-western Uganda and where community malaria prevalence is 4-23% malaria was an infrequent cause of sepsis in adults. The ability to detect parasitaemia was maximized through use of PCR technology. Using this sensitive laboratory method, only 8 out of 216 patients were diagnosed with malaria. In contrast, bacteraemia was found in 41 (19%) patients. Additionally, tuberculosis was a definitive diagnosis for 24 (11%) and presumptive diagnosis for 61 (28%) of these patients. When treating adult septic patients in similar regions a thorough evaluation of non-malaria diagnoses with particular emphasis on identifying bacteraemia and tuberculosis is recommended.Plasmodium lactate dehydrogenase; LED-FM: Light-emitting diode fluorescent microscopy; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; CD: Cluster of differentiation; IQR: Interquartile range; RR: Relative risk; CI: Confidence interval; EIR: Entomological inoculation rate; MTb: Mycobacterium tuberculosis.SSA: Sub-Saharan Africa; HIV: Human immunodeficiency virus; RDT: Rapid diagnostic test; PCR: Polymerase chain reaction; \MRRH: Mbarara Regional Referral Hospital; MUST: Mbarara University of Science and Technology; WBC: White blood cell concentration; GCS: Glasgow coma scale; HRP-II: Histidine-rich protein II; pLDH: The authors declare that they have no competing interests.MAA helped design the study, enrolled patients, analysed the data, and helped to draft the manuscript. MJS analysed the data and helped to draft the manuscript. DN, AN, MN, and JM-A performed laboratory analyses including malaria PCR and bacterial cultures and helped to draft the manuscript. RM and LAW helped to design the study, analyse the data, and draft the manuscript. YB helped to design the study, supervised the malaria PCR work, helped to analyse the data, and helped to draft the manuscript. CCM conceived of and designed the study, coordinated the study, helped to analyse the data, and drafted the manuscript. All authors read and approved the final manuscript.


Its incidence . varies between 10 and 45% in different reports and is seen more in adults than in children. Jaundice may vary from mild to very severe. However, clinical signs of hepatic encephalopathy (such as liver flaps) are never seen unless there is presence of concomitant viral hepatitis . Our case is a 6-year-old female child presented with fever, jaundice, and anasarca. Peripheral smear showed trophozoites and schizonts of Plasmodium (P.) vivax and trophozoites and gametocytes of P. falciparum. Viral markers for hepatitis were negative. She developed fulminant hepatic failure and expired after 26 hours of admission.Malaria remains an overwhelming problem in the tropical developing countries, with 300 to 500 million new cases and about a million deaths per year . Malaria is a potentially life-threatening disease in the tropics. Jaundice is one of the severe manifestations of  Hepatic dysfunction in malaria has been known for many years, but hepatic encephalopathy is unusual. According to the World Health Organization (WHO), apart from jaundice, signs of hepatic dysfunction are unusual. Jaundice has been found to be more common in falciparum as compared to vivax malaria. Hazra et al. found an association of jaundice in 40% and 9.09% of cases with P. falciparum and P. vivax, respectively, from Calcutta [vivax malaria from Colombia have reported an incidence of jaundice in 15% [falciparum malaria while they did not find jaundice in vivax malaria [Malaria remains a major problem in many parts of the world. Approximately 500 million people are affected annually, and approximately 3 million, mostly children, die of ach year , 2. AccoSix-year-old female child, first product of nonconsanguineous marriage resident of New Delhi, presented with fever for four days, yellowish discoloration of eyes, and urine for two days, progressive swelling of whole body starting from feet for one day. Fever was moderate to high grade, intermittent in type associated with chills and rigor subsided with medication and profuse sweating. She was having high coloured urine for two days. Progressive swelling of whole body for last one day starting from feet then involved the whole body. Stools were normal in colour. There was no history of black coloured stools, vomiting, bleeding from any site, and any change in conscious level, jaundice in the past, any history of blood transfusion, and intravenous medication in the past or any history of drug intake. There was no history suggestive of respiratory tract infections, skin infection, decreased micturition, and reddish coloured urine. There was no significant past, antenatal, perinatal, or family history. The child was fully immunized and was receiving adequate calories and proteins for her age.On examination, the child was conscious, cooperative, and orientated. At admission her pulse was 78 per minute, respiratory rate 26 per minute, and blood pressure 98/68 mm of mercury. There were severe pallor, and icterus. Lymph nodes were not palpable. On abdominal examination liver was palpable 4 cms below costal margin with span of 10 cm the spleen was palpable 1 cms below costal margin. Her respiratory, cardiovascular (CVS), and central nervous system (CNS) examination was unremarkable.3 per μL with polymorphs 54%, lymphocytes 42%, monocyte 4%, platelet count 11×103 per μL and erythrocyte sedimentation rate 8 mm/1st hour by Westergren method. Blood sugar was 84 mg/dL. Total serum bilirubin was 27.5 mg/dL, indirect fraction of 18 mg/dL. SGOT was 495 and SGPT 210. Serum total protein was 2.8 gram (gm) and albumin 1 gm per 100 mL of blood. Blood urea nitrogen was 189 mg/dL, creatinine 0.6 mg/dL, sodium 136 meq/L, potassium 4.1 meq/L. Prothrombin time was 17.3/12 with INR of 1.44. Viral markers  were negative. Chest X-ray was normal and ultrasonography abdomen showed hepatosplenomegaly. On peripheral smear trophozoites and schizonts of P. vivax and trophozoites and gametocytes of P. falciparum were seen. Rapid malarial antigen test was positive. Arterial blood gas analysis showed pH 7.45, partial pressure (PP) of oxygen 104, PP of carbon dioxide 45, and standard bicarbonate 28 meq/L. Urine routine and microscopy were normal. She was given injection of Artesunate 2.4 gm per kilogram body weight, along with cefotaxime and vitamin K. She received fresh frozen plasma, packed red cell, platelets along with inotropes. Within five hours of admission she developed profuse upper gastrointestinal bleeding, along with deterioration of consciousness. She went into shock, then cardiorespiratory arrest from which she could not be revived.On investigation haemoglobin was 4.9 g/dL, total leucocyte count 10.6 × 10P. falciparum malaria. After the initial stage (preerythrocyte schizogony), merozoites are released into the blood stream. They do not have exoerythrocytic schizogony. But in the patients of severe malaria, the liver may be involved to different extents. In patients with severe malarial infection, the incidence of jaundice is reported to be 2.58% only [falciparum malaria indicates a more severe illness with higher incidence of complications.Malaria is a major health problem in many of the developing and tropical countries. Although rare, severe malaria must be distinguished from viral FHF. This can have profound implications for the line of treatment and ultimate patient outcome. The liver is the first organ to be affected in a case of 58% only . PresencDirect causes: malarial hepatitis, intravascular hemolysis of parasitized RBC, and septicemic hepatitis.Indirect causes: microangiopathic hemolysis associated with DIC, G6PD-related hemolysis, and anti-malarial-drug induction.Unrelated causes: coexisting acute viral hepatitis.P. falciparum infection. There have been occasional reports of mixed infection with P. vivax [P. falciparum, resulting in malarial hepatitis. The management of malarial hepatitis is not different from the management of severe malarial infection. In endemic areas, it is important to have a high degree of suspicion of severe infection with malarial parasite presenting as acute febrile illness with hepatic dysfunction. The other conditions prevalent in tropical countries like acute viral infections leading to hepatitis, enteric fever and leptospirosis should be figured in the differential diagnosis. Since the clinical presentation may not differ much, laboratory help may be needed to establish the etiology of malarial hepatitis. Routine, single peripheral smear examination may not be sufficient, so repeated smears are required to pick up the diagnosis. Treatment of severe malaria is artesunate or quinine intravenously till the child becomes conscious thereafter given orally for a total of seven days.Hyperbilirubinaemia is mostly of unconjugated type. Often jaundice is mild, and total serum bilirubin is below 5 mg/dL, but at times bilirubin rises beyond 50 mg/dL. Enzymes are usually raised (within 3 to 8 times); alanine transaminase (ALT) never reaches to the level of viral hepatitis. 5′ nucleotidases and GGT concentrations may be moderately elevated. Hypoalbuminaemia is also seen in these patients. Prothrombin time may be moderately prolonged. The literature on the hepatic involvement in malaria has largely shown severe infection with P. vivax  and hepafalciparum malaria.Jaundice is one of the common severe manifestations of Altered consciousness in malarial hepatitis, although uncommon, may make the distinction between malarial hepatitis and viral FHF difficult.Failure to recognize this uncommon presentation often leads to delayed diagnosis, resulting in poor outcome.P. falciparum may masquerade as FHF. Simple clinical and laboratory parameters can easily distinguish severe malaria from FHF.Severe malaria due to Early diagnosis of malarial FHF has profound implications because the cornerstone of therapy is quinine, whereas liver transplantation may be required for FHF in selected patients.


This paper reviews specific issues surrounding RDT use in field settings and presents results of research investigating how to interpret "faint test bands" on ParaCheck Pf® testing. Primary outcome variables were sensitivity, specificity and proportion misdiagnosed by ParaCheck Pf® and local microscopy. ParaCheck Pf® "faint bands" were classified as both true positives or true negatives during evaluation to determine appropriate clinical interpretation. Multivariate logistic regression adjusted for age and gender was conducted to determine odds of misdiagnosis for local microscopy and ParaCheck Pf®.A multi-phase cross-sectional study was conducted at a remote hospital in the northern Tanzanian highlands. Capillary blood samples were taken from consenting participants (n = 319) for blood smear and ParaCheck Pf® tests resulted in a "faint band" and 94.20% corresponded with true negatives. When ParaCheck Pf® "faint bands" were classified as positive, specificity was 75.5% (95% CI = 70.3% - 80.6%) as compared to 98.9% (95% CI = 97.0% - 99.8%) when classified as negative. The odds of misdiagnosis by local microscopy for those > 5 years as compared to those ≤ 5 years are 0.370 . In contrast, even when ParaCheck Pf® faint bands are considered positive, the odds of misdiagnosis by ParaCheck Pf® for those > 5 years as compared to those ≤ 5 years are 0.837 .Overall, 23.71% of all ParaCheck PfWe provide compelling evidence that in areas of low transmission, "faint bands" should be considered a negative test when used to inform clinical decision-making. Correct interpretation of RDT test bands in a clinical setting plays a central role in successful malaria surveillance, appropriate patient management and most importantly reducing misdiagnosis. Current recommendations of effective, yet expensive artemisinin-based combination therapies (ACT) for malaria in Sub-Saharan Africa have increased the importance of laboratory-confirmed diagnosis -3. The cPlasmodium species detection, paired with high specificity necessary to successfully reduce malaria associated morbidity and mortality and accurately diagnose other tropical diseases [Rapid Diagnostic Tests (RDTs) for malaria have substantial potential to help address these issues, especially in resource poor areas . In ordediseases . This sudiseases .However, RDTs are not a perfect solution and there is growing concern regarding their effectiveness, quality, accuracy and perceived value. This paper reviews some of the current literature on challenges associated with RDTs and presents the results of research investigating the interpretation of "faint bands" which are a problematic feature of some malaria RDTs ,11,12.® detects histidine rich protein 2 (HRP2) produced uniquely by Plasmodium falciparum [® is consistently reported to be above 90% in hospital and field based studies, specificity varies from as low as 52% up to 99.5% [® false positives can be attributed to: reading the test after fifteen minutes, application of too much blood, inappropriate dilution/concentration of buffer, contamination of the sample, backflow of sample and conjugate leading to entrapment at the detection line, high titers of non-specific anti-bodies due to viral infection or the presence of rheumatoid factor [® also may have implications for producing false positives during practical interpretation.Since the introduction of RDTs in the early 1990's, there have been over 100 published trial reports . One of lciparum ,14-21. Ato 99.5% . Variabito 99.5% . Beyond d factor ,14,22. T® (bands that can only be seen in good light) and classified them true positives using concordance values between two HRP2 RDTs [® results in various malaria transmission settings.In previous reports from a hyper-endemic area of Uganda, Kyabayinze et al. (2008) identified "faint test bands" in ParaCheck PfRP2 RDTs . Aside f® against microscopy in an area of low malaria transmission in the Tanzanian highlands and determine whether, in this setting "faint bands" are most frequently associated with true positives or negatives. In addition, we provide a comparison of malaria misdiagnosis based on local microscopy as compared to ParaCheck Pf® and use these findings to propose potential implications for areas of low malaria transmission.In this paper, we evaluate the sensitivity and specificity of ParaCheck PfThis study was conducted at a rural hospital in the Ngorongoro Conservation Area (NCA) located within the highlands of northern Tanzania where the temperature can range from 2°C to 35°C annually. The hospital laboratory relies on generator power for electricity and sunlight through open windows is the major source of lighting. Altitude in the NCA ranges from 1000 m to over 3000 m placing the majority of the population in a low transmission zone for malaria. Based on hospital outpatient records, the reported prevalence of malaria at the hospital was over 40%, but it is anticipated that the actual prevalence of malaria in the NCA is estimated to be 9.3% . Interpr® as compared to local hospital microscopy and external quality control (QC) performed at the Bugando University College of Health Sciences (BUCHS) in Mwanza, Tanzania. Study design was based on the World Health Organization (WHO) Evaluation of diagnostic tests for infectious diseases: general principles [® sensitivity, specificity and the appearance of faint bands (see Appendix A for questionnaire).A multi-phase cross-sectional study was conducted to determine accuracy of ParaCheck Pfinciples  and the inciples . A demog2 P(1-P)/d2, where n = sample size, Z = Z statistic for a level of confidence, P = expected prevalence or proportion and d = precision was used for calculations [Sample size was calculated using 95% confidence, an expected malaria prevalence of 9.3%  and preculations . The respatients presenting with clinical features of malaria sent for microscopy investigations after giving informed consent.any women with known or suspected pregnancy, patients with severe and/or complicated malaria, and patients with severe and complicated co-morbidities as defined by the clinical health officers. Patients were excluded if they had received anti-malarial therapy at the hospital in the last 2 weeks.Using convenience sampling , researc®test results.Following provision of informed consent, patient demographics  and health seeking behaviors  were recorded using a previously pilot tested and validated questionnaire . In addition they all have been attending refresher courses to improve their skills in malaria microscopy. At both sites, the WHO standard operating procedures for microscopy were followed .®  was used for the qualitative detection of Plasmodium falciparum. The tests were stored in a cool and dry location for the duration of the trial until use, and each package was examined for the integrity of the desiccant. Standard protocol was followed by trained research personnel, where 5 μl of fresh capillary blood was transferred using the provided loop applicator to the test window and then 6 drops of sample buffer were added to the buffer well. Once a ParaCheck Pf® package was opened, it was used immediately. Trained research staff interpreted RDT results for exactly 15 minutes following the addition of the buffer solution as recommended by the manufacturer. In all cases, interpretation of ParaCheck Pf® results was overseen and confirmed by the lead author. In order to ensure integrity of interpretation, all research personnel involved in ParaCheck Pf® interpretation were blinded to both the results of microscopy as well as the clinical decision.ParaCheck PfP. falciparum. A negative result was indicated by the presence of a control band and the absence of a test band. A "faint band" was identified using the same definition as Kyabayinze et al. (2008), where "faint bands" are those that are only visible in good light. Good light is defined as viewing the ParaCheck Pf® test in direct sunlight . A test was considered invalid when the control line did not appear or the test failed to clear remaining blood from the test area. The final diagnosis and further patient management and treatment were solely based on microscopy results.The presence of a band in both the control and test line indicates a positive test for After completion of standard WHO microscopy procedures , a bloodTimers were used to ensure that tests were read at exactly 15 minutes. In the case of a "faint band" a second observer was consulted to determine the presence of a "true faint band". "Faint bands" were considered present only if both observers agreed.Ethical approval from the Conjoint Health Ethics Board (CHREB) at the University of Calgary and the National Institute of Medical Research (NIMR) in Tanzania as well as a research permit required in Tanzania from the Commission on Science and Technology (COSTECH) were received.® by coding the test result as a misdiagnosis when there was a disagreement with the external QC result and the test result . Logistic regression adjusting for age and gender was conducted to determine the odds of misdiagnosis using ParaCheck Pf® or local microscopy. All confidence intervals are calculated using the Exact-Method.All data were entered into Microsoft Excel and checked by a second researcher for consistency against original data sources, then analysis was done using STATA version 11.0. The variable misdiagnosis was created for both local microscopy and ParaCheck PfFor prevalence calculations, the population is defined as patients who are suspected of malaria by a clinical officer or physician at the outpatient department of the local rural hospital. The resulting prevalence estimates are shown in Table A total of 319 study participants were divided into three study groups: 1) malaria-like symptoms ≤5 years (n = 98), 2) malaria-like symptoms > 5 years (n = 122) and 3) a control group presenting with non-malaria-like symptoms > 5 years (n = 71). Due to inconsistencies and missing data, the sample size used for analysis was n = 291. Of the 291 participants, there were approximately equal numbers of males and females , a larger proportion of patients were > 5 years (n = 193), waited longer than 1 day to present at the hospital (n = 225) and a small proportion had to travel longer than 3 hours to get to the hospital (n = 40).The external QC readers were regarded as the gold standard for this evaluation. Evaluation of external QC reader inter-rater reliability resulted in 97.25% agreement . There were 8 cases out of 291 that the QC readers did not agree. These cases were then excluded from calculations that use true positives or true negatives. The proportion of true positives determined by agreement between both external QC readers was 2.10% (6/283).® tests resulted in a "faint band" . Only 4.34% (3/69) of "faint bands" corresponded with a true positive, and 94.20% (65/69) of "faint bands" corresponded with true negatives . In contrast, when ParaCheck Pf® test bands were classified as negative the inter-rater agreement with the QC gold standard was 97.53% .Overall, 23.71% of all ParaCheck Pf® test result did not agree with microscopy QC results. When "faint bands" were classified as positive tests, the proportion of ParaCheck Pf® misdiagnosis was 23.4% (95% CI = 18.5%-28.3%)(Table ® decreased to only 0.353% (95% CI = -0.33%-1.0%) .Misdiagnosis of malaria is defined as any outcome where the local hospital microscopy or ParaCheck Pf® "faint bands" were classified as true positives, specificity was 75.5% (95% CI = 70.3%-80.6%) as compared to 98.9% (95% CI = 97.0%-99.8%) when "faint bands" were classified as negative. For blood slide parasitemia results > 600 parasites/μL  the sensitivity was 100% for both the "faint band" negative and positive classifications. When the "faint bands" were classified as negative, 3 out of 69 patients with parasitemia values ≤ 600 parasites/μL were not detected and became false negatives.When ParaCheck PfThe sensitivity of blood smears performed by the local hospital was 83.3% (95% CI = 35.9%-99.6%) and specificity was 54.4% (95% CI = 48.4%-60.3%). All values were calculated using the external QC microscopy as the "gold standard".Prior to the start of our research, the local hospital records estimated the prevalence of malaria in patients presenting with malaria-like symptoms at the out-patient department to be 46% in 2008 . This isPlasmodium falciparum [Further, in this study correct interpretation of ParaCheck Pf "faint bands" as negative was found to be associated with a decrease in misdiagnosis  as compared to local microscopy. These results may indicate that the use of RDTs will provide an objective parasitological tool to accurately diagnose malaria in children and adults. As the current WHO Guidelines for malaria diagnosis and treatment still promote presumptive diagnosis of malaria for children < 5 years in areas where parasite-based diagnosis is not available, RDTs may have important implications for reducing over-diagnosis in resource poor areas . Moreovelciparum ,31.® and potentially other RDTs should be interpreted [http://www.wpro.who.int/sites/rdt), we assert that this is an important consideration in areas of low transmission.In the current clinical environment where training and experience results in presumptive malaria treatment and belief that "a negative blood slide does not rule out malaria" it is essential for training algorithms to clarify how to interpret "faint bands" occurring in ParaCheck Pferpreted . Here, wIn this setting, if faint bands are interpreted as true positives and the patient is treated with anti-malarial drugs, this may result in the wrong disease being treated and augment morbidity and unnecessary drug expenses. At the health system level, incorrect interpretation may lead to decreased trust of RDTs, create perceptions of treatment failures and result in an over-estimation of the prevalence of malaria in the area. The consequences are a reinforcement of the culture of presumptive treatment and inaccurate treatment of patients. Especially for areas of low transmission, it is vital to have an accurate prevalence estimate in order to track epidemiological trends and the potential emergence of epidemics due to factors such as climate change.® uses a loop applicator with the interior of the loop applicator being calibrated for 5 μl. In field settings the method of blood application is usually dropping the blood onto the loop, leading to the formation of a bubble above the loop structure. The result is the addition of greater than 5 μl to the test strip. Application of too much blood has been identified previously as a contributing factor to false positive results, although the authors did not discuss band intensity [® RDT, has been previously shown to remain in the bloodstream up to 60 days following anti-malaria treatment leading to false positive test results [Although we have not conducted targeted studies to determine the precise underlying reason that "faint bands" appear we propose the following hypothesis based on previous explanations of false positives. ParaCheck Pfntensity . Further results . Future It may be possible that "faint bands" are due to low-level parasitemias that are commonly found in areas of low prevalence and often not detectable by microscopy, ,10, but ® categorized three patients with parasitemias < 600 parasites/μL as false negatives when "faint bands" were considered negative. The failure to treat these true positive cases will have important ramifications for the individual patient and their families (for example delayed treatment may lead to the development of serious disease and ultimately death of the patient). The risks associated with both over and under treatment of malaria are serious and have been previously discussed [Furthermore, it is important to remember that ParaCheck Pfiscussed -34. So, ®, when interpreted correctly is a useful tool for accurately estimating prevalence in a defined population and reducing misdiagnosis of children under the age of five in a low transmission setting. Discerning how to interpret "faint bands" and the resulting impacts on malaria misdiagnosis in areas of medium to high transmission and may give different results and therefore we advocate for further research that will add to the growing body of knowledge regarding RDTs and their inherent strengths and limitations.This research shows that in an area of low malaria transmission, over 94% of "faint bands" are associated with negative microscopy results. Additionally, ParaCheck PfACT: artemisinin-based combination therapy; BUCHS: Bugando University College of Health Sciences; CHREB: Conjoint Health Ethics Board; COSTECH: Commission on Science and Technology; HRP2: histidine rich protein 2; NIMR: National Institute of Medical Research; NCA: Ngorongoro Conservation Area; QC: Quality Control; RDT: rapid diagnostic test; WHO: World Health Organization.The authors declare that they have no competing interests.LKA planned the study, conducted the field research, data analysis and interpretation and wrote the manuscript. JMH contributed to study planning, was involved in the field research and reviewed the manuscript. GD planned the study, conducted field research and reviewed the manuscript. JH was involved in the field research, data analysis and writing of the manuscript. MM contributed to study planning, was involved in field research and reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/308/prepubQuestionnaire: Malaria and Rapid Diagnostic Tests. This questionnaire was designed to support the testing of the ParaCheck Pf® through collection of demographic information on study participants as well as investigating their reported malaria health-seeking behaviors. In collaboration with our research personnel, a clinical officer or physician at the local hospital administered either in KiSwahili or Maa the questionnaire to consenting participants. The questionnaire was pilot tested for validity and reliability. During translation the questions were translated into KiSwahili by author MM and then back translated into English by another local research assistant.Click here for file


WHO now recommends test-based management of malaria (TBMM) across all age-groups. This implies artemisinin-based combination treatment (ACT) should be restricted to rapid diagnostic test (RDT)-positive cases. This is a departure from what caregivers in rural communities have been used to for many years.We conducted a survey among caregivers living close to 32 health centres in six districts in rural Ghana and used logistic regression to explore factors likely to influence caregiver acceptability of RDT based case management and concern about the denial of ACT on account of negative RDT results. Focus group discussions were conducted to explain the quantitative findings and to elicit further factors.A total of 3047 caregivers were interviewed. Nearly all (98%) reported a preference for TBMM over presumptive treatment. Caregivers who preferred TBMM were less likely to be concerned about the denial of ACT to their test-negative children . Compared with caregivers who had never secured national health insurance cover, caregivers who had valid  or expired  insurance cover were more likely to be concerned about the denial of ACT to their RDT-negative children. Major factors that promote TBMM acceptability include the perception that a blood test at health centre level represents improvement in the quality of care, leads to improvement in treatment outcomes, and offers opportunity for better communication between health workers and caregivers. Acceptability is also enhanced by engaging caregivers in the procedures of the test. Apprehensions about negative health worker attitude could however undermine acceptance.Test (RDT)-based management of malaria in under-five children is likely to be acceptable to caregivers in rural Ghana. The quality of caregiver-health worker interaction needs to be improved if acceptability is to be sustained. Presumptive management of malaria was practiced for many years in low-income countries because of the lack of laboratory facilities, the availability of cheap antimalarials and the need to avert deaths through early initiation of treatment Conversely, arguments opposing the shift to test-based management of malaria have included: there is insufficient evidence that malaria transmission is declining and will remain on the decline; the inadequacy of the health systems in malaria-endemic countries to ensure continuous availability of quality-assured RDTs; there is insufficient evidence on the safety of restricting ACT to test-positive cases; and that a policy of test-based management of malaria will not necessarily lead to improvement in the management of non-malarial febrile illnesses For the policy of test-based management of malaria to be applied in under-five children, it must be incorporated into the Integrated Management of Childhood Illnesses (IMCI) http://www.ghana.gov.gh/census/phc2010.pdf). It has a gross domestic product per capita of US$1,325. Life expectancy at birth is estimated at 63.8 years. Infant mortality and under-five mortality rates are estimated at 50 and 111 deaths per 1000 live births respectively Ghana has an estimated population of 24.2 million and an annual population growth rate of 2.4%.  has a cohort of 3063 children attending 16 health centres that are implementing test (with RDT)-based management of malaria and 16 other health centres that are implementing the presumptive approach. We took advantage of this trial to embed a study to assess the acceptability of RDT-based management of malaria among caregivers of children enrolled in the trial. The study, which we report here, involved a combination of quantitative (survey using a structured questionnaire) and qualitative (focus group discussions-FGDs) approaches.A large, cluster randomised-controlled trial evaluating the effect of restricting ACT to RDT-positive cases in under-five children is currently ongoing in six districts of the Brong Ahafo Region of Ghana. The trial  estimated at 269 infective bites per person per year Prior to the commencement of the clinical trial, caregivers of children less than two years of age, living within 2 kilometres of 32 health centres in the six districts were interviewed in their homes by trained field supervisors, using a structured questionnaire. The health centres were selected using probability proportional to out-patient attendance in the year preceding the survey. The list of caregivers of potentially-eligible children was generated from the database of the KHRC Demographic Surveillance System (DSS) using simple random sampling. The target was to interview 100 caregivers per health centre, and to enrol their children into the clinical trial.The questionnaire consisted of structured questions on the demographic characteristics, household assets, household characteristics , ITN ownership and use, and acceptability of RDT-based management of malaria. The household assets and characteristics used in the questionnaire were similar to those used in the 2008 Ghana Demographic and Health Survey A total of 3200 caregivers were targeted to be interviewed. This estimation was based upon statistical considerations that were made for the clinical trial. With this sample size however, it was possible to estimate the level of acceptability of RDT-based management of malaria based upon responses to the questions with a precision of 1.5%, at 95% confidence level, given a population of about 10,000 eligible caregivers and assuming a level of acceptability of RDT-based management of malaria of 50%.The data was doubled-entered, validated and cleaned using FoxPro version 9.0 and exported into Stata version 11 for analysis. Using data on 11 household assets and 5 characteristics of the dwelling of caregivers, principal component analysis was used to generate wealth quintiles Twelve to fifteen months (between June 2010 and August 2010) into the implementation of the trial, we conducted FGDs with caregivers of children enrolled in the trial to assess the acceptability of RDT-based management of malaria. Participants were caregivers of children in the intervention arm of the trial who had visited one of the study health centres in the preceding 6 weeks. As a part of the protocol of the clinical trial all children in the intervention arm had rapid tests for malaria and haemoglobin levels performed when they attended health facilities. In order to obtain as varied a perspective as possible, caregiver FGDs were formed according to the results of RDTs on their children  and the locations of their residences . Forty-six caregivers participated in the FGDs, with each FGD comprising 6 to 8 participants.Factors that emerged from preliminary analysis of the survey data such as the apparently high caregiver acceptance of RDT-based management and the reaction towards ACT denial following a test-negative result were included in the FGD topic guide. Other related behavioural and contextual issues that emerged in the course of the clinical trial were also included in the guide. Exploring the influence of health insurance status on attitudes towards the denial of ACT (following an RDT negative result) was inadvertently excluded from the FGD guide and this is a limitation of the study. The guide was piloted among women living outside the study area and therefore not participants in the trial or FGDs. All the FGDs were conducted in convenient locations within the communities; away from the health facilities. They were conducted in the local dialect and moderated by experienced social scientists from the Kintampo Health Research Centre. Caregivers did not know the moderators prior to the day of interview and they were only introduced as workers of the research centre who had come to facilitate the discussions. Each item on the FGD guide was explored to the point when no new issues emerged.All the FGDs were tape-recorded, transcribed and translated into English. The information was supplemented with notes taken during the discussions. A framework approach was adopted in the coding and analysis of the data to explain the findings of the quantitative survey. Coding was performed manually by two independent investigators. These investigators were an experienced social scientist and a post-graduate student supervised by the social scientist. Although a framework approach was adopted in the analysis, this included content analysis to gauge how dominant perceptions were, to identify emergent themes and to recognize differences in perception between groups in the FGDs. Quotes that typified consensus, or were atypical yet relevant were extracted and are reported here. They are presented here with an indication of the characteristics of the group from which the comment was made .A total of 3047 caregivers were interviewed in the survey. This fell short of the target 3200 caregivers because some health centres were situated in communities that had less than 100 children under two years of age who lived within 2 kilometres of the centre. The median age of caregivers was 28 years (range 19–45). The majority (76%) were aged between 19 and 35 years. Nearly all (99%) caregivers were mothers who were in either traditional or orthodox marriages. About a third (29%) had had no formal education. Only 50% of caregivers lived in households that had access to piped water. For 676 (22.2%) caregivers the main sources of water were springs, rivers and streams.Nearly all (98%) caregivers indicated a preference for RDT-based management of malaria over presumptive treatment. The remaining 2% either wanted the diagnosis of malaria to be based on clinical judgement or were unsure of their preference. Compared with caregivers in traditional marriages, caregivers who were single were more likely to consider RDT-based management of malaria acceptable  . About 9Forty-six caregivers participated in the six FGDs that were conducted. Each FGD was made up of 6–8 caregivers who lived around one health centre. Health centres were purposively sampled to obtain a mix of centres sited in typically rural and semi-rural settings. All discussants were the biological mothers of the children they had sent to the health centre in the preceding 6 weeks. The median age of caregivers was 28 years (range 19–45).Based upon the FGDs, the factors that positively influenced caregivers to prefer RDT-based management of malaria were: the perception that it represented improvement in the quality of care at the health centre level; the belief that it provided objective assessment of what was wrong with the child; led to favourable clinical outcomes; and afforded opportunity for interaction between the health workers and caregivers.Most caregivers were highly enthusiastic about the availability of a test for malaria at the health centres. They frequently referred to the RDT kit as a “machine” that was not available previously but is now available. Its availability was perceived to represent improvement in the quality of services delivered at the health centres in terms of both accurate diagnosis and treatment.“What I am very happy about it that at first it was in the big hospital that they would take the child's blood to see what is in it. Even there at times they won't take it but now they do it here and it's good for them to do it to know what is wrong with the child. That is what I am happy about!”..“At first because there was no blood testing they weren't giving proper medicines to the children”..Most caregivers agreed with the notion that the blood test result provided objective evidence on the condition of the child. It was felt that this relieved them (caregivers) of the burden of responsibility when reporting the symptoms of the child at the facility.“I want him [fieldworker] to take the blood and use the machine so tell what the child's problem is. If I were to say what the problem was, I could get it wrong”..“After the test he [fieldworker] writes the result on the paper and sends it to the doctor. That way, when you meet the doctor, you don't have too much to say. What is written on the paper says it all for you”..Favourable clinical outcome was perceived by many caregivers to be an outcome of use of the “machine” to guide prescriptions.“Previously whenever I brought the child to the health centre, they will give her medicine without any test and the child remained ill. Now they take her blood and do the test before saying what the child's problem is. With that when they give medicines, the child gets well”.semirural]..“He (fieldworker) uses it to check if you have malaria. It is like the one they use in pregnant women. If he drops the blood on the paper and two lines appear it means you have malaria, but if only one line appears, it means you don't have malaria. I asked about it and he showed it to me”..The awareness among some caregivers that fever could be due to causes other than malaria, and how RDT made such distinction clearer, also promoted a positive appreciation of RDT-based management of malaria.“At first we thought that all fever was caused by malaria but now that they use the test we know otherwise”..“I like the test because the fever might not be due to malaria. It could due to teething or something else. You may think it is malaria but the test may show otherwise”..“When he takes the blood, he puts it in a glass and puts a paper on it and puts it in a machine before he tells you if the blood has reduced or not”..The few caregivers who had misgivings about RDT-based management of malaria raised issues related to the effect of blood draw and apprehension that the test could rather be a test for HIV. Comments to these effects were however few.“When I went there and he said he was checking the blood I wasn't happy because the child was sick. When I looked at beneath the child's eyes, I saw that it was white  and I didn't know how he (fieldworker) was not going to be able to replace the blood that he was taking”..“On my visit, I was worried about the quantity of blood that was being drawn, and how it will be replaced in the body. However the fieldworker explained it to me and I accepted it”..“I haven't personally asked the worker to explain it to me. But I learnt that part of the lines shows red and part shows green. If it shows red, it is AIDS but if it shows green then it is malaria. That is my mind”..Rejection of RDT-based management of malaria was also influenced by caregivers' pre-conceived notions of what could be the cause of a child's illness. The dominant notion was that the presence of fever was necessarily indicative of malaria and therefore warranted malaria treatment. In addition caregivers' prior knowledge that a child had been bitten by a mosquito in the days leading to the illnesses led them to hold to the notion that the child's illness ought to be malaria. A negative RDT result in such instances made caregivers to believe that the result was probably false.“I don't trust the result of the RDT because it tends to say there is no malaria even when there high fever”..“I don't also believe it [RDT] because there are lots of mosquitoes in my room. At times when I wake up from bed, I see blood stains on the bed sheet which is as a results of the mosquito bite but any time I take the child to the hospital they say there is no malaria in the blood. The way the mosquitoes bite us in the room I don't understand why they always say that there is no malaria in the child's blood but what can I do, it is the doctor who has spoken”..Most caregivers did not consider the denial of ACT on account of a negative RDT result a problem. Based on discussions on whether the denial of ACT based on a negative RDT was a problem, the major factors that enhanced acceptance of this strategy were caregiver confidence in the “infallibility” of RDTs, the perception of favourable outcome of treatments that are based on the RDT results, and the knowledge that fever could have other causes.“If the machine tells me that is malaria, I believe it is malaria”..“When my child's RDT test was negative they did not give ACT. They gave other drugs and I thought the child will not recover on those drugs. To my surprise, when I gave the drugs to my child, he recovered”..“If perhaps I made up my mind that the child had malaria, but when I got here, the machine said the child did not have malaria and therefore they did not give malaria drug, I will accept it because it is the machine that knows better”..Some of the few caregivers who indicated they would be worried if their child was denied ACT said they would adopt alternative ACT-seeking behaviour such as procuring ACT from the drug store.“If they don't give me the malaria drugs, I will buy some from the drug store when I get home”..Across all the six FGDs, caregivers spent a considerable amount of time complaining about the attitude of health workers and how actions such as shouting and blaming caregivers for the ailments of their children made them apprehensive about speaking up during consultation. With each topic in the FGDs caregivers found a way to discuss the negative attitude of health workers. Complaints about the attitude of health workers dominated a significant amount of the discussions overall:“At times the way they [health workers] will ask you about the duration of the child's illness is not nice. They appear to be in hurry and won't take their time. She [health worker] will say, you have allowed the child to be sick for a long time. What do you want us to do for the child? [Turning to co-discussants and rhetorically asking] If I knew what could be done for the child, would I have brought the child? Their attitude is very bad”..“The clinician at the health centre easily gets angry. If he asks you a question and you talk a lot, he gets angry. My child was sick and I explained that the child had fever, cough and rashes. The man got so angry that he didn't write the drugs he was supposed to write for us. He wrote only 2 drugs for me”..“If you meet a good nurse she would pamper you but if you are not lucky and you meet one who is perhaps already angry from home, she will shout at you. She would be angry as she writes your prescription and accuse you of not taking good care of the child”..Caregivers whose children tested RDT-negative on their last visit and were therefore denied ACT, made comments that reflected a higher level of anxiety about treatment outcome than caregivers whose children tested positive and were therefore given ACT. No clear differences were observed between groups that are resident in rural and semirural areas.We used a sequential approach to apply both quantitative and qualitative methods to explore the acceptability of RDT-based management of malaria among caregivers in rural Ghana. By using this approach we were able to explain and contextualise some of the findings of the survey, as well as allow for the emergence of new themes during the qualitative study. Although the two methods were applied at different stages of caregivers' experience with RDT-based management of malaria, most of the findings of the qualitative study were consistent with the findings of the survey.In trying to understand the high level of acceptability of RDT-based management of malaria and its related factors, we find the attitude of caregivers to be consistent with the theory of rational expectation, which states that individuals make choices based on their rational outlook, available information and past experiences In terms of rational outlook, caregiver acceptability of RDT-based management of malaria was enhanced by the perception that confirming malaria diagnosis before giving ACT will lead to favourable clinical outcome while management on the basis of clinical judgement alone was speculative and led to unfavourable clinical outcome. Improved clinical outcome is likely to be the major expectation of caregivers as the new strategy is rolled out within the routine health system. Whether their expectations will be met remains to be seen. Currently health care providers cannot, with confidence, assure caregivers that RDT-based management of malaria  will lead to improved clinical outcome since the scientific evidence for this remains weak In the context of the clinical trial within which this study was conducted, caregivers appeared quite satisfied with the information fieldworkers provided about RDT-based management of malaria and the procedure adopted. Given that caregivers made very few references to information obtained from other sources, it appears the perception formed of RDT-based management of malaria was based largely on information provided by the fieldworkers and other health workers. As RDT-based management of malaria becomes more firmly established, perceptions of its advantages and disadvantages will be also be influenced by information obtained from other caregivers, family members and other members of the community. Sustaining the apparently high level of acceptability of the intervention will depend how vigorous and consciously health workers educate caregivers about the test and its place in the management of childhood illnesses in general.In traditional African society, procedures associated with blood draw easily lead to rumours that have the potential to impede adherence to interventions The findings of the study suggest that with appropriate education, test-based management of malaria using RDT can be implemented with very little or no resistance from caregivers in rural Ghana. The high level of acceptance has similarly been reported in studies in Uganda and Tanzania The belief that RDT-based management of malaria at primary care level represented improvement in the quality of care was similarly reported in another study in Uganda New themes that emerged from the FGDs provided indication as to other ways through which acceptability of RDT-based management of malaria may be further enhanced. As part of the protocol of the clinical trial, fieldworkers simultaneously conducted rapid blood test to determine haemoglobin level. From the FGDs, it was apparent that this contributed to caregivers' acceptability of RDT-based management of malaria. Another feature that appeared to have left a positive impression on the minds of caregivers was the opportunity the performance of these tests offered for educative interaction between fieldworkers and caregivers. The opportunity for them to be part of the procedure, for example waiting to see the emergence or otherwise of the “two lines”  in the window of the test kit was exciting and positive.vis-a-vis adopting alternative ACT-seeking behaviour should be explored in future research.The finding that caregivers who had health insurance cover  were more likely to be worried if their children were denied ACT suggested an expectation that they were entitled to ACT and the denial of this was the denial of something that was a matter of right. Unfortunately we did not explore this issue further in the focus group discussions. The association between being insured under the national health insurance scheme and accepting the denial of ACT The fact that caregivers think the RDT has obviated the need for them to be exact when reporting the symptoms of the child suggest a degree of uneasiness of caregivers when they consult health workers. It was therefore not surprising that caregivers spent a considerable part of the discussions lamenting the negative attitude of health workers. Poor health worker attitude could become the greatest threat to caregivers' acceptability of RDT-based management of malaria and other health interventions in rural communities. Similar issues on health worker attitudes were reported from a study in the neighbouring Ashanti Region of Ghana that was related to the use of RDTs for intermittent screening for malaria in pregnancy The fact that the assessment of caregiver perception of RDT-based management of malaria was conducted within the context of a clinical trial imposes some important limitations on the interpretation of the findings. The studies were conducted at the time when caregivers were either expecting their children to be enrolled into the clinical trial or had their children already enrolled in the trial. Despite assurances to the contrary, it is conceivable that some caregivers had apprehensions about possible negative repercussions for the care of their children if they gave responses or raised issues that were less favourable towards the intervention.The fieldworkers who performed the RDTs at the health centres received salary from the research centre. They received an uninterrupted supply of all the logistics that were required and also had fewer children to attend to each day. It is reasonable to suppose that these fieldworkers were better motivated than routine health workers. Routine health services in Ghana and other malaria-endemic sub-Saharan African countries are characterized by high patient to health worker ratios, inadequate logistics and lowly-motivated staff Although we had constituted groups in the FGDs on the basis of the RDT results of their children during their last visit, and also on the basis of their areas of residence, very little discernible differences were identified during the discussions. This could have been due to the fact that their children had had different RDT results on occasions prior to their last visits.Within the context of an on-going clinical trial, we assessed the acceptability of RDT-based management of malaria among caregivers of under-five children in rural Ghana. We found that although RDT-based management of malaria represented a departure from practices that caregivers attending health centres in rural Ghana are used to, the tests were seen as an improvement in the quality of care and therefore acceptable. Acceptability will however be further enhanced if RDT-based management of malaria leads to obvious improvement in clinical outcomes, and tests are conducted with interactive provider education and caregiver participation. Guidelines currently being developed to guide the process of incorporating RDT-based management of malaria into IMCI should consider how caregivers may be actively engaged in the procedures of the test.


There has been much debate about the appropriate statistical methodology for the evaluation of malaria field studies and the challenges in interpreting data arising from these trials.The present paper describes, for a pivotal phase III efficacy of the RTS, S/AS01 malaria vaccine, the methods of the statistical analysis and the rationale for their selection. The methods used to estimate efficacy of the primary course of vaccination, and of a booster dose, in preventing clinical episodes of uncomplicated and severe malaria, and to determine the duration of protection, are described. The interpretation of various measures of efficacy in terms of the potential public health impact of the vaccine is discussed.The methodology selected to analyse the clinical trial must be scientifically sound, acceptable to regulatory authorities and meaningful to those responsible for malaria control and public health policy.NCT00866619Clinicaltrials.gov  Plasmodium falciparum parasite. This phase III trial is designed to determine the efficacy of the vaccine against clinical malaria in young children to support the file submission for regulatory review by European and African regulatory authorities. Additionally, a broad range of secondary efficacy endpoints are included to evaluate the full potential direct impact of the vaccine on child health. These include severe malaria and malaria-specific mortality as well as all-cause hospitalization and mortality.In May 2009, the first large scale, phase III efficacy trial assessing a candidate malaria vaccine was launched across seven countries in sub-Saharan Africa. The vaccine candidate RTS, S/AS01 targets the pre-erythrocytic stage of the Malaria is a common disease in sub-Saharan children and so it may at first appear relatively simple to design trials to demonstrate the effects of interventions with reasonable sample sizes. However, the design of such trials comes with specific statistical challenges. All individuals are not at the same level or risk of malaria disease; transmission varies widely across trial sites. Malaria control measures are being scaled-up across sub-Saharan Africa, but at differing rates and coverage. Statistical methods must be able to adjust for the confounding effect of explanatory variables and take into account the fact that multiple events within the same individual are not independent.Phase II data suggest that while RTS, S/AS01 offers approximately 50% protection against clinical malaria disease, the likely mechanism of protection is reduction in the risk of disease associated with an infectious bite in all individuals rather than complete protection in 50% of individuals. A vaccine with this kind of effect is often referred to as a "leaky vaccine". A consequence of the use of a leaky malaria vaccine is that over time both vaccinated and comparator children will continue to experience malaria episodes and that in high transmission areas following sufficient exposure and extended follow-up, all individuals will experience an infection, regardless of whether they were vaccinated or not. However, this does not mean that vaccination would not provide public health benefit, since the number of episodes could be significantly reduced as well as the risk of complications.In endemic areas, young children may experience several episodes of malaria per year and gradually acquire immunity against malaria disease through repeated exposure to parasites. Naturally acquired immunity is predominantly directed to the blood stage of the infection, in contrast to the vaccine, which induces protection through an effect on the pre-erythrocytic stage. Vaccine trials are conducted superimposed on this background making long-term vaccine efficacy difficult to disentangle from protection acquired by exposure to infection.Vaccine efficacy is calculated as VE = 1 - R where R may be a ratio of risks, rates or hazards. All of these methods have been used to license vaccines. The choice of method is dependent upon the data, but it is also important that the output is meaningful to the end user: those making vaccine policy decisions and eventually parents. In this trial, vaccine efficacy is calculated using comparison of risks, rates, or hazards depending on the endpoint. The purpose of this paper is to set out the statistical methods that will be used to estimate the efficacy of RTS, S/AS01. The choice of statistical analysis for endpoints in the RTS, S/AS01 phase III trial is used to illustrate the advantages and disadvantages of each method.Haemophilus influenzae type B and oral polio vaccines).The RTS, S/AS01 pivotal phase III trial is a double-blind (observer-blind), randomized, controlled study conducted in 11 centres located across seven countries in sub-Saharan Africa enrolling children in two age categories: 6-12 weeks and 5-17 months old with a minimum of 6,000 subjects in both age categories up to a maximum of 16,000 children and infants overall. In both age categories, the subjects are randomized to one of the three study groups in a 1:1:1 ratio; (i) RTS/SAS01 given in a 0,1,2-month schedule + a RTS, S/AS01 booster dose at Month 20 (group R3R); (ii) RTS/SAS01 given in a 0,1,2-month schedule + a booster dose of control vaccine at Month 20 (group R3C); (iii) control vaccine given in a 0,1,2-month schedule + a booster dose of control vaccine at Month 20 (group C3C) [The full list of efficacy endpoints is given in Table The co-primary endpoints are efficacy against clinical malaria for 12 months post Dose 3 in both the 6-12 weeks and 5-17 months age groups. The analysis of efficacy will be carried out when 6,000 children in an age category have been enrolled and followed up for 12 months post Dose 3. Vaccine efficacy (VE) against first or only episodes will be determined using a standard formula: VE = 100 × (1 - HR)% where HR is the hazard ratio estimated from Cox regression model. Following an episode of malaria, an individual will be removed from the risk set and subjects who are lost to follow-up, who are withdrawn, or who die will be included up to the date of loss, withdrawal or death [The primary analysis will be stratified for study site and unadjusted for other covariates; however, vaccine efficacy estimates adjusted for covariates will also be presented. As there are two co-primary endpoints, 97.5% confidence intervals (CIs) on VE will be calculated, ensuring an overall 2-sided 5% alpha-level. Therefore, p-values lower than 0.025 will be considered significant for the primary endpoints. Assuming at least 5400 evaluable subjects (randomized 2:1), an attack rate in controls of 10/100 per child year at risk (cyr) over the follow-up period from 2 weeks post Dose 3 to 1 year post Dose 3 and a true vaccine efficacy of 30%, the study has 90% power to detect a lower limit of the 97.5% CI around estimated VE above 0%. All secondary endpoints will be evaluated using 95% CIs.VE against all episodes will be calculated by an incidence rate ratio (IRR) using a negative binomial regression model controlling for interdependence between episodes within the same subject. The 95% CI and p-values on VE estimates, defined as 100 × (1 - IRR)%, will be calculated from this model. Events occurring within 14 days following a malaria episode meeting the malaria case definition under evaluation will not contribute, to minimize counting treatment failures as an episode resulting from a new infection and the total time at risk will be adjusted.VE against severe malaria will be evaluated when 250 subjects have reported an episode of severe malaria meeting the primary case definition. Recruitment of 250 affected subjects provides 90% power to detect 30% VE with a lower limit of 95% CI above 0%. The primary analysis will compare the pooled RTS, S/AS01 groups versus the control group (R3R + R3C vs C3C). VE against severe malaria will be estimated as 1 - RR where RR is the risk ratio  together with 95% CIs. Analysis of all events will be performed in a similar way as for clinical malaria disease. Table The benefit of the booster dose will be evaluated by the comparison of RTS, S/AS01 recipients receiving a booster dose compared to control booster (R3R vs R3C) over the 12 months following booster dose. Children receiving a primary schedule with or without booster dose will be compared to controls as well (R3R vs C3C and R3C vs C3C).At 18 and 30 months post primary immunization, a cross sectional survey will sample all participating children to assess vaccine efficacy against prevalent parasitaemia and anaemia. This will be estimated by the prevalence ratio with 95%CI.The primary efficacy analysis will be performed on the according to protocol (ATP) population over the period starting 14 days post Dose 3. The ATP population includes all subjects meeting the inclusion criteria who received all vaccinations according to protocol and contribute to time at risk starting 14 days post dose 3. The choice to perform the primary analysis on the ATP population reflects the thought that the main outcome is to describe vaccine efficacy for a vaccinated population. However, analyses will also be conducted on a modified intention to treat (ITT) population, which includes all subjects who received at least one dose of vaccine. The ITT population will be analysed by randomization assignment and start time at risk from the day of first vaccination. Subjects randomized but not vaccinated are not under efficacy surveillance.P. falciparum. Fever in these children may be caused by disease other than malaria. In order to improve specificity, cases in this trial are defined as children who are unwell and have presented to the clinic with fever and parasite density above a threshold value [Case definitions - malaria is characterized by fever caused by parasitaemia. In malaria endemic regions it is not uncommon for children to carry asymptomatic infections with ld value . A commold value .The percentage of children free of malaria by the end of the trial can be compared but efficacy will depend on the level of incidence and the duration of the trial so this measure of efficacy is difficult to compare between trials and it does not reflect the reduction in disease burden due to vaccination. As a primary analysis, vaccine efficacy is estimated by evaluating the hazard ratio of first or only episodes, allowing evaluation of the direct effect of the vaccine. This is in line with the hypothesized mechanism of RTS, S/AS vaccination, namely that rather than providing complete protection to half of the individuals , it protects by reducing the risk of disease following an infectious bite by a certain proportion for all individuals. The Cox model allows for different levels of baseline risk across study sites and can include baseline covariates. Under the assumption that vaccine efficacy is not different across study sites and the hazard ratio is constant over time, it is a good method to reduce variability and control for confounding, particularly as data for the primary endpoint will be pooled across multiple transmission settings. Also, analysis on vaccine efficacy by study site is a secondary analyses planned for by the protocol. This methodology is well established and has been broadly used to evaluate previous malaria interventions -13 and tVaccine efficacy is also calculated against all episodes of clinical malaria. The methodology used to do this must take into account the fact that the risk in all individuals is not equal; some individuals are more susceptible than others related to host factors such as genetic background, health and behaviour or to the level of exposure to bites by infectious mosquitoes in the community. Moreover, the risk of malaria may not be constant over subsequent episodes. Vaccine efficacy is calculated by comparing incidence rates using a negative binomial regression model allowing for individual heterogeneity arising from non-independence of multiple episodes within the same subject. This model accounts for heterogeneity among individuals and considers non-independent multiple episodes ,16. VaccUnlike uncomplicated clinical malaria, which is a relatively common disease from which patients usually recover completely with treatment, severe malaria is a rare event associated with a high case fatality rate even in the presence of optimal treatment and repeated episodes in the same individual are uncommon . As suchVaccine efficacy against severe malaria disease will be analysed as a risk ratio, comparing the proportion of children affected by at least one episode of severe malaria in each group. The key advantage of this measure is that it is readily interpretable; the vaccine reduces the risk of severe disease over the specified time frame by a proportion. This methodology is also used for each of the endpoints, which share the same features of being rare and life-threatening Table .In a secondary analysis, all episodes of severe malaria will be investigated in the same way as all episodes of clinical malaria.The assessment of the evolution of vaccine efficacy against clinical malaria over time is the most challenging aspect of the analysis; it is not a limitation of the statistical methodology but of the data set itself. Two analytic methods will be used to evaluate the evolution of protection against clinical malaria over the 30-month follow up period. The first will compare the study groups R3C and C3C from the beginning to the end of the study and the second will estimate vaccine efficacy over shorter time periods; from 14 days post dose 3 to 17 months and from 18 months to 30 months. To evaluate duration of protection, analyses of all events is more informative than first or only episodes.The challenge lies in the interpretation of the results that these analyses will provide because neither methodology is able to disentangle the effects of vaccine induced immunity and naturally acquired protection. In most parts of Africa, children acquire a high level of protection against the severe forms of malaria in early childhood as a result of repeated exposure to blood stage parasites. It is expected that the vaccinees also acquire immunity as a result of exposure to infection. The difficulty is that there is no immunological assay that can tell whether vaccinees have acquired natural immunity at the same rate as controls and allow adjustment for this in the analysis. If natural immunity is acquired at a similar rate in controls and vaccines, then estimates of vaccine efficacy will approximate true vaccine efficacy, but if vaccinees and controls acquire natural immunity at a different rate, estimates will not reflect true vaccine efficacy. Although efficacy estimates based on first or only episodes may not be very informative of duration of protection, the Cox proportional hazards method allows to investigate whether the proportionality of hazards remains constant over time (i.e. that observed vaccine effect remains constant) by examining cumulative incidence plots, Schoenfeld residuals and models with time-dependent covariates. If there is no support from the data that the HR changes over time, one could assume a constant effect over the follow-up. However, the opposite is not true: violating the proportionality of hazards assumption does not necessarily mean waning efficacy. Violating a proportionality of hazards assumption in the presence of declining attack rates may suggest decreasing susceptibility or heterogeneity in exposure rather than waning vaccine efficacy. The approach of breaking the surveillance period into shorter time periods, sometimes described as "resetting the clock", is intuitive. However, the interpretation remains complicated by the fact that the control group is no longer directly comparable in the second time period due to different exposure in the first time period and the concomitant development of natural immunity.As relevant to policy makers as the evolution of vaccine efficacy over time, is the presentation of results addressing the fundamental hypothesis underlying the development strategy for RTS, S/AS01. The hypothesis is that a partially efficacious vaccine would be of great value in endemic areas, if it was able to be given to young children and provide them with protection against the severe life-threatening forms of the disease until they had acquired natural protection. This can be assessed by presenting an analysis of the number of cases averted by the vaccine over time.A covariate can be defined as a qualitative factor or a quantitative variable that is expected to have an influence on study endpoint(s) to be analysed. Covariates can be highly variable and may include disease characteristics, social or economic factors and geographic or demographic data .Study centres participating in this trial represent a range of malaria transmission intensities across sub-Saharan Africa, including the intense seasonal transmission of West Africa and perennial transmission in East Africa. Other factors might influence the pattern of malaria disease in a population and may be taken into account as covariates for data analyses. The importance to controlling for heterogeneity in malaria risk to avoid underestimating vaccine efficacy has been highlighted earlier .All the covariates collected in this trial have known associations with malaria risk. Cox regression allows for seasonal variation of malaria incidence and adding study centres as a covariate allows for different levels of baseline risk and different patterns of seasonality. The trial collected a number of potential covariates including age, bed net use -24, distIn conclusion, the analytic methods selected to analyse the results of the RTS, S/AS01 phase III clinical trial must be scientifically sound, acceptable to regulatory authorities and meaningful to those responsible for malaria control and public health policy. These needs have been taken into account and resulted in a list of primary and secondary endpoints, each with their appropriate analytic methods. Finally, an understanding of the biological action and full benefit/risk profile of the vaccine will only be gained by looking at the full picture that is painted by the entire range of predefined endpoints and planned analyses.ML and AL are employees of GSK Biologicals. AL holds stock options in GSK Biologicals. ML, JA, JW, BM, PB, and AL declare their institution received a grant from MVI for the clinical trial described in this manuscript. ML and AL declare their institution has received grants from MVI for previous clinical trials. PB declares his institution has grants pending from MVI. JA, JW, and PB declare receiving travel funds from MVI for travel related to this clinical trial. JA declares his institution received financial compensation from MVI for administrative support. AM declares no potential conflicts of interest.All authors contributed to the development of this manuscript through discussions and document review. ML led the writing of the manuscript coordinating the incorporation of all reviewer comments. All authors critically contributed to the discussions on statistical methodology. All authors read and approved the final manuscript.