Background Malaria continues to claim one to two million lives a year, mainly those of children in sub-Saharan Africa. Reduction in mortality depends, in part, on improving the quality of hospital care, the training of healthcare workers and improvements in public health. This study examined the prognostic indicators of severe falciparum malaria in Gabonese children.
Background
Malaria continues to claim one to two million lives a year, mainly those of children in sub-Saharan Africa. Reduction in mortality depends, in part, on improving the quality of hospital care, the training of healthcare workers and improvements in public health. This study examined the prognostic indicators of severe falciparum malaria in Gabonese children.Methods An observational study examining the clinical presentations and laboratory features of severe malaria was conducted at the Centre Hospitalier de Libreville, Gabon over two years. Febrile children aged from 0 to 10 years with  Plasmodium falciparum  infection and one or more features of severe malaria were enrolled.
Methods
An observational study examining the clinical presentations and laboratory features of severe malaria was conducted at the Centre Hospitalier de Libreville, Gabon over two years. Febrile children aged from 0 to 10 years with  Plasmodium falciparum  infection and one or more features of severe malaria were enrolled.Plasmodium falciparumResults Most children presenting with severe falciparum malaria were less than 5 years (92.3% of 583 cases). Anaemia was the most frequent feature of severe malaria (67.8% of cases), followed by respiratory distress (31%), cerebral malaria (24%) hyperlactataemia (16%) and then hypoglycaemia (10%). Anaemia was more common in children under 18 months old, while cerebral malaria usually occurred in those over 18 months. The overall case fatality rate was 9%. The prognostic indicators with the highest case fatality rates were coma/seizures, hyperlactataemia and hypoglycaemia, and the highest case fatality rate was in children with all three of these features.
Results
Most children presenting with severe falciparum malaria were less than 5 years (92.3% of 583 cases). Anaemia was the most frequent feature of severe malaria (67.8% of cases), followed by respiratory distress (31%), cerebral malaria (24%) hyperlactataemia (16%) and then hypoglycaemia (10%). Anaemia was more common in children under 18 months old, while cerebral malaria usually occurred in those over 18 months. The overall case fatality rate was 9%. The prognostic indicators with the highest case fatality rates were coma/seizures, hyperlactataemia and hypoglycaemia, and the highest case fatality rate was in children with all three of these features.Conclusions Prompt and appropriate, classification and treatment of malaria helps identify the most severely ill children and aids early and appropriate management of the severely ill child.
Conclusions
Prompt and appropriate, classification and treatment of malaria helps identify the most severely ill children and aids early and appropriate management of the severely ill child.

Introduction Each year 500 million infections and up to 2.7 millions deaths are attributable to malaria [ 1 ], about 90% of these deaths occur in children in sub-Saharan Africa [ 2 ]. Eighty percent of the deaths occur during the first 24 hours following admission [ 3 - 5 ]. Despite a better understanding of pathophysiology and management of malaria, childhood mortality remains unacceptably high [ 6 ]. The acquisition of malaria immunity is closely linked to the level of transmission and severe  Plasmodium falciparum  infection is very rare after the age of 5 years in highly endemic areas. Presentations of severe malaria are different at different ages and in areas with different levels of transmission. In Gabon, a country of about 1.2 million people, malaria is the main cause of neurological, haematological and infectious emergencies at the Centre Hospitalier de Libreville (CHL), the country's tertiary referral centre [ 7 ]. Malaria transmission is hyperendemic and perennial with an entomological inoculation rate of 50 per person-year [ 8 ]. Previous studies in Africa have shown three frequent presentations of severe falciparum malaria: cerebral malaria, metabolic malaria (hyperlactaemia, acidosis or respiratory distress) and severe anaemia [ 3 , 5 , 9 ]. The case fatality rate of severe anaemia, however, is low and in some studies it is not an independent predictor of death [ 9 , 10 ]. In Africa, malaria mortality remains high for a number of reasons including limited access to healthcare and increased drug resistance [ 6 ]. Better classification of severe malaria could aid clinicians caring for children with severe malaria to avoid diagnostic delays, identify the children most likely to die and thus improve management by targeting resources to the sickest children. This prospective observational study was designed to determine the clinical and laboratory features that identify those children most severely ill with malaria.
Introduction
Each year 500 million infections and up to 2.7 millions deaths are attributable to malaria [ 1 ], about 90% of these deaths occur in children in sub-Saharan Africa [ 2 ]. Eighty percent of the deaths occur during the first 24 hours following admission [ 3 - 5 ]. Despite a better understanding of pathophysiology and management of malaria, childhood mortality remains unacceptably high [ 6 ]. The acquisition of malaria immunity is closely linked to the level of transmission and severe  Plasmodium falciparum  infection is very rare after the age of 5 years in highly endemic areas. Presentations of severe malaria are different at different ages and in areas with different levels of transmission. In Gabon, a country of about 1.2 million people, malaria is the main cause of neurological, haematological and infectious emergencies at the Centre Hospitalier de Libreville (CHL), the country's tertiary referral centre [ 7 ]. Malaria transmission is hyperendemic and perennial with an entomological inoculation rate of 50 per person-year [ 8 ]. Previous studies in Africa have shown three frequent presentations of severe falciparum malaria: cerebral malaria, metabolic malaria (hyperlactaemia, acidosis or respiratory distress) and severe anaemia [ 3 , 5 , 9 ]. The case fatality rate of severe anaemia, however, is low and in some studies it is not an independent predictor of death [ 9 , 10 ]. In Africa, malaria mortality remains high for a number of reasons including limited access to healthcare and increased drug resistance [ 6 ]. Better classification of severe malaria could aid clinicians caring for children with severe malaria to avoid diagnostic delays, identify the children most likely to die and thus improve management by targeting resources to the sickest children. This prospective observational study was designed to determine the clinical and laboratory features that identify those children most severely ill with malaria.Plasmodium falciparumMethods This study was carried out in the CHL between 1st August 2000 and 31st July 2002. The CHL is the largest public hospital in the country, situated in Libreville, the capital city (pop. 500,000). Services at the CHL include an Emergency Department (20 beds), a Paediatrics Unit (80 beds) and an Intensive Care Unit (12 beds). Ethical permission for the study was granted by the Gabonese Ministry of Health. Febrile children were referred the study team and seen on admission by a Malaria Clinical Research Unit (MCRU) clinician, summary data were recorded on a  pro-forma  sheet. A blood sample (2 ml) was drawn (anti-coagulated with EDTA) for quantitative examination of a blood film for malarial parasites using the Lambaréné method [ 11 ], measurement of haemoglobin concentration, white cell count, platelet count (STKS, Coulter Corporation) and blood lactate and glucose concentrations. Within 15 minutes of blood sampling, blood lactate and glucose concentrations were measured using Accusport™ hand held analyser (Bohringer, Manheim, Inc., Germany) and One-touch Analyzer (LifeScan, Inc., USA) [ 11 ]. Blood films were defined as negative if there were no asexual forms of  P. falciparum  in 100 high power fields of a thick film. The schematic process of the inclusion is shown in Figure  1 . Figure 1 Schematic process of the screening of febrile children in the Centre Hospitalier de Libreville, Gabon. Febrile children (or those with history of fever in the last 48 hours) were considered for inclusion in the study if they were: aged 0 to 10 years of age (inclusive), had malaria (> 2 asexual forms of  P. falciparum  seen on blood film) and had one or more of the following features of severity: [ 9 , 12 , 13 ]: Blantyre coma score (BCS) ≤ 2 defining cerebral malaria, repeated observed seizures (3 or more observed in 24 hours), lactate concentration in whole blood or capillary blood ≥ 5 mmol/L, glucose concentration in whole blood or capillary blood ≤ 2.2 mmol/L, severe anaemia (haemoglobin concentration of < 5 g/dL) and/or haematocrit concentration < 15%). Children were excluded from the study if informed consent was not obtained from a relative or if an alternative diagnosis was made clinically or by investigation (such as cerebrospinal fluid examination, chest radiography or blood culture). Respiratory distress was defined as the presence of one or more of these features [ 14 ]: abnormalities in respiratory rate (according to the age), rhythm (Kussmaul's or Cheyne-Stokes's breathing) and signs of distress such as nasal flaring, intercostal or subcostal recession. Management All children enrolled were hospitalised and treated with parenteral quinine (12.5 mg salt/kg/day, intravenous; Quinimax*, Sanofi-Synthelabo, France) without a loading dose, followed by oral quinine when tolerated. Pyrexial children received paracetamol suppositories (60 mg/kg/day, rectal; Efferalgan™, Bristol- UPSA, France). Seizures were controlled with diazepam (0.3 mg/kg, iv or 0.5 mg/kg rectal; Valium™, Roche, France). Severe anaemia was corrected by transfusion of packed red cells (15 ml/kg over 4 hours) screened for blood borne infections. Hypoglycaemia was treated with a slow intravenous injection of hypertonic glucose 40 %(Braun, Germany) at a dose of 1 ml/kg. Nasal oxygen at 6 1/minute was given to children with respiratory distress. Follow up An MCRU clinician performed a full physical examination daily until discharge for each child. Laboratory assessments including parasitaemia, blood glucose and lactate were performed during hospitalization as necessary. The outcome (survived, death) was recorded. Statistical analysis Statistical analysis was carried out with Epi info 6.04 (ENSP-Epiconcept- InVS, Corp.) and Stata Statistical Software (version 7.0, Stata Corporation, College Station, Texas, USA). Normality of data distribution was checked using either Shapiro-Wilks or Kolmogrov -Smirnov test. Normally distributed data were analysed by two-tailed Student's T test and non-normally distributed data with the Mann-Whitney U statistic. Proportions were compared with χ 2  tests with Yates' correction or Fisher's exact test. ANOVA test were used for multiple comparisons of variances, with Tukey's  post hoc  test. Assessments of prognostic factors were conducted with logistic regression model. A  p  value < 0.05 was considered as significant. Specific prevalence for each subgroup has been defined as the ratio of the number of cases observed in this sub-group over the population of this same sub-group. Hyperlactataemia is usually defined as a blood lactate concentration of ≥ 5 mmol/L. As the Accusport has been found to have poor agreement with the gold standard YSI 2300 [ 11 ] or YSI 1500 sport [ 15 ], a definition of hyperlactataemia as blood lactate concentration ≥ 10 mmol/L was used to increase the specificity in the analysis.
Methods
This study was carried out in the CHL between 1st August 2000 and 31st July 2002. The CHL is the largest public hospital in the country, situated in Libreville, the capital city (pop. 500,000). Services at the CHL include an Emergency Department (20 beds), a Paediatrics Unit (80 beds) and an Intensive Care Unit (12 beds). Ethical permission for the study was granted by the Gabonese Ministry of Health.Febrile children were referred the study team and seen on admission by a Malaria Clinical Research Unit (MCRU) clinician, summary data were recorded on a  pro-forma  sheet. A blood sample (2 ml) was drawn (anti-coagulated with EDTA) for quantitative examination of a blood film for malarial parasites using the Lambaréné method [ 11 ], measurement of haemoglobin concentration, white cell count, platelet count (STKS, Coulter Corporation) and blood lactate and glucose concentrations. Within 15 minutes of blood sampling, blood lactate and glucose concentrations were measured using Accusport™ hand held analyser (Bohringer, Manheim, Inc., Germany) and One-touch Analyzer (LifeScan, Inc., USA) [ 11 ]. Blood films were defined as negative if there were no asexual forms of  P. falciparum  in 100 high power fields of a thick film. The schematic process of the inclusion is shown in Figure  1 .pro-formaP. falciparumFebrile children (or those with history of fever in the last 48 hours) were considered for inclusion in the study if they were: aged 0 to 10 years of age (inclusive), had malaria (> 2 asexual forms of  P. falciparum  seen on blood film) and had one or more of the following features of severity: [ 9 , 12 , 13 ]: Blantyre coma score (BCS) ≤ 2 defining cerebral malaria, repeated observed seizures (3 or more observed in 24 hours), lactate concentration in whole blood or capillary blood ≥ 5 mmol/L, glucose concentration in whole blood or capillary blood ≤ 2.2 mmol/L, severe anaemia (haemoglobin concentration of < 5 g/dL) and/or haematocrit concentration < 15%). Children were excluded from the study if informed consent was not obtained from a relative or if an alternative diagnosis was made clinically or by investigation (such as cerebrospinal fluid examination, chest radiography or blood culture).P. falciparumRespiratory distress was defined as the presence of one or more of these features [ 14 ]: abnormalities in respiratory rate (according to the age), rhythm (Kussmaul's or Cheyne-Stokes's breathing) and signs of distress such as nasal flaring, intercostal or subcostal recession.Management All children enrolled were hospitalised and treated with parenteral quinine (12.5 mg salt/kg/day, intravenous; Quinimax*, Sanofi-Synthelabo, France) without a loading dose, followed by oral quinine when tolerated. Pyrexial children received paracetamol suppositories (60 mg/kg/day, rectal; Efferalgan™, Bristol- UPSA, France). Seizures were controlled with diazepam (0.3 mg/kg, iv or 0.5 mg/kg rectal; Valium™, Roche, France). Severe anaemia was corrected by transfusion of packed red cells (15 ml/kg over 4 hours) screened for blood borne infections. Hypoglycaemia was treated with a slow intravenous injection of hypertonic glucose 40 %(Braun, Germany) at a dose of 1 ml/kg. Nasal oxygen at 6 1/minute was given to children with respiratory distress.
Management
All children enrolled were hospitalised and treated with parenteral quinine (12.5 mg salt/kg/day, intravenous; Quinimax*, Sanofi-Synthelabo, France) without a loading dose, followed by oral quinine when tolerated. Pyrexial children received paracetamol suppositories (60 mg/kg/day, rectal; Efferalgan™, Bristol- UPSA, France). Seizures were controlled with diazepam (0.3 mg/kg, iv or 0.5 mg/kg rectal; Valium™, Roche, France). Severe anaemia was corrected by transfusion of packed red cells (15 ml/kg over 4 hours) screened for blood borne infections. Hypoglycaemia was treated with a slow intravenous injection of hypertonic glucose 40 %(Braun, Germany) at a dose of 1 ml/kg. Nasal oxygen at 6 1/minute was given to children with respiratory distress.Follow up An MCRU clinician performed a full physical examination daily until discharge for each child. Laboratory assessments including parasitaemia, blood glucose and lactate were performed during hospitalization as necessary. The outcome (survived, death) was recorded.
Follow up
An MCRU clinician performed a full physical examination daily until discharge for each child. Laboratory assessments including parasitaemia, blood glucose and lactate were performed during hospitalization as necessary. The outcome (survived, death) was recorded.Statistical analysis Statistical analysis was carried out with Epi info 6.04 (ENSP-Epiconcept- InVS, Corp.) and Stata Statistical Software (version 7.0, Stata Corporation, College Station, Texas, USA). Normality of data distribution was checked using either Shapiro-Wilks or Kolmogrov -Smirnov test. Normally distributed data were analysed by two-tailed Student's T test and non-normally distributed data with the Mann-Whitney U statistic. Proportions were compared with χ 2  tests with Yates' correction or Fisher's exact test. ANOVA test were used for multiple comparisons of variances, with Tukey's  post hoc  test. Assessments of prognostic factors were conducted with logistic regression model. A  p  value < 0.05 was considered as significant. Specific prevalence for each subgroup has been defined as the ratio of the number of cases observed in this sub-group over the population of this same sub-group. Hyperlactataemia is usually defined as a blood lactate concentration of ≥ 5 mmol/L. As the Accusport has been found to have poor agreement with the gold standard YSI 2300 [ 11 ] or YSI 1500 sport [ 15 ], a definition of hyperlactataemia as blood lactate concentration ≥ 10 mmol/L was used to increase the specificity in the analysis.
Statistical analysis
Statistical analysis was carried out with Epi info 6.04 (ENSP-Epiconcept- InVS, Corp.) and Stata Statistical Software (version 7.0, Stata Corporation, College Station, Texas, USA). Normality of data distribution was checked using either Shapiro-Wilks or Kolmogrov -Smirnov test. Normally distributed data were analysed by two-tailed Student's T test and non-normally distributed data with the Mann-Whitney U statistic. Proportions were compared with χ 2  tests with Yates' correction or Fisher's exact test. ANOVA test were used for multiple comparisons of variances, with Tukey's  post hoc  test. Assessments of prognostic factors were conducted with logistic regression model. A  p  value < 0.05 was considered as significant. Specific prevalence for each subgroup has been defined as the ratio of the number of cases observed in this sub-group over the population of this same sub-group. Hyperlactataemia is usually defined as a blood lactate concentration of ≥ 5 mmol/L. As the Accusport has been found to have poor agreement with the gold standard YSI 2300 [ 11 ] or YSI 1500 sport [ 15 ], a definition of hyperlactataemia as blood lactate concentration ≥ 10 mmol/L was used to increase the specificity in the analysis.post hocpResults Demographic and clinical data During the study period (1st August 2000-31st July 2002), 8,036 febrile children were screened for malaria at the hospital. The data for 7,980 of these children were analysed: 4,368 male (54.5%) and 3,612 female (45.5%). Seventy-five percent these were less than 5 years of age. 3,156 (39.3%) of the 8,036 febrile children screened in the MCRU had a positive blood film for  P. falciparum . A lower prevalence of malaria was seen in children aged< 6 months (3.7%, n = 118, p < 0.001). Specific prevalence of malaria rises after the 6 first months of life until it reaches a maximum at 47 months (47.5%), after which it declines again. Severe anaemia was most frequent in children less than 24 months old with 68 % of the cases of severe malarial anaemia occurring before this age. In contrast, the highest specific prevalence of cerebral malaria was found in children aged > 12 months. Sixty five percent of cases of cerebral malaria occurred in children aged between 12–48 months. Characteristics of severe malaria The admission clinical, laboratory and parasitological characteristics of the 583 children with severe malaria are shown in the Figure  2 . Two hundred and ninety nine of the severe malaria cases were male (51.3%) and 536 (92%) of the children with severe malaria were less than 5 years old. A history of vomiting, seizures and anti-malarial treatment before admission were reported in 322 (55.2%), 267 (45.8%) and 315 (54%) of the children with severe malaria respectively. Despite significant fluctuations in rainfall, the number of malaria cases per month was sustained during the study period, confirming the perennial transmission of malaria in this region. Figure 2 Admission characteristics of the study population (All results are mean ± SD except those specified. Severe anaemia was the commonest feature of severe malaria present in 395 (67.8%,) of the children. Neurological presentations (either coma or repeated convulsions) were present in 228 (39.2%) of the children. Respiratory distress occurred in 181 (31%) of the children ; hyperlactataemia in 73 (15.7%) and hypoglycaemia in 33 (6.2%) of the children with severe malaria. Hyperparasitaemia (> = 20% of circulating infected red blood cells) was relatively rare, occurring in only 24 (4.1%) of the children with severe malaria. Renal failure, acute pulmonary oedema and spontaneous bleeding are uncommon complications of childhood malaria [ 5 , 9 , 10 ] and were not seen in this study. Circulatory collapse was not found alone in this study and was not considered in the statistical analysis. One hundred and forty six (25%) children with severe malaria were afebrile on admission to hospital. Case rate fatality Figure  3  shows a Venn diagram summarizing clinical and laboratory features of severe malaria and case fatality rates. Figure 3 Venn diagram of features of 463 cases with a complete dataset of clinical measures. Case fatality rates shown in brackets. Of 583 patients, 52 died (40% male), giving an overall case fatality rate of 8.9%. Seven children were lost to follow-up. Forty seven deaths (90%) occurred within the first 24 hours after admission. Neurological sequelae were present in 27 (5 %) of the 531 survivors. The case fatality rate was significantly higher in females than males (10.9% vs.6.9%, p = 0.006). Children who died were older than those who survived (mean (SD) = 35.4 (41.9) vs. 25.0 (18.2) months, p = 0.0009). Of the 52 deaths in the course of the study, 32 (61.5%) presented with cerebral malaria, 32 (61.5%) presented with respiratory distress, and 20 (50% of the 40 with measurements) had hyperlactataemia. Thirty (60%) of the children who died presented with convulsions, and 13 (25%) with hypoglycaemia. Twenty four (46.2%) of those who died had severe anaemia. Mortalities in each sub-group demonstrated that hyperparasitaemia (1 death in 24, 4%) and severe anaemia (24 deaths in 395, i.e. 6%) had a better prognosis than cerebral malaria (32 deaths in 142, i.e. 22.5%) and hypoglycaemia (15 deaths in 60, i.e. 25%). Thirty-two (17.7%) of the 181 patients with RD died, as did 30 (11%) of the 264 patients with convulsions and 20 (27.4 %) of the 73 patients with hyperlactataemia. A multiple logistic regression model identified coma (OR = 3.6, 95% CI = 1.8–7.1, p < 0.001), hyperlactataemia (OR = 6.98, 95% CI = 3.5–13.8, p = 0.0001), respiratory distress (OR = 2.0, 95% CI = 1.0–3.9, p = 0.033) and hypoglycaemia (OR = 4.0, 95% CI = 1.7–9.4, p = 0.001) as independent predictors of a fatal outcome. Severe anaemia, hyperparasitaemia and thrombocytopaenia were not shown to be predictors of death (Figure  4 ). Figure 4 Prognostic indicators at the time of admission. OR – odds ratio, SD – standard deviation.
Results
Demographic and clinical data During the study period (1st August 2000-31st July 2002), 8,036 febrile children were screened for malaria at the hospital. The data for 7,980 of these children were analysed: 4,368 male (54.5%) and 3,612 female (45.5%). Seventy-five percent these were less than 5 years of age. 3,156 (39.3%) of the 8,036 febrile children screened in the MCRU had a positive blood film for  P. falciparum . A lower prevalence of malaria was seen in children aged< 6 months (3.7%, n = 118, p < 0.001). Specific prevalence of malaria rises after the 6 first months of life until it reaches a maximum at 47 months (47.5%), after which it declines again. Severe anaemia was most frequent in children less than 24 months old with 68 % of the cases of severe malarial anaemia occurring before this age. In contrast, the highest specific prevalence of cerebral malaria was found in children aged > 12 months. Sixty five percent of cases of cerebral malaria occurred in children aged between 12–48 months.
Demographic and clinical data
During the study period (1st August 2000-31st July 2002), 8,036 febrile children were screened for malaria at the hospital. The data for 7,980 of these children were analysed: 4,368 male (54.5%) and 3,612 female (45.5%). Seventy-five percent these were less than 5 years of age. 3,156 (39.3%) of the 8,036 febrile children screened in the MCRU had a positive blood film for  P. falciparum . A lower prevalence of malaria was seen in children aged< 6 months (3.7%, n = 118, p < 0.001). Specific prevalence of malaria rises after the 6 first months of life until it reaches a maximum at 47 months (47.5%), after which it declines again. Severe anaemia was most frequent in children less than 24 months old with 68 % of the cases of severe malarial anaemia occurring before this age. In contrast, the highest specific prevalence of cerebral malaria was found in children aged > 12 months. Sixty five percent of cases of cerebral malaria occurred in children aged between 12–48 months.P. falciparumCharacteristics of severe malaria The admission clinical, laboratory and parasitological characteristics of the 583 children with severe malaria are shown in the Figure  2 . Two hundred and ninety nine of the severe malaria cases were male (51.3%) and 536 (92%) of the children with severe malaria were less than 5 years old. A history of vomiting, seizures and anti-malarial treatment before admission were reported in 322 (55.2%), 267 (45.8%) and 315 (54%) of the children with severe malaria respectively. Despite significant fluctuations in rainfall, the number of malaria cases per month was sustained during the study period, confirming the perennial transmission of malaria in this region. Figure 2 Admission characteristics of the study population (All results are mean ± SD except those specified. Severe anaemia was the commonest feature of severe malaria present in 395 (67.8%,) of the children. Neurological presentations (either coma or repeated convulsions) were present in 228 (39.2%) of the children. Respiratory distress occurred in 181 (31%) of the children ; hyperlactataemia in 73 (15.7%) and hypoglycaemia in 33 (6.2%) of the children with severe malaria. Hyperparasitaemia (> = 20% of circulating infected red blood cells) was relatively rare, occurring in only 24 (4.1%) of the children with severe malaria. Renal failure, acute pulmonary oedema and spontaneous bleeding are uncommon complications of childhood malaria [ 5 , 9 , 10 ] and were not seen in this study. Circulatory collapse was not found alone in this study and was not considered in the statistical analysis. One hundred and forty six (25%) children with severe malaria were afebrile on admission to hospital.
Characteristics of severe malaria
The admission clinical, laboratory and parasitological characteristics of the 583 children with severe malaria are shown in the Figure  2 . Two hundred and ninety nine of the severe malaria cases were male (51.3%) and 536 (92%) of the children with severe malaria were less than 5 years old. A history of vomiting, seizures and anti-malarial treatment before admission were reported in 322 (55.2%), 267 (45.8%) and 315 (54%) of the children with severe malaria respectively. Despite significant fluctuations in rainfall, the number of malaria cases per month was sustained during the study period, confirming the perennial transmission of malaria in this region.Severe anaemia was the commonest feature of severe malaria present in 395 (67.8%,) of the children. Neurological presentations (either coma or repeated convulsions) were present in 228 (39.2%) of the children. Respiratory distress occurred in 181 (31%) of the children ; hyperlactataemia in 73 (15.7%) and hypoglycaemia in 33 (6.2%) of the children with severe malaria. Hyperparasitaemia (> = 20% of circulating infected red blood cells) was relatively rare, occurring in only 24 (4.1%) of the children with severe malaria.Renal failure, acute pulmonary oedema and spontaneous bleeding are uncommon complications of childhood malaria [ 5 , 9 , 10 ] and were not seen in this study. Circulatory collapse was not found alone in this study and was not considered in the statistical analysis. One hundred and forty six (25%) children with severe malaria were afebrile on admission to hospital.Case rate fatality Figure  3  shows a Venn diagram summarizing clinical and laboratory features of severe malaria and case fatality rates. Figure 3 Venn diagram of features of 463 cases with a complete dataset of clinical measures. Case fatality rates shown in brackets. Of 583 patients, 52 died (40% male), giving an overall case fatality rate of 8.9%. Seven children were lost to follow-up. Forty seven deaths (90%) occurred within the first 24 hours after admission. Neurological sequelae were present in 27 (5 %) of the 531 survivors. The case fatality rate was significantly higher in females than males (10.9% vs.6.9%, p = 0.006). Children who died were older than those who survived (mean (SD) = 35.4 (41.9) vs. 25.0 (18.2) months, p = 0.0009). Of the 52 deaths in the course of the study, 32 (61.5%) presented with cerebral malaria, 32 (61.5%) presented with respiratory distress, and 20 (50% of the 40 with measurements) had hyperlactataemia. Thirty (60%) of the children who died presented with convulsions, and 13 (25%) with hypoglycaemia. Twenty four (46.2%) of those who died had severe anaemia. Mortalities in each sub-group demonstrated that hyperparasitaemia (1 death in 24, 4%) and severe anaemia (24 deaths in 395, i.e. 6%) had a better prognosis than cerebral malaria (32 deaths in 142, i.e. 22.5%) and hypoglycaemia (15 deaths in 60, i.e. 25%). Thirty-two (17.7%) of the 181 patients with RD died, as did 30 (11%) of the 264 patients with convulsions and 20 (27.4 %) of the 73 patients with hyperlactataemia. A multiple logistic regression model identified coma (OR = 3.6, 95% CI = 1.8–7.1, p < 0.001), hyperlactataemia (OR = 6.98, 95% CI = 3.5–13.8, p = 0.0001), respiratory distress (OR = 2.0, 95% CI = 1.0–3.9, p = 0.033) and hypoglycaemia (OR = 4.0, 95% CI = 1.7–9.4, p = 0.001) as independent predictors of a fatal outcome. Severe anaemia, hyperparasitaemia and thrombocytopaenia were not shown to be predictors of death (Figure  4 ). Figure 4 Prognostic indicators at the time of admission. OR – odds ratio, SD – standard deviation.
Case rate fatality
Figure  3  shows a Venn diagram summarizing clinical and laboratory features of severe malaria and case fatality rates.Of 583 patients, 52 died (40% male), giving an overall case fatality rate of 8.9%. Seven children were lost to follow-up. Forty seven deaths (90%) occurred within the first 24 hours after admission.Neurological sequelae were present in 27 (5 %) of the 531 survivors. The case fatality rate was significantly higher in females than males (10.9% vs.6.9%, p = 0.006). Children who died were older than those who survived (mean (SD) = 35.4 (41.9) vs. 25.0 (18.2) months, p = 0.0009).Of the 52 deaths in the course of the study, 32 (61.5%) presented with cerebral malaria, 32 (61.5%) presented with respiratory distress, and 20 (50% of the 40 with measurements) had hyperlactataemia.Thirty (60%) of the children who died presented with convulsions, and 13 (25%) with hypoglycaemia. Twenty four (46.2%) of those who died had severe anaemia. Mortalities in each sub-group demonstrated that hyperparasitaemia (1 death in 24, 4%) and severe anaemia (24 deaths in 395, i.e. 6%) had a better prognosis than cerebral malaria (32 deaths in 142, i.e. 22.5%) and hypoglycaemia (15 deaths in 60, i.e. 25%). Thirty-two (17.7%) of the 181 patients with RD died, as did 30 (11%) of the 264 patients with convulsions and 20 (27.4 %) of the 73 patients with hyperlactataemia.A multiple logistic regression model identified coma (OR = 3.6, 95% CI = 1.8–7.1, p < 0.001), hyperlactataemia (OR = 6.98, 95% CI = 3.5–13.8, p = 0.0001), respiratory distress (OR = 2.0, 95% CI = 1.0–3.9, p = 0.033) and hypoglycaemia (OR = 4.0, 95% CI = 1.7–9.4, p = 0.001) as independent predictors of a fatal outcome. Severe anaemia, hyperparasitaemia and thrombocytopaenia were not shown to be predictors of death (Figure  4 ).Discussion Malaria remains a serious health problem in sub-Sahara Africa. It was the most common reason for neurological emergencies during 2001 at the CHL [ 7 ]. This study was designed to describe the epidemiology, clinical and laboratory presentations of severe falciparum malaria in childhood presenting to CHL, in order to improve the diagnosis, classification and appropriate management of malaria. It is not possible to exclude absolutely all children with alternative diagnoses on clinical examination and simple investigation alone, which is a problem shared by all other similar studies on severe malaria [ 3 - 5 , 9 , 10 , 14 , 16 - 18 ]. The small numbers with alternative diagnoses should not affect the conclusions of this or other studies. Most cases (92%) of severe malaria were in children less than 5 years old. Similar observations have been made in another group of children hospitalized for malaria in Gabon [ 19 ]. Elsewhere, severe malaria tends to occur in older children [ 20 ]. Differences in the age of presentation of severe malaria may be the result of lower background immunity or other undefined variables [ 21 ]. The study confirms the stable and perennial transmission of malaria in Gabon, which contrasts with reports from other countries in West Africa where malaria transmission is predominantly at the end of the long rainy season [ 22 , 23 ]. Fever is a characteristic feature of  P. falciparum  infection, but a sizeable proportion of these children (25%) with severe malaria were afebrile on admission as observed elsewhere [ 23 ]. Self-medication with antipyretic or antimalarial agents was common (about 50% of the children) and may contribute to this finding. There are obvious implications for the diagnosis of malaria, which may be underestimated using clinical criteria alone. Severe anaemia was the most frequent feature of severity in this study, but was associated with decreased mortality. A similar observation in a recent Ghanaian study showed a better outcome in children with severe anaemia [ 17 ]. These findings confirm that severe malaria anaemia has a lower case fatality rate than other complications of severe malaria, consistent with several other studies where severe anaemia was not an independent predictor of in-hospital mortality [ 9 , 10 , 18 ]. The case fatality rate of severe anaemia without other markers of severe malaria is 1 to 2%, where blood transfusion is available [ 3 , 9 , 10 , 14 , 18 ] raising questions about the value of severe anaemia as a defining feature of the syndrome of severe malaria. Despite the increasing toll of HIV infection, and the continuing burden of diarrhoeal disease, malnutrition and respiratory tract infections, malaria remains a major cause of childhood death in endemic regions [ 1 ]. The overall case fatality rate of severe malaria in the study was 8.9% (52 deaths/583 cases), which is in keeping with studies from other geographic areas, where case fatality rates range between 8 and 40% [ 4 , 5 , 9 , 14 , 16 , 20 , 22 , 24 , 25 ]. Most of these deaths (90% in this study) occurred in the first twenty-four hours of hospital admission, a finding also in keeping with other studies [ 5 ]. The independent prognostic indicators in this study were cerebral malaria, respiratory distress, hypoglycaemia and hyperlactataemia. These observations are entirely consistent with a large number of studies where the independent predictors of a fatal outcome in malaria are impaired consciousness and metabolic dysfunction (as measured by hyperlactataemia, hypoglycaemia, acidosis or respiratory distress) [ 3 , 5 , 9 , 10 , 14 , 16 - 18 ]. The metabolic complications of malaria are complex and a number of interrelated measures have been used in different studies. Severe malaria is associated with a metabolic acidosis [ 16 ] and hyperlactataemia [ 5 ]. Respiratory distress has been associated with acidosis and hyperlactataemia in some studies [ 26 ]. These features of metabolic malaria probably all result from increased anaerobic metabolism due to tissue hypoxia [ 27 ]. Estimates of the prevalence of hypoglycaemia have been reported in Africa, ranging from 8% to 34% [ 28 , 29 ]. In severe childhood malaria hypoglycaemia results from impaired gluconeogenesis and increased tissue demand for glucose [ 27 , 28 ] and quinine induced hyperinsulinaemia. Blood glucose concentrations should be monitored in all children hospitalised for malaria especially those who receive quinine. The definition of hyperlactataemia used in this study was a blood lactate concentration higher than the conventional cut-off (≥ 5 mmol/L). This was necessary because of the limitations of the analyser used, but probably means that the frequency of true hyperlactataemia was underestimated. The Accusport™ analyser used has been shown to have poor agreement with "gold standard" machines [ 11 , 15 , 30 ]. Hyperlactataemia is a frequent and serious complication of severe malaria in childhood [ 5 , 9 , 10 ], which may be due microcirculatory sequestration of parasitized erythrocytes resulting in increased production of lactate by anaerobic glycolysis [ 31 ]. A recent study showed a correlation between hyperlactataemia and high plasma glutamine levels in severe malaria. This correlation may reflect impaired gluconeogenesis [ 31 ]. Lactate disposal is proportional to blood lactate concentration and can be increased by dichloroacetate [ 27 , 32 ]. Lactic acidosis, as confirmed in this study, is an established strong predictor of a fatal outcome in falciparum malaria in African children [ 5 , 9 , 10 ] and may prove a target for further interventional studies to improve survival. Respiratory distress was present in 31% of these children. This is higher than the frequency reported in other studies of severe malaria: 4.9% in Burkina [ 20 ], 6.4% in Togo [ 22 ] and 13.7% in Kenya [ 14 ]. These differences may partly be explained by low inter-observer agreement for this variable, geographical variations in disease pattern as well as differing definitions of severe malaria. Results from many studies consistently show that respiratory distress is a life-threatening syndrome in childhood malaria [ 14 , 33 ]. Respiratory distress was significantly associated with both hyperlactataemia and cerebral malaria in this study. The Blantyre coma score has long been established in children as a good indicator of cerebral dysfunction in malaria [ 3 ] and has enabled better standardization of studies on cerebral malaria in African children. The case-fatality rate associated with cerebral malaria (22%) is similar to that in Gambian children (27%) [ 4 ] but is higher than that observed for Kenyan (17%) [ 14 ] and Malawian children (15%) [ 3 ]. It has been postulated that with the higher the level of malaria transmission, immunity is acquired earlier, perhaps altering the presentation of severe malaria from predominantly a cerebral syndrome to that of severe anaemia [ 34 , 35 ]. The clinical and laboratory presentations of severe malaria are described in a hospitalized population of children in Gabon. The severe cases are likely to be only the "tip of the iceberg", many children living far from health care units may die whilst travelling to the nearest hospital. Most deaths from malaria occurred in the first 24 hours of admission, which highlights the need for early recognition of the most severely ill children. Early diagnosis and classification of severe malaria would allow appropriate management, including basic adjunctive therapy such as to prevent hypoglycaemia, and better use of scarce healthcare resources. Together these improvements could contribute to a reduction in the intractably high mortality due to the disease.
Discussion
Malaria remains a serious health problem in sub-Sahara Africa. It was the most common reason for neurological emergencies during 2001 at the CHL [ 7 ]. This study was designed to describe the epidemiology, clinical and laboratory presentations of severe falciparum malaria in childhood presenting to CHL, in order to improve the diagnosis, classification and appropriate management of malaria. It is not possible to exclude absolutely all children with alternative diagnoses on clinical examination and simple investigation alone, which is a problem shared by all other similar studies on severe malaria [ 3 - 5 , 9 , 10 , 14 , 16 - 18 ]. The small numbers with alternative diagnoses should not affect the conclusions of this or other studies. Most cases (92%) of severe malaria were in children less than 5 years old. Similar observations have been made in another group of children hospitalized for malaria in Gabon [ 19 ]. Elsewhere, severe malaria tends to occur in older children [ 20 ]. Differences in the age of presentation of severe malaria may be the result of lower background immunity or other undefined variables [ 21 ]. The study confirms the stable and perennial transmission of malaria in Gabon, which contrasts with reports from other countries in West Africa where malaria transmission is predominantly at the end of the long rainy season [ 22 , 23 ].Fever is a characteristic feature of  P. falciparum  infection, but a sizeable proportion of these children (25%) with severe malaria were afebrile on admission as observed elsewhere [ 23 ]. Self-medication with antipyretic or antimalarial agents was common (about 50% of the children) and may contribute to this finding. There are obvious implications for the diagnosis of malaria, which may be underestimated using clinical criteria alone.P. falciparumSevere anaemia was the most frequent feature of severity in this study, but was associated with decreased mortality. A similar observation in a recent Ghanaian study showed a better outcome in children with severe anaemia [ 17 ]. These findings confirm that severe malaria anaemia has a lower case fatality rate than other complications of severe malaria, consistent with several other studies where severe anaemia was not an independent predictor of in-hospital mortality [ 9 , 10 , 18 ]. The case fatality rate of severe anaemia without other markers of severe malaria is 1 to 2%, where blood transfusion is available [ 3 , 9 , 10 , 14 , 18 ] raising questions about the value of severe anaemia as a defining feature of the syndrome of severe malaria.Despite the increasing toll of HIV infection, and the continuing burden of diarrhoeal disease, malnutrition and respiratory tract infections, malaria remains a major cause of childhood death in endemic regions [ 1 ]. The overall case fatality rate of severe malaria in the study was 8.9% (52 deaths/583 cases), which is in keeping with studies from other geographic areas, where case fatality rates range between 8 and 40% [ 4 , 5 , 9 , 14 , 16 , 20 , 22 , 24 , 25 ]. Most of these deaths (90% in this study) occurred in the first twenty-four hours of hospital admission, a finding also in keeping with other studies [ 5 ].The independent prognostic indicators in this study were cerebral malaria, respiratory distress, hypoglycaemia and hyperlactataemia. These observations are entirely consistent with a large number of studies where the independent predictors of a fatal outcome in malaria are impaired consciousness and metabolic dysfunction (as measured by hyperlactataemia, hypoglycaemia, acidosis or respiratory distress) [ 3 , 5 , 9 , 10 , 14 , 16 - 18 ]. The metabolic complications of malaria are complex and a number of interrelated measures have been used in different studies. Severe malaria is associated with a metabolic acidosis [ 16 ] and hyperlactataemia [ 5 ]. Respiratory distress has been associated with acidosis and hyperlactataemia in some studies [ 26 ]. These features of metabolic malaria probably all result from increased anaerobic metabolism due to tissue hypoxia [ 27 ].Estimates of the prevalence of hypoglycaemia have been reported in Africa, ranging from 8% to 34% [ 28 , 29 ]. In severe childhood malaria hypoglycaemia results from impaired gluconeogenesis and increased tissue demand for glucose [ 27 , 28 ] and quinine induced hyperinsulinaemia. Blood glucose concentrations should be monitored in all children hospitalised for malaria especially those who receive quinine.The definition of hyperlactataemia used in this study was a blood lactate concentration higher than the conventional cut-off (≥ 5 mmol/L). This was necessary because of the limitations of the analyser used, but probably means that the frequency of true hyperlactataemia was underestimated. The Accusport™ analyser used has been shown to have poor agreement with "gold standard" machines [ 11 , 15 , 30 ].Hyperlactataemia is a frequent and serious complication of severe malaria in childhood [ 5 , 9 , 10 ], which may be due microcirculatory sequestration of parasitized erythrocytes resulting in increased production of lactate by anaerobic glycolysis [ 31 ]. A recent study showed a correlation between hyperlactataemia and high plasma glutamine levels in severe malaria. This correlation may reflect impaired gluconeogenesis [ 31 ]. Lactate disposal is proportional to blood lactate concentration and can be increased by dichloroacetate [ 27 , 32 ]. Lactic acidosis, as confirmed in this study, is an established strong predictor of a fatal outcome in falciparum malaria in African children [ 5 , 9 , 10 ] and may prove a target for further interventional studies to improve survival. Respiratory distress was present in 31% of these children. This is higher than the frequency reported in other studies of severe malaria: 4.9% in Burkina [ 20 ], 6.4% in Togo [ 22 ] and 13.7% in Kenya [ 14 ].These differences may partly be explained by low inter-observer agreement for this variable, geographical variations in disease pattern as well as differing definitions of severe malaria. Results from many studies consistently show that respiratory distress is a life-threatening syndrome in childhood malaria [ 14 , 33 ]. Respiratory distress was significantly associated with both hyperlactataemia and cerebral malaria in this study. The Blantyre coma score has long been established in children as a good indicator of cerebral dysfunction in malaria [ 3 ] and has enabled better standardization of studies on cerebral malaria in African children. The case-fatality rate associated with cerebral malaria (22%) is similar to that in Gambian children (27%) [ 4 ] but is higher than that observed for Kenyan (17%) [ 14 ] and Malawian children (15%) [ 3 ]. It has been postulated that with the higher the level of malaria transmission, immunity is acquired earlier, perhaps altering the presentation of severe malaria from predominantly a cerebral syndrome to that of severe anaemia [ 34 , 35 ].The clinical and laboratory presentations of severe malaria are described in a hospitalized population of children in Gabon. The severe cases are likely to be only the "tip of the iceberg", many children living far from health care units may die whilst travelling to the nearest hospital. Most deaths from malaria occurred in the first 24 hours of admission, which highlights the need for early recognition of the most severely ill children. Early diagnosis and classification of severe malaria would allow appropriate management, including basic adjunctive therapy such as to prevent hypoglycaemia, and better use of scarce healthcare resources. Together these improvements could contribute to a reduction in the intractably high mortality due to the disease.Authors' contributions AD is a MCRU clinician. He participated to the study and wrote the article. PN conducted the study for his MD thesis. RT and MB are paediatricians who participated in the study. TP is a UK collaborator who participated in the study and helped write the article. MM was involved with the intensive care of the children. UMR participated in the study as a MCRU clinician. JJ helped to write the article. EK did all the statistical analysis. ENM, PK, SK and MK coordinated the realization of the study and edited the final version approved by all authors.
Authors' contributions
AD is a MCRU clinician. He participated to the study and wrote the article. PN conducted the study for his MD thesis. RT and MB are paediatricians who participated in the study. TP is a UK collaborator who participated in the study and helped write the article. MM was involved with the intensive care of the children. UMR participated in the study as a MCRU clinician. JJ helped to write the article. EK did all the statistical analysis. ENM, PK, SK and MK coordinated the realization of the study and edited the final version approved by all authors.


Background Pneumonia remains the leading cause of death in young children. The poor specificity of chest radiographs (CXRs) to diagnose pneumococcal pneumonia may underestimate the efficacy of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia.
Background
Pneumonia remains the leading cause of death in young children. The poor specificity of chest radiographs (CXRs) to diagnose pneumococcal pneumonia may underestimate the efficacy of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia.Methods and Findings The efficacy of nine-valent pneumococcal conjugate vaccine among children not infected with HIV (21%; 95% confidence interval, 1%–37%) increased when CXR-confirmed pneumonia was associated with serum C-reactive protein of 120 mg/l (12mg/dl) or more and procalcitonin of 5.0 ng/ml or more (64%; 95% confidence interval, 23%–83%). Similar results were observed in children infected with HIV.
Methods and Findings
The efficacy of nine-valent pneumococcal conjugate vaccine among children not infected with HIV (21%; 95% confidence interval, 1%–37%) increased when CXR-confirmed pneumonia was associated with serum C-reactive protein of 120 mg/l (12mg/dl) or more and procalcitonin of 5.0 ng/ml or more (64%; 95% confidence interval, 23%–83%). Similar results were observed in children infected with HIV.Conclusion C-reactive protein and procalcitonin improve the specificity of CXR to diagnose pneumococcal pneumonia and may be useful for the future evaluation of the effectiveness of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia.
Conclusion
C-reactive protein and procalcitonin improve the specificity of CXR to diagnose pneumococcal pneumonia and may be useful for the future evaluation of the effectiveness of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia.

Introduction While pneumonia remains the leading cause of death in children, the absence of sensitive and specific tools to make an etiological diagnosis is a major limitation to our understanding of the efficacy of vaccines against pneumonia. Blood cultures lack sensitivity, and cultures from lung aspirates may be influenced by delayed presentation, antecedent antibiotic therapy, difficulties finding an accessible site to aspirate, lack of skills in performing the procedure, and the perception of both parents and clinicians that the procedure is too invasive [ 1 , 2 ]. Procalcitonin, at a low threshold (≥0.25 ng/ml), has been shown to be useful in directing the use of antibiotics in adults with pneumonia [ 3 ], and a recent meta-analysis concludes that procalcitonin may offer some advantages over C-reactive protein (CRP) for discriminating bacterial from nonbacterial infections [ 4 ]. Most studies support the observation that children with bacterial infections have higher levels of CRP and procalcitonin than those with viral infections [ 4 , 5 , 6 ]. Our recent observation that many children with viral-associated pneumonia have a bacterial super-infection [ 7 ], however, suggests that a high level of CRP and procalcitonin may be associated with unrecognised bacterial co-infection in a child with an established viral aetiology for pneumonia. This may also explain why some studies have found procalcitonin and CRP not to be useful in distinguishing between bacterial and “viral” pneumonia [ 4 , 8 ]. Based on the postulate that the most likely chest radiograph (CXR) manifestation of pneumococcal pneumonia is alveolar consolidation, we reported the efficacy of a nine-valent pneumococcal conjugate vaccine (PnCV) in reducing CXR-confirmed pneumonia based on definitions recommended by a World Health Organization working group [ 9 ]. The observed reduction in the incidence of CXR-confirmed pneumonia in the intent-to-treat analyses in PnCV recipients not infected with HIV (20%; 95% confidence interval [CI], 2% to 35%) and infected with HIV (13%; 95% CI, −7% to 29%) likely underestimated the reduction in the incidence of pneumococcal pneumonia [ 10 ]. The reason for this is that the outcome measure (i.e., CXR-confirmed pneumonia) is not highly specific for pneumococcal pneumonia. Consequently, it is likely that many of the CXR-confirmed pneumonia episodes were not pneumococcal in origin and therefore could not have been prevented by the vaccine under evaluation. We thus evaluated the usefulness of procalcitonin and CRP to improve the specificity of CXR-confirmed pneumonia as an endpoint in vaccine efficacy trials. This analysis was not a primary objective of the study, and is therefore an hypothesis-generating analysis, which should be tested as an a priori hypothesis in other study settings.
Introduction
While pneumonia remains the leading cause of death in children, the absence of sensitive and specific tools to make an etiological diagnosis is a major limitation to our understanding of the efficacy of vaccines against pneumonia. Blood cultures lack sensitivity, and cultures from lung aspirates may be influenced by delayed presentation, antecedent antibiotic therapy, difficulties finding an accessible site to aspirate, lack of skills in performing the procedure, and the perception of both parents and clinicians that the procedure is too invasive [ 1 , 2 ].Procalcitonin, at a low threshold (≥0.25 ng/ml), has been shown to be useful in directing the use of antibiotics in adults with pneumonia [ 3 ], and a recent meta-analysis concludes that procalcitonin may offer some advantages over C-reactive protein (CRP) for discriminating bacterial from nonbacterial infections [ 4 ]. Most studies support the observation that children with bacterial infections have higher levels of CRP and procalcitonin than those with viral infections [ 4 , 5 , 6 ]. Our recent observation that many children with viral-associated pneumonia have a bacterial super-infection [ 7 ], however, suggests that a high level of CRP and procalcitonin may be associated with unrecognised bacterial co-infection in a child with an established viral aetiology for pneumonia. This may also explain why some studies have found procalcitonin and CRP not to be useful in distinguishing between bacterial and “viral” pneumonia [ 4 , 8 ].Based on the postulate that the most likely chest radiograph (CXR) manifestation of pneumococcal pneumonia is alveolar consolidation, we reported the efficacy of a nine-valent pneumococcal conjugate vaccine (PnCV) in reducing CXR-confirmed pneumonia based on definitions recommended by a World Health Organization working group [ 9 ]. The observed reduction in the incidence of CXR-confirmed pneumonia in the intent-to-treat analyses in PnCV recipients not infected with HIV (20%; 95% confidence interval [CI], 2% to 35%) and infected with HIV (13%; 95% CI, −7% to 29%) likely underestimated the reduction in the incidence of pneumococcal pneumonia [ 10 ]. The reason for this is that the outcome measure (i.e., CXR-confirmed pneumonia) is not highly specific for pneumococcal pneumonia. Consequently, it is likely that many of the CXR-confirmed pneumonia episodes were not pneumococcal in origin and therefore could not have been prevented by the vaccine under evaluation.We thus evaluated the usefulness of procalcitonin and CRP to improve the specificity of CXR-confirmed pneumonia as an endpoint in vaccine efficacy trials. This analysis was not a primary objective of the study, and is therefore an hypothesis-generating analysis, which should be tested as an a priori hypothesis in other study settings.Methods The methods of the randomised, double-blind, placebo-controlled trial, the objectives of which were to measure PnCV efficacy against invasive pneumococcal disease and CXR-confirmed pneumonia, have been published [ 10 ] ( Protocol S1 ). Briefly stated, the study included 39,836 children, including an estimated 6.47% infected with HIV [ 7 ], and was performed in Soweto, South Africa. Children were randomised to receive either a nine-valent PnCV conjugated to CRM 197  (Wyeth-Lederle Vaccines and Pediatrics, Pearl River, New York, United States) or a placebo at 6, 10, and 14 wk of age. The nine-valent PnCV included serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F (i.e., vaccine serotypes) [ 10 ]. We now further measured procalcitonin and CRP levels in serum obtained within 12 h of hospitalisation among children with CXR-confirmed alveolar consolidation in the intent-to-treat analysis. CXRs were requested for any child hospitalised with a clinical diagnosis of lower respiratory tract infection according to the diagnosis of the attending study-physician. CRP values were determined at the time of admission by immunoturbidometry (717 Automated Analyzer, Boehringer Mannheim/Hitachi, Mannheim, Germany) and were reported in milligrams per liter with a lower threshold set at 3 mg/l or lower. In those children in whom CRP was not measured on admission to hospital, archived serum obtained within 12 h of admission and stored at −70 °C was used for analysis. The archived serum samples were also used for measuring quantitative procalcitonin levels using the LUMItest PCT assay (BRAHMS Diagnostica, Berlin, Germany). The CRP and procalcitonin tests were performed using commercially available assays according to the manufacturers' recommendations at the National Health and Laboratory Services, Johannesburg, South Africa. Bacterial Cultures Blood was cultured for bacterial growth on admission and processed using the BacT/Alert microbial detection system (Organon Teknika, Durham, North Carolina, United States). Isolates of  Streptococcus pneumoniae  were serotyped using the quellung method at the Respiratory and Meningeal Pathogens Research Unit and the results validated at the Statens Serum Institute in Copenhagen, Denmark [ 10 ]. Statistics Data were analysed using S
TATA version 8.0 (StataCorp, College Station, Texas, United States) and Epi Info version 6.04d (Centers for Disease Control and Prevention, Atlanta, Georgia, United States). Vaccine efficacy was calculated using Epi Info based on the formula: vaccine efficacy (percent) = [(ARU − ARV)/ARU] × 100, where ARU is attack rate in unvaccinated individuals and ARV is attack rate in vaccinated indivduals. The vaccine-attributable reduction in disease (VAR) was estimated by measuring the difference in incidence rate between vaccine and placebo recipients and expressed per 100,000 child years of observation. A  p- value of 0.05 or lower was considered significant.
 All analyses were performed on an intent-to-treat basis, which included the first event of CXR-confirmed pneumonia in any child who had received at least a single dose of study vaccine. Vaccine efficacy calculations were also performed for bacteremic pneumococcal pneumonia that included all first episodes of pneumonia associated with growth of  S. pneumoniae  of any serotype from blood. Ethical Considerations The efficacy study and subsequent study evaluating the role of procalcitonin and CRP in children with CXR-confirmed pneumonia were approved by the Ethics Committee for Research on Human Subjects, University of the Witwatersrand, South Africa ( Protocol S2 ). Guardians gave informed consent for the collection of serum and its use for diagnostic assays that may improve the diagnosis of pneumonia ( Protocol S3 ).
Methods
The methods of the randomised, double-blind, placebo-controlled trial, the objectives of which were to measure PnCV efficacy against invasive pneumococcal disease and CXR-confirmed pneumonia, have been published [ 10 ] ( Protocol S1 ). Briefly stated, the study included 39,836 children, including an estimated 6.47% infected with HIV [ 7 ], and was performed in Soweto, South Africa. Children were randomised to receive either a nine-valent PnCV conjugated to CRM 197  (Wyeth-Lederle Vaccines and Pediatrics, Pearl River, New York, United States) or a placebo at 6, 10, and 14 wk of age. The nine-valent PnCV included serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F (i.e., vaccine serotypes) [ 10 ]. We now further measured procalcitonin and CRP levels in serum obtained within 12 h of hospitalisation among children with CXR-confirmed alveolar consolidation in the intent-to-treat analysis. CXRs were requested for any child hospitalised with a clinical diagnosis of lower respiratory tract infection according to the diagnosis of the attending study-physician.CRP values were determined at the time of admission by immunoturbidometry (717 Automated Analyzer, Boehringer Mannheim/Hitachi, Mannheim, Germany) and were reported in milligrams per liter with a lower threshold set at 3 mg/l or lower. In those children in whom CRP was not measured on admission to hospital, archived serum obtained within 12 h of admission and stored at −70 °C was used for analysis. The archived serum samples were also used for measuring quantitative procalcitonin levels using the LUMItest PCT assay (BRAHMS Diagnostica, Berlin, Germany). The CRP and procalcitonin tests were performed using commercially available assays according to the manufacturers' recommendations at the National Health and Laboratory Services, Johannesburg, South Africa.Bacterial Cultures Blood was cultured for bacterial growth on admission and processed using the BacT/Alert microbial detection system (Organon Teknika, Durham, North Carolina, United States). Isolates of  Streptococcus pneumoniae  were serotyped using the quellung method at the Respiratory and Meningeal Pathogens Research Unit and the results validated at the Statens Serum Institute in Copenhagen, Denmark [ 10 ].
Bacterial Cultures
Blood was cultured for bacterial growth on admission and processed using the BacT/Alert microbial detection system (Organon Teknika, Durham, North Carolina, United States). Isolates of  Streptococcus pneumoniae  were serotyped using the quellung method at the Respiratory and Meningeal Pathogens Research Unit and the results validated at the Statens Serum Institute in Copenhagen, Denmark [ 10 ].Statistics Data were analysed using S
TATA version 8.0 (StataCorp, College Station, Texas, United States) and Epi Info version 6.04d (Centers for Disease Control and Prevention, Atlanta, Georgia, United States). Vaccine efficacy was calculated using Epi Info based on the formula: vaccine efficacy (percent) = [(ARU − ARV)/ARU] × 100, where ARU is attack rate in unvaccinated individuals and ARV is attack rate in vaccinated indivduals. The vaccine-attributable reduction in disease (VAR) was estimated by measuring the difference in incidence rate between vaccine and placebo recipients and expressed per 100,000 child years of observation. A  p- value of 0.05 or lower was considered significant.
 All analyses were performed on an intent-to-treat basis, which included the first event of CXR-confirmed pneumonia in any child who had received at least a single dose of study vaccine. Vaccine efficacy calculations were also performed for bacteremic pneumococcal pneumonia that included all first episodes of pneumonia associated with growth of  S. pneumoniae  of any serotype from blood.
Statistics
Data were analysed using S
TATA version 8.0 (StataCorp, College Station, Texas, United States) and Epi Info version 6.04d (Centers for Disease Control and Prevention, Atlanta, Georgia, United States). Vaccine efficacy was calculated using Epi Info based on the formula: vaccine efficacy (percent) = [(ARU − ARV)/ARU] × 100, where ARU is attack rate in unvaccinated individuals and ARV is attack rate in vaccinated indivduals. The vaccine-attributable reduction in disease (VAR) was estimated by measuring the difference in incidence rate between vaccine and placebo recipients and expressed per 100,000 child years of observation. A  p- value of 0.05 or lower was considered significant.p-All analyses were performed on an intent-to-treat basis, which included the first event of CXR-confirmed pneumonia in any child who had received at least a single dose of study vaccine. Vaccine efficacy calculations were also performed for bacteremic pneumococcal pneumonia that included all first episodes of pneumonia associated with growth of  S. pneumoniae  of any serotype from blood.Ethical Considerations The efficacy study and subsequent study evaluating the role of procalcitonin and CRP in children with CXR-confirmed pneumonia were approved by the Ethics Committee for Research on Human Subjects, University of the Witwatersrand, South Africa ( Protocol S2 ). Guardians gave informed consent for the collection of serum and its use for diagnostic assays that may improve the diagnosis of pneumonia ( Protocol S3 ).
Ethical Considerations
The efficacy study and subsequent study evaluating the role of procalcitonin and CRP in children with CXR-confirmed pneumonia were approved by the Ethics Committee for Research on Human Subjects, University of the Witwatersrand, South Africa ( Protocol S2 ). Guardians gave informed consent for the collection of serum and its use for diagnostic assays that may improve the diagnosis of pneumonia ( Protocol S3 ).Results In children not infected with HIV with CXR-confirmed pneumonia, procalcitonin and CRP values were available for 132 (78.1%) of 169 vaccine recipients and 167 (78.8%) of 212 placebo recipients ( p =  0.88). The point estimate of vaccine efficacy in this cohort of children for whom serum was available was 21% ( p =  0.04; 95% CI 1%–37%), not significantly different from that determined in the original study (20%,  p =  0.03; 95% CI 3%–35%). At CRP levels of 120 mg/l or more in conjunction with the primary CXR-confirmed outcome measure, the estimate of vaccine efficacy increased to 38% ( p =  0.05); at procalcitonin levels of 5 ng/ml or more, the estimated efficacy increased to 46% ( p =  0.04); and if both conditions were met, the estimated efficacy increased to 64% ( p  = 0.006) ( Table 1 ). Table 1 CRPand Procalcitonin Improve the Specificity of CXR to Measure the Efficacy of PnCV in the Prevention of Pneumococcal Pneumonia CXR-confirmed alveolar consolidation based on World Health Organization guidelines for interpretation. Point estimates differ from those previously published because only children with CXR-confirmed pneumonia for whom serum for CRP and procalcitonin measurements were available are included
 a  CXR-AC, CXR-confirmed alveolar consolidation; PCT, procalcitonin
 b  Pneumonia associated with growth from blood of  S. pneumoniae,  of any serotype
 c  Pneumonia associated with growth from blood of  S. pneumoniae  of vaccine-serotype-specific bacteremic pneumonia Among children infected with HIV with CXR-confirmed pneumonia, procalcitonin and CRP values were available for 139 (76.4%) of 182 vaccine recipients and 153 (73.2%) of 209 placebo recipients ( p =  0.47). Whereas vaccine efficacy was not significant for CXR-confirmed pneumonia (9%;  p =  0.38), vaccine efficacy was significant when measured against CXR-confirmed pneumonia in conjunction with a CRP level of 120 mg/l or more (35%;  p =  0.03) or a procalcitonin level of 5 ng/ml or more (33%;  p =  0.04). As observed in children not infected with HIV, the point efficacy estimate in children infected with HIV with CXR-confirmed pneumonia was greatest if both CRP and procalcitonin were at or above 120 mg/l and 5 ng/ml, respectively (52%;  p =  0.004;  Table 1 ). The sensitivity of CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml in detecting vaccine efficacy against pneumococcal pneumonia was analysed by comparing the VAR per 100,000 child years to that observed using an outcome of bacteremic pneumococcal pneumonia. In children not infected with HIV, the combined outcome of CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml (VAR = 37) identified 5.3-fold more cases of pneumonia that were prevented by vaccination than was identified by bacteremic pneumococcal pneumonia (VAR = 7) and 4.1-fold more cases than was identified by vaccine-serotype-specific bacteremic pneumococcal pneumonia (VAR = 9). Similarly, CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml in children infected with HIV (VAR = 772) identified 1.6-fold more cases of pneumonia that were prevented by vaccination than was identified by pneumococcal bacteremic pneumonia (VAR = 483) and 2.2-fold more cases than was identified by vaccine-serotype-specific bacteremic pneumococcal pneumonia (VAR = 344). Among the children not infected with HIV with CXR-confirmed pneumonia, the likelihood of a difference between placebo and vaccine recipients was significantly increased by the presence of a CRP level of 120 mg/l or more and a procalcitonin level of 5 ng/ml or more (odds ratio = 1.37; 95% CI, 1.09–1.73;  p =  0.027). This was true also among children infected with HIV (odds ratio = 1.41; 95% CI, 1.14–1.75;  p =  0.005).
Results
In children not infected with HIV with CXR-confirmed pneumonia, procalcitonin and CRP values were available for 132 (78.1%) of 169 vaccine recipients and 167 (78.8%) of 212 placebo recipients ( p =  0.88). The point estimate of vaccine efficacy in this cohort of children for whom serum was available was 21% ( p =  0.04; 95% CI 1%–37%), not significantly different from that determined in the original study (20%,  p =  0.03; 95% CI 3%–35%). At CRP levels of 120 mg/l or more in conjunction with the primary CXR-confirmed outcome measure, the estimate of vaccine efficacy increased to 38% ( p =  0.05); at procalcitonin levels of 5 ng/ml or more, the estimated efficacy increased to 46% ( p =  0.04); and if both conditions were met, the estimated efficacy increased to 64% ( p  = 0.006) ( Table 1 ).p =p =p =p =p =pCRPand Procalcitonin Improve the Specificity of CXR to Measure the Efficacy of PnCV in the Prevention of Pneumococcal Pneumonia
CXR-confirmed alveolar consolidation based on World Health Organization guidelines for interpretation. Point estimates differ from those previously published because only children with CXR-confirmed pneumonia for whom serum for CRP and procalcitonin measurements were available are includeda  CXR-AC, CXR-confirmed alveolar consolidation; PCT, procalcitoninb  Pneumonia associated with growth from blood of  S. pneumoniae,  of any serotypeS. pneumoniae,c  Pneumonia associated with growth from blood of  S. pneumoniae  of vaccine-serotype-specific bacteremic pneumoniaAmong children infected with HIV with CXR-confirmed pneumonia, procalcitonin and CRP values were available for 139 (76.4%) of 182 vaccine recipients and 153 (73.2%) of 209 placebo recipients ( p =  0.47). Whereas vaccine efficacy was not significant for CXR-confirmed pneumonia (9%;  p =  0.38), vaccine efficacy was significant when measured against CXR-confirmed pneumonia in conjunction with a CRP level of 120 mg/l or more (35%;  p =  0.03) or a procalcitonin level of 5 ng/ml or more (33%;  p =  0.04). As observed in children not infected with HIV, the point efficacy estimate in children infected with HIV with CXR-confirmed pneumonia was greatest if both CRP and procalcitonin were at or above 120 mg/l and 5 ng/ml, respectively (52%;  p =  0.004;  Table 1 ).p =p =p =p =p =The sensitivity of CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml in detecting vaccine efficacy against pneumococcal pneumonia was analysed by comparing the VAR per 100,000 child years to that observed using an outcome of bacteremic pneumococcal pneumonia. In children not infected with HIV, the combined outcome of CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml (VAR = 37) identified 5.3-fold more cases of pneumonia that were prevented by vaccination than was identified by bacteremic pneumococcal pneumonia (VAR = 7) and 4.1-fold more cases than was identified by vaccine-serotype-specific bacteremic pneumococcal pneumonia (VAR = 9). Similarly, CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml in children infected with HIV (VAR = 772) identified 1.6-fold more cases of pneumonia that were prevented by vaccination than was identified by pneumococcal bacteremic pneumonia (VAR = 483) and 2.2-fold more cases than was identified by vaccine-serotype-specific bacteremic pneumococcal pneumonia (VAR = 344).Among the children not infected with HIV with CXR-confirmed pneumonia, the likelihood of a difference between placebo and vaccine recipients was significantly increased by the presence of a CRP level of 120 mg/l or more and a procalcitonin level of 5 ng/ml or more (odds ratio = 1.37; 95% CI, 1.09–1.73;  p =  0.027). This was true also among children infected with HIV (odds ratio = 1.41; 95% CI, 1.14–1.75;  p =  0.005).p =p =Discussion Our results show that the true efficacy of the PnCV against pneumococcal pneumonia may be underestimated if one relies solely on a radiological diagnosis of pneumococcal pneumonia. The lack of specificity of CXRs for inferring bacterial versus non-bacterial etiology of pneumonia has been previously described [ 11 , 12 ]. The specificity of CXR-confirmed pneumonia as an outcome measure of efficacy of PnCV to prevent pneumococcal pneumonia was improved when it was analysed together with indirect evidence suggestive of bacterial infection, namely, elevated CRP and procalcitonin levels. This outcome measure of CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml, while more specific than CXR-confirmed pneumonia alone, will not be 100% accurate because pneumonia associated with other bacteria, including that due to pneumococcal serotypes against which the PnCV has no effect, may well present in a similar manner. An alternate explanation for our results, rather than that of improved specificity in diagnosing pneumococcal pneumonia, may be that the criteria of CXR-confirmed pneumonia coupled with a CRP level of 120 mg/l or more and a procalcitonin level of 5 ng/ml or more detected more severe disease against which the vaccine may have been more efficacious. Although we are unaware of data to support that CRP and procalcitonin are elevated in those with severe pneumonia, the absence of such data supporting this interpretation does not rule out this possibility. Importantly, the levels of CRP and procalcitonin used in this report are designed for specificity rather than sensitivity; it would be inappropriate to use the same threshold values of CRP or procalcitonin during the course of routine clinical practice when managing individuals suspected of having bacterial pneumonia. In that instance, it would be more appropriate to use thresholds that have a high sensitivity, to ensure that all children with possible bacterial infection are adequately treated [ 3 , 4 , 8 ]. Procalcitonin and CRP were equally useful in children infected and not infected with HIV to improve the specificity of the pneumococcal pneumonia efficacy endpoint in the vaccine trial among children with CXR-confirmed pneumonia. This is particularly important, as our initial observation of a non-significant reduction in CXR-confirmed pneumonia was attributed to the complexity of etiology of pneumonia, and thus the even poorer specificity of CXRs for identifying pneumococcal pneumonia in children infected with HIV compared to children not infected with HIV [ 13 , 14 ]. The current study thus indicates that the specificity of CXR-confirmed pneumonia for diagnosing pneumococcal pneumonia may also be improved in children with HIV through the concurrent use of procalcitonin and CRP. A distinction must be made between measures of vaccine efficacy for which the increased specificity of CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml over CXR-confirmed pneumonia alone is apparent, and studies of the burden of disease prevented (VAR), which are optimised when vaccine sensitivity is maximised. Our data show that the VAR is greater for CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml than that calculated using the very highly specific endpoints of serotype-specific or all-serotype pneumococcal bacteremic pneumonia. The best estimates of VAR will, however, be provided by endpoints that sacrifice some specificity for maximal sensitivity. Such studies include less specific clinical endpoints and are outside of the scope of the current analysis [ 7 ]. Our findings have implications for the planning of future studies aimed at evaluating the effectiveness of pneumococcal vaccines against bacterial pneumonia. Our data suggest that the outcome measure of CXR-confirmed pneumonia together with elevated CRP and procalcitonin levels may be more accurate as a surrogate of pneumococcal pneumonia than CXRs on their own. The increased specificity of this endpoint may allow smaller sample sizes for future vaccine efficacy/effectiveness studies. Based on the incidence of CXR-confirmed pneumonia in this report (167 [0.9%] of 18,626), a sample size of 80,058 children were required to detect the 20% reduction in CXR-confirmed pneumonia with 80% power and an alpha of 0.05. The actual power for this outcome in this study was thus only 46.8%. Using the more specific outcome measure of CXR-confirmed pneumonia with a CRP level of 120 mg/l or more and a procalcitonin level of 5 ng/ml or more, the sample size required to detect the observed 64% reduction in outcome was 44,734, i.e., 56% of the sample size required when measuring vaccine efficacy against CXR-confirmed pneumonia alone, and the power of the current study would have been increased from 46.8% to 71.5%. Our findings therefore suggest that CXR-confirmed pneumonia coupled with serological markers of CRP and procalcitonin is a more specific marker of pneumococcal pneumonia and may therefore provide a closer estimate of the efficacy of the PnCV against pneumococcal pneumonia.
Discussion
Our results show that the true efficacy of the PnCV against pneumococcal pneumonia may be underestimated if one relies solely on a radiological diagnosis of pneumococcal pneumonia. The lack of specificity of CXRs for inferring bacterial versus non-bacterial etiology of pneumonia has been previously described [ 11 , 12 ]. The specificity of CXR-confirmed pneumonia as an outcome measure of efficacy of PnCV to prevent pneumococcal pneumonia was improved when it was analysed together with indirect evidence suggestive of bacterial infection, namely, elevated CRP and procalcitonin levels. This outcome measure of CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml, while more specific than CXR-confirmed pneumonia alone, will not be 100% accurate because pneumonia associated with other bacteria, including that due to pneumococcal serotypes against which the PnCV has no effect, may well present in a similar manner.An alternate explanation for our results, rather than that of improved specificity in diagnosing pneumococcal pneumonia, may be that the criteria of CXR-confirmed pneumonia coupled with a CRP level of 120 mg/l or more and a procalcitonin level of 5 ng/ml or more detected more severe disease against which the vaccine may have been more efficacious. Although we are unaware of data to support that CRP and procalcitonin are elevated in those with severe pneumonia, the absence of such data supporting this interpretation does not rule out this possibility.Importantly, the levels of CRP and procalcitonin used in this report are designed for specificity rather than sensitivity; it would be inappropriate to use the same threshold values of CRP or procalcitonin during the course of routine clinical practice when managing individuals suspected of having bacterial pneumonia. In that instance, it would be more appropriate to use thresholds that have a high sensitivity, to ensure that all children with possible bacterial infection are adequately treated [ 3 , 4 , 8 ].Procalcitonin and CRP were equally useful in children infected and not infected with HIV to improve the specificity of the pneumococcal pneumonia efficacy endpoint in the vaccine trial among children with CXR-confirmed pneumonia. This is particularly important, as our initial observation of a non-significant reduction in CXR-confirmed pneumonia was attributed to the complexity of etiology of pneumonia, and thus the even poorer specificity of CXRs for identifying pneumococcal pneumonia in children infected with HIV compared to children not infected with HIV [ 13 , 14 ]. The current study thus indicates that the specificity of CXR-confirmed pneumonia for diagnosing pneumococcal pneumonia may also be improved in children with HIV through the concurrent use of procalcitonin and CRP.A distinction must be made between measures of vaccine efficacy for which the increased specificity of CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml over CXR-confirmed pneumonia alone is apparent, and studies of the burden of disease prevented (VAR), which are optimised when vaccine sensitivity is maximised. Our data show that the VAR is greater for CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml than that calculated using the very highly specific endpoints of serotype-specific or all-serotype pneumococcal bacteremic pneumonia. The best estimates of VAR will, however, be provided by endpoints that sacrifice some specificity for maximal sensitivity. Such studies include less specific clinical endpoints and are outside of the scope of the current analysis [ 7 ].Our findings have implications for the planning of future studies aimed at evaluating the effectiveness of pneumococcal vaccines against bacterial pneumonia. Our data suggest that the outcome measure of CXR-confirmed pneumonia together with elevated CRP and procalcitonin levels may be more accurate as a surrogate of pneumococcal pneumonia than CXRs on their own. The increased specificity of this endpoint may allow smaller sample sizes for future vaccine efficacy/effectiveness studies. Based on the incidence of CXR-confirmed pneumonia in this report (167 [0.9%] of 18,626), a sample size of 80,058 children were required to detect the 20% reduction in CXR-confirmed pneumonia with 80% power and an alpha of 0.05. The actual power for this outcome in this study was thus only 46.8%. Using the more specific outcome measure of CXR-confirmed pneumonia with a CRP level of 120 mg/l or more and a procalcitonin level of 5 ng/ml or more, the sample size required to detect the observed 64% reduction in outcome was 44,734, i.e., 56% of the sample size required when measuring vaccine efficacy against CXR-confirmed pneumonia alone, and the power of the current study would have been increased from 46.8% to 71.5%.Our findings therefore suggest that CXR-confirmed pneumonia coupled with serological markers of CRP and procalcitonin is a more specific marker of pneumococcal pneumonia and may therefore provide a closer estimate of the efficacy of the PnCV against pneumococcal pneumonia.Supporting Information Protocol S1 Protocol of the Original Phase 3 Study (250 KB DOC). Click here for additional data file. Protocol S2 Ethics Committee Letter of Approval (378 KB DOC). Click here for additional data file. Protocol S3 Informed Consent Form for Original Phase 3 Study (24 KB DOC). Click here for additional data file. Patient Summary Background Pneumonia is the leading cause of death in children worldwide. Pneumonia can be caused by different bacteria and viruses, and there are no easy diagnostic tests to find out which bacterium or virus has caused the disease in a particular patient. This not only causes problems with prescribing the best treatment, but also makes it hard to evaluate vaccines that might protect against some causes of the disease but not others. Why Was This Study Done? The researchers involved in this study have evaluated vaccines against a particular bacterium (called  Pneumococcus ) that is the leading cause of pneumonia in children. They have begun to test these vaccines in children, but were looking for more specific ways to distinguish cases of pneumonia caused by this particular bacterium from those caused by other bacteria or viruses. What Did the Researchers Do? They had previously done a trial for a vaccine that relied on chest X-rays to diagnose pneumonia. They had also collected blood from children who had participated in the trial and had become sick with pneumonia. They now checked those blood samples for two markers that indicate a bacterial infection and re-analyzed the study. What Did They Find? The vaccine was able to protect children—to some extent—against pneumonia. The vaccine appeared to offer greater protection against pneumonia when the pneumonia was diagnosed by a combination of X-rays and high levels of the two blood markers than when the illness was diagnosed just with a chest X-ray. What Does This Mean? These results raise the possibility that a combined test (chest X-ray plus two blood markers) is better at assessing whether the pneumonia vaccine works than just a chest X-ray alone. What Next? Because of the way this study was done—adding a specific analysis to a clinical trial after it was completed, rather than planning to test a hypothesis from the outset—it cannot be considered as proof of the idea tested. The results suggest that it is worth testing whether the combined diagnosis is more specific for pneumococcal pneumonia than chest X-rays alone, but new studies are needed to resolve the issue. More Information Online The Global Alliance for Vaccines and Immunization (GAVI):  http://www.vaccinealliance.org/
 GAVI Web page “Call for Intensified Research after Pneumococcus Trial Surprises”:  http://www.vaccinealliance.org/Resources_Documents/Immunization_Focus/Download/update.php
 World Health Organization (WHO) Web site on vaccines:  http://www.who.int/vaccines/
 WHO Web page “Pneumococcal Vaccines”:  http://www.who.int/vaccines/en/olddocs/pneumococcus.shtml )
Supporting Information
Protocol of the Original Phase 3 Study
(250 KB DOC).Click here for additional data file.Ethics Committee Letter of Approval
(378 KB DOC).Click here for additional data file.Informed Consent Form for Original Phase 3 Study
(24 KB DOC).Click here for additional data file.Patient Summary Background Pneumonia is the leading cause of death in children worldwide. Pneumonia can be caused by different bacteria and viruses, and there are no easy diagnostic tests to find out which bacterium or virus has caused the disease in a particular patient. This not only causes problems with prescribing the best treatment, but also makes it hard to evaluate vaccines that might protect against some causes of the disease but not others. Why Was This Study Done? The researchers involved in this study have evaluated vaccines against a particular bacterium (called  Pneumococcus ) that is the leading cause of pneumonia in children. They have begun to test these vaccines in children, but were looking for more specific ways to distinguish cases of pneumonia caused by this particular bacterium from those caused by other bacteria or viruses. What Did the Researchers Do? They had previously done a trial for a vaccine that relied on chest X-rays to diagnose pneumonia. They had also collected blood from children who had participated in the trial and had become sick with pneumonia. They now checked those blood samples for two markers that indicate a bacterial infection and re-analyzed the study. What Did They Find? The vaccine was able to protect children—to some extent—against pneumonia. The vaccine appeared to offer greater protection against pneumonia when the pneumonia was diagnosed by a combination of X-rays and high levels of the two blood markers than when the illness was diagnosed just with a chest X-ray. What Does This Mean? These results raise the possibility that a combined test (chest X-ray plus two blood markers) is better at assessing whether the pneumonia vaccine works than just a chest X-ray alone. What Next? Because of the way this study was done—adding a specific analysis to a clinical trial after it was completed, rather than planning to test a hypothesis from the outset—it cannot be considered as proof of the idea tested. The results suggest that it is worth testing whether the combined diagnosis is more specific for pneumococcal pneumonia than chest X-rays alone, but new studies are needed to resolve the issue. More Information Online The Global Alliance for Vaccines and Immunization (GAVI):  http://www.vaccinealliance.org/
 GAVI Web page “Call for Intensified Research after Pneumococcus Trial Surprises”:  http://www.vaccinealliance.org/Resources_Documents/Immunization_Focus/Download/update.php
 World Health Organization (WHO) Web site on vaccines:  http://www.who.int/vaccines/
 WHO Web page “Pneumococcal Vaccines”:  http://www.who.int/vaccines/en/olddocs/pneumococcus.shtml )
Patient Summary
Background Pneumonia is the leading cause of death in children worldwide. Pneumonia can be caused by different bacteria and viruses, and there are no easy diagnostic tests to find out which bacterium or virus has caused the disease in a particular patient. This not only causes problems with prescribing the best treatment, but also makes it hard to evaluate vaccines that might protect against some causes of the disease but not others. Why Was This Study Done? The researchers involved in this study have evaluated vaccines against a particular bacterium (called  Pneumococcus ) that is the leading cause of pneumonia in children. They have begun to test these vaccines in children, but were looking for more specific ways to distinguish cases of pneumonia caused by this particular bacterium from those caused by other bacteria or viruses. What Did the Researchers Do? They had previously done a trial for a vaccine that relied on chest X-rays to diagnose pneumonia. They had also collected blood from children who had participated in the trial and had become sick with pneumonia. They now checked those blood samples for two markers that indicate a bacterial infection and re-analyzed the study. What Did They Find? The vaccine was able to protect children—to some extent—against pneumonia. The vaccine appeared to offer greater protection against pneumonia when the pneumonia was diagnosed by a combination of X-rays and high levels of the two blood markers than when the illness was diagnosed just with a chest X-ray. What Does This Mean? These results raise the possibility that a combined test (chest X-ray plus two blood markers) is better at assessing whether the pneumonia vaccine works than just a chest X-ray alone. What Next? Because of the way this study was done—adding a specific analysis to a clinical trial after it was completed, rather than planning to test a hypothesis from the outset—it cannot be considered as proof of the idea tested. The results suggest that it is worth testing whether the combined diagnosis is more specific for pneumococcal pneumonia than chest X-rays alone, but new studies are needed to resolve the issue. More Information Online The Global Alliance for Vaccines and Immunization (GAVI):  http://www.vaccinealliance.org/
 GAVI Web page “Call for Intensified Research after Pneumococcus Trial Surprises”:  http://www.vaccinealliance.org/Resources_Documents/Immunization_Focus/Download/update.php
 World Health Organization (WHO) Web site on vaccines:  http://www.who.int/vaccines/
 WHO Web page “Pneumococcal Vaccines”:  http://www.who.int/vaccines/en/olddocs/pneumococcus.shtml )

Background Pneumonia is the leading cause of death in children worldwide. Pneumonia can be caused by different bacteria and viruses, and there are no easy diagnostic tests to find out which bacterium or virus has caused the disease in a particular patient. This not only causes problems with prescribing the best treatment, but also makes it hard to evaluate vaccines that might protect against some causes of the disease but not others.
Background
Pneumonia is the leading cause of death in children worldwide. Pneumonia can be caused by different bacteria and viruses, and there are no easy diagnostic tests to find out which bacterium or virus has caused the disease in a particular patient. This not only causes problems with prescribing the best treatment, but also makes it hard to evaluate vaccines that might protect against some causes of the disease but not others.Why Was This Study Done? The researchers involved in this study have evaluated vaccines against a particular bacterium (called  Pneumococcus ) that is the leading cause of pneumonia in children. They have begun to test these vaccines in children, but were looking for more specific ways to distinguish cases of pneumonia caused by this particular bacterium from those caused by other bacteria or viruses.
Why Was This Study Done?
The researchers involved in this study have evaluated vaccines against a particular bacterium (called  Pneumococcus ) that is the leading cause of pneumonia in children. They have begun to test these vaccines in children, but were looking for more specific ways to distinguish cases of pneumonia caused by this particular bacterium from those caused by other bacteria or viruses.PneumococcusWhat Did the Researchers Do? They had previously done a trial for a vaccine that relied on chest X-rays to diagnose pneumonia. They had also collected blood from children who had participated in the trial and had become sick with pneumonia. They now checked those blood samples for two markers that indicate a bacterial infection and re-analyzed the study.
What Did the Researchers Do?
They had previously done a trial for a vaccine that relied on chest X-rays to diagnose pneumonia. They had also collected blood from children who had participated in the trial and had become sick with pneumonia. They now checked those blood samples for two markers that indicate a bacterial infection and re-analyzed the study.What Did They Find? The vaccine was able to protect children—to some extent—against pneumonia. The vaccine appeared to offer greater protection against pneumonia when the pneumonia was diagnosed by a combination of X-rays and high levels of the two blood markers than when the illness was diagnosed just with a chest X-ray.
What Did They Find?
The vaccine was able to protect children—to some extent—against pneumonia. The vaccine appeared to offer greater protection against pneumonia when the pneumonia was diagnosed by a combination of X-rays and high levels of the two blood markers than when the illness was diagnosed just with a chest X-ray.What Does This Mean? These results raise the possibility that a combined test (chest X-ray plus two blood markers) is better at assessing whether the pneumonia vaccine works than just a chest X-ray alone.
What Does This Mean?
These results raise the possibility that a combined test (chest X-ray plus two blood markers) is better at assessing whether the pneumonia vaccine works than just a chest X-ray alone.What Next? Because of the way this study was done—adding a specific analysis to a clinical trial after it was completed, rather than planning to test a hypothesis from the outset—it cannot be considered as proof of the idea tested. The results suggest that it is worth testing whether the combined diagnosis is more specific for pneumococcal pneumonia than chest X-rays alone, but new studies are needed to resolve the issue.
What Next?
Because of the way this study was done—adding a specific analysis to a clinical trial after it was completed, rather than planning to test a hypothesis from the outset—it cannot be considered as proof of the idea tested. The results suggest that it is worth testing whether the combined diagnosis is more specific for pneumococcal pneumonia than chest X-rays alone, but new studies are needed to resolve the issue.More Information Online The Global Alliance for Vaccines and Immunization (GAVI):  http://www.vaccinealliance.org/
 GAVI Web page “Call for Intensified Research after Pneumococcus Trial Surprises”:  http://www.vaccinealliance.org/Resources_Documents/Immunization_Focus/Download/update.php
 World Health Organization (WHO) Web site on vaccines:  http://www.who.int/vaccines/
 WHO Web page “Pneumococcal Vaccines”:  http://www.who.int/vaccines/en/olddocs/pneumococcus.shtml )
More Information Online
The Global Alliance for Vaccines and Immunization (GAVI):  http://www.vaccinealliance.org/GAVI Web page “Call for Intensified Research after Pneumococcus Trial Surprises”:  http://www.vaccinealliance.org/Resources_Documents/Immunization_Focus/Download/update.phpWorld Health Organization (WHO) Web site on vaccines:  http://www.who.int/vaccines/WHO Web page “Pneumococcal Vaccines”:  http://www.who.int/vaccines/en/olddocs/pneumococcus.shtml )