Feasibility of flow cytometry for measurements of Plasmodium falciparum parasite burden in studies in areas of malaria endemicity by use of bidimensional assessment of YOYO-1 and autofluorescence

The detection and quantification of Plasmodium falciparum in studies of malaria endemicity primarily relies upon microscopy. High-throughput quantitative methods with less subjectivity and greater reliability are needed for investigational studies. The staining of parasitized erythrocytes with YOYO-1 for flow cytometry bears great potential as a tool for assessing malaria parasite burden. Capillary blood was collected from children presenting to the pediatric ward of the Manhiça District Hospital in Mozambique for parasitemia assessment by thick and thin blood films, flow cytometry (YOYO-1_530/585), and quantitative real-time PCR (qRT-PCR). Whole blood was fixed and stained with YOYO-1 for acquisition on a cytometer to assess the frequency of infected erythrocyte events. qRT-PCR was used as the gold standard for the detection of P. falciparum. The YOYO-1_530/585 method was as sensitive and specific as conventional microscopy (area under the receiver operating characteristic, 0.9 for both methods). The interrater mean difference for YOYO-1_530/585 was near zero. Parasite density using flow cytometry and complete blood counts returned density estimates with a mean difference 2.2 times greater than results by microscopy (confidence interval, 1.46 to 3.60) but with limits of agreement between 10 times lower and 50 times higher than those of microscopy. The YOYO-1_530/585 staining pattern was established exactly as demonstrated in animal models, but the assay was limited by the lack of appropriate negative-control samples for establishing background levels and the definition of positives in areas in which malaria is endemic. YOYO-1_530/585 is a high-throughput tool with great potential if the limitations of negative controls and heterogeneous levels of background signal can be overcome.     

Plasmodium falciparum-Specific Cellular Immune Responses after Immunization with the RTS,S/AS02D Candidate Malaria Vaccine in Infants Living in an Area of High Endemicity in Mozambique

Results from clinical trials in areas where malaria is endemic have shown that immunization with RTS,S/AS02A malaria vaccine candidate induces partial protection in adults and children and cellular effector and memory responses in adults. For the first time in a malaria vaccine trial, we sought to assess the cell-mediated immune responses to RTS,S antigen components in infants under 1 year of age participating in a clinical phase I/IIb trial of RTS,S/AS02D in Mozambique. Circumsporozoite protein (CSP)-specific responses were detected in approximately half of RTS,S-immunized infants and included gamma interferon (IFN-γ), interleukin-2 (IL-2), and combined IL-2/IL-4 responses. The median stimulation indices of cytokine-producing CD4⁺ and CD8⁺ cells were very low but significantly higher in RTS,S-immunized infants than in infants that received the comparator vaccine. Protection against subsequent malarial infection tended to be associated with a higher percentage of individuals with CSP-specific IL-2 in the supernatant (P = 0.053) and with higher CSP-specific IFN-γ-producing CD8⁺ T-cell responses (P = 0.07). These results report for the first time the detection of malaria-specific cellular immune responses after vaccination of infants less than 1 year of age and pave the way for future field studies of cellular immunity to malaria vaccine candidates.Malaria remains one of the major world heath problems affecting between 200 and 400 million people annually and causing 2 to 3 million deaths, mostly children and pregnant women living in sub-Saharan Africa (37). Infections by Plasmodium falciparum, one of the four species of plasmodia that affect humans, cause 80 to 90% of the malaria cases and are responsible for 95% of all malaria-associated deaths (14). Since most of the worldwide malaria burden is due to P. falciparum, efforts for prevention and eradication of malaria have focused on this parasite, and a P. falciparum-customized malaria vaccine is one of most promising tools (12, 25, 26).The most abundant and immunogenic antigen on the surface of Plasmodium sporozoites is the circumsporozoite protein (CSP), which is a target for vaccine development (9, 10, 17, 27). In vaccines based on irradiated sporozoites and CSP in human and mouse models, antibodies to circulating sporozoites, followed by cell-mediated responses to the protein after invasion of hepatocytes, have been described as crucial for the generation of protective responses (7, 11, 13, 28, 29).RTS,S is a subunit malaria vaccine candidate based on the CSP of P. falciparum that has been under study for many years. The chimeric vaccine contains a portion of the NANP-repeats, all four NVDP-repeats, and the complete carboxyl-terminal region of CSP suggested to be targets for humoral and cellular immunity, along with the amino-terminal region of HbsAg (HBS) (16). The malaria vaccine candidate RTS,S (GlaxoSmithKline, Rixensart, Belgium) formulated with the adjuvant system AS01 or AS02 has proven to confer partial protective immunity against malaria infection in malaria-naive adults (20, 21, 41), as well as in adults and infants in areas where malaria is endemic (2-6). Clinical safety, immunogenicity, and efficacy trials in infants and children have shown RTS,S/AS02 to be safe and protective and to induce high antibody titers (2, 4, 6, 34).Although the induction of a CSP-specific humoral response after RTS,S vaccination has been well described, the generation of cellular immune responses has not yet been addressed in infants or young children immunized with the RTS,S vaccine candidate. In adults, protection conferred by the RTS,S vaccine has been associated with acquisition of strong antibody and cellular responses to the CSP fragment of RTS,S (20, 22). Malaria naive volunteers immunized with RTS,S/AS02 frequently develop strong proliferative and IFN-γ-producing T-cell responses to peptides representing T-cell epitopes (Th2R and Th3R) present in the vaccine (22). A correlation between protection against experimental challenge and the CSP-specific production of IFN-γ by CD4⁺ and CD8⁺ T cells has been described in a limited number of individuals (42).Current efforts are under way to proceed to phase III clinical trials with the RTS,S vaccine, despite no currently identified immune correlates of protection for vaccination with RTS,S in infants or young children. The present study was integrated into a phaseI/IIb clinical trial of the RTS,S/AS02D candidate vaccine in infants in a rural area of malaria endemicity in Mozambique (4). We sought here to examine the cellular responses in infants vaccinated with RTS,S/AS02D and further the development of assays for use in malaria vaccine trials in infants and young children, the population most vulnerable to severe malaria.     

Haematological and biochemical indices in young African children: in search of reference intervals

INTRODUCTION: The reference intervals of haematological and biochemical indices currently used in Africa are derived from data collected from populations living in industrialized countries. Few studies have been performed in Africa questioning the validity of these values when applied to local African populations. OBJECTIVE: To provide reference intervals of haematological [haemoglobin (Hb), white blood cells (WBC), haematocrit (Htc) and platelets] and biochemical indices (ALT, creatinine and bilirubin) for children aged 1-4 from a rural area of southern Mozambique. METHODS: Reference intervals were developed using the 2.5 and 97.5 centiles. Partition tests were performed to evaluate age and gender differences. Quantile regression models were estimated for those variables in which age partition was recommended. Deviances from linearity in the estimated models were evaluated using fractional polynomials of first or second degree. Agreement to classify normality, using the estimated reference intervals or values in use in a western paediatric hospital, was made using the kappa statistic. RESULTS: Reference intervals for Hb, WBC, Htc, platelets, ALT and creatinine show significant differences by age. Gender differences were observed for creatinine values, while for bilirubin there were no significant differences for age or gender. Estimated Hb and Htc reference intervals in African children were lower than the accepted western ones, while ALT values were higher in the former. Agreement between normal classification, using the estimated intervals or the western values, was from slight to fair. CONCLUSIONS: Reference intervals of haematological and biochemical indices based on results from western individuals of the same age are not in agreement with the estimated values for African children. These observed values should not be used as a rule to define normality, but are the ones usually found in this population where anaemia, malaria and high mortality rates are also common.     

Etiology of diarrhea in children younger than 5 years of age admitted in a rural hospital of southern Mozambique

Diarrhea is one of the main causes of morbidity and mortality among children in sub-Saharan Africa and one of the main causes of hospital admissions in rural areas. Stool samples were collected from 529 children admitted with diarrhea to the Manhi?a District Hospital (September 2000 to September 2001) and processed to detect bacterial enteropathogens, parasites, and virus. Diarrheagenic Escherichia coli, isolated from 120 samples (22.6%; enteroaggregative [corrected] [9.6%], enterotoxigenic [6.8%], enteropathogenic [corrected] [4.3%], and verotoxigenic [1.9%]) was the most frequently isolated pathogen, followed by Ascaris lumbricoides (9.3%). Others detected included Salmonella spp. and Giardia lamblia (2.5% each) and Campylobacter spp. (1.7%). A. lumbricoides (92% versus 8%; P<0.001) and Strongyloides stercolaris (100% versus 0%; P=0.008) were most frequently isolated in children older than 12 months of age. Resistance to trimethoprim-sulphametoxazole and ampicillin was high. Etiologic data on diarrheal diseases and susceptibility patterns of diarrheal pathogens are important tools for clinical management and control strategic planning.     

Safety and immunogenicity of the RTS,S/AS02A candidate malaria vaccine in children aged 1-4 in Mozambique

BACKGROUND: The development of a malaria vaccine remains a public health priority for sub-Saharan Africa. RTS,S/AS02A candidate malaria vaccine has been shown to be safe and immunogenic in previous studies in adults and staggered dose-escalation studies in children in The Gambia. However, genetic features and the intensity of malaria transmission may modify the safety and immune response of a vaccine. OBJECTIVE: We carried out a phase I, double-blind randomized controlled trial in 60 children aged 1-4 in Mozambique to evaluate the safety, reactogenicity and immunogenicity of the paediatric vaccine dose (fixed 25 microg RTS,S in 0.25 ml) of RTS,S/AS02A, prior to undertaking a planned larger phase IIb proof-of-concept of efficacy study in the same population. METHOD: Children were randomized to receive either RTS,S/AS02A or Engerix-B vaccine. Monitoring of safety and reactogenicity included detailed clinical and laboratory analyses and assessment of adverse events (AEs). RESULTS: The RTS,S/AS02A was found to be safe and well tolerated. Serious adverse events were balanced between both groups and none was related to vaccination. The frequency of adverse events reported with RTS, S/AS02A was comparable to previous studies in children. Grade 3 AEs were infrequent (one case of pain, one of fever in each group and some swelling greater than 20 mm in diameter), transient and resolved without sequelae. RTS,S/AS02A was highly immunogenic for anti-circumsporozoite protein antibody response and induced a strong anti-hepatitis-B surface antigen response.     

Severe malaria and concomitant bacteraemia in children admitted to a rural Mozambican hospital

Objectives  To describe the prevalence, aetiology and prognostic implications of coexisting invasive bacterial disease in children admitted with severe malaria in a rural Mozambican Hospital.
Methods  Retrospective study of data systematically collected from June 2003 to May 2007 in a rural Mozambican hospital, from all children younger than 5 years admitted with severe malaria.
Results  Seven thousand and forty-three children were admitted with a diagnosis of malaria. 25.2% fulfilled the criteria for severe malaria. 5.4% of the children with severe malaria and valid blood culture results had a concomitant bacteraemia. Case fatality rates of severe malaria cases rose steeply when bacteraemia was also present (from 4.0% to 22.0%, P  < 0.0001), and bacteraemia was an independent risk factor for death among severe malaria patients (adjusted OR 6.2, 95% CI 2.8–13.7, P  = 0.0001). Streptococcus pneumoniae , Gram-negative bacteria, Staphilococcus aureus and non-typhoid Salmonella (NTS) were the most frequently isolated microorganisms among severe malaria cases. Their frequency and associated case fatality rates (CFR) varied according to age and to syndromic presentation. Streptococcus pneumoniae had a relatively low CFR, but was consistently associated with severe malaria syndromes, or anaemia severity groups. No clear-cut relationship between malarial anaemia and NTS bacteraemia was found.
Conclusions  The coexistence of malaria and invasive bacterial infections is a frequent and life-threatening condition in many endemic African settings. In Mozambique, S. pneumoniae is the leading pathogen in this interaction, possibly as a consequence of the high HIV prevalence in the area. Measures directed at reducing the burden of both those infections are urgently needed to reduce child mortality in Africa.     

Acute bacterial meningitis among children, in Manhiça, a rural area in Southern Mozambique

INTRODUCTION: Acute bacterial meningitis (ABM) is one of the most severe diseases in Sub-Saharan Africa. Although data for the continent is very limited, more than one million cases are estimated per year, with mortality and life-long sequelae occurring in 50% of these cases. METHODS: As part of the clinical management of children admitted to the Manhi?a District Hospital, information on cases of ABM was recorded. We analysed data from June 1998 to November 2003. RESULTS: During the study period, 475 cerebrospinal-fluid (CSF) samples were collected from 20,173 children <15 years of age admitted to hospital. Culture results confirmed 71 (15%) cases of ABM. The most prevalent bacterial aetiologies were Streptotoccus pneumoniae (pneumococcus, n=31), Haemophilus influenzae (n=13) and Neisseria meningitis (n=8). Other important bacteria were Streptococcus sp. (n=7), Salmonella sp. (n=4) and Staphylococcus aureus (n=3). Crude incidence rates of ABM and pneumococcal meningitis were 20/100,000 and 10/100,000 children-year-at-risk, respectively. Incidences were more than three times higher in the <1 year age group. Overall case fatality rate was 36%, and was highest for H. influenzae and pneumococcal meningitis (55% and 45%, respectively, p=0.044). Pneumococcal susceptibility was 81% for oxacillin and 93% for chloramphenicol. For H. influenzae isolates, susceptibility was 54% for ampicillin and 62% for chloramphenicol. CONCLUSIONS: S. pneumoniae and H. influenzae are the main aetiologies responsible for the high burden of morbidity and mortality associated with ABM in rural Mozambique. These findings are important to evaluate treatment guidelines and potential impact of control measures.